U.S. patent application number 10/052545 was filed with the patent office on 2002-10-03 for human melanocyte stimulating hormone receptor polypeptide and dna.
This patent application is currently assigned to WA Pharm AB. Invention is credited to Chhajlani, Vijay, Wikberg, Jarl.
Application Number | 20020142392 10/052545 |
Document ID | / |
Family ID | 27220753 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142392 |
Kind Code |
A1 |
Wikberg, Jarl ; et
al. |
October 3, 2002 |
Human melanocyte stimulating hormone receptor polypeptide and
DNA
Abstract
Novel DNA fragments encoding novel polypeptide having properties
of melantropic hormone receptors, especially DNA fragments encoding
melanocyte stimulating hormone receptors (MSH receptors), as well
as polypeptide which are MSH receptors, are disclosed. The use and
engineering of melanotropic hormone receptor DNA and polypeptide
for production of monoclonal antibodies for diagnostic and
therapeutic purposes, as well as the engineering of drugs, cell
lines, vectors and DNA for therapeutic and diagnostic purposes are
also disclosed. Also disclosed are methods for therapy and
diagnosis of malignant melanoma, skin cancer, vitiligo, pyretic
condition, inflammatory condition, nociceptive condition, catatonic
condition, impaired memory condition, reduced or increased skin
tanning, pigmentation condition, epilepsy and nerve damage, using
the DNA fragments, polypeptide and antibodies. Methods for
selecting substances which interact with the receptors are also
disclosed.
Inventors: |
Wikberg, Jarl; (Umea,
SE) ; Chhajlani, Vijay; (Uppsala, SE) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Assignee: |
WA Pharm AB
|
Family ID: |
27220753 |
Appl. No.: |
10/052545 |
Filed: |
January 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10052545 |
Jan 23, 2002 |
|
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08387805 |
Feb 21, 1995 |
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08387805 |
Feb 21, 1995 |
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PCT/DK93/00273 |
Aug 20, 1993 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 16/2869 20130101;
C07K 2319/00 20130101; C07K 14/72 20130101; A61K 38/00 20130101;
C07K 2317/34 20130101; A61P 29/00 20180101; A61P 17/16 20180101;
A61P 25/28 20180101; A61P 35/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C07K 014/72; C12P
021/02; C12N 005/06; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 21, 1992 |
DK |
1046/92 |
Sep 10, 1992 |
DK |
1118/92 |
May 5, 1993 |
DK |
0528/93 |
Claims
1. A DNA fragment having the nucleotide sequence shown in SEQ ID
NO:1 or an analogue or subsequence thereof which 1) has a homology
with the DNA sequence shown in SEQ ID NO:1 of at least 50%, and/or
2) encodes a polypeptide, the amino acid sequence of which is at
least 50% homologous with the amino acid sequence shown in SEQ ID
NO:2, and/or 3) encodes a polypeptide which binds an antibody which
is also bound by an MSH receptor, and/or 4) encodes a polypeptide
which is an MSH receptor or which has the same binding capacity as
an MSH receptor.
2. A DNA fragment having the nucleotide sequence shown in SEQ ID
NO:15 or an analogue or subsequence thereof which 1) has a homology
with the DNA sequence shown in SEQ ID NO:15 of at least 50%, and/or
2) encodes a polypeptide, the amino acid sequence of which is at
least 50% homologous with the amino acid sequence shown in SEQ ID
NO:16, and/or 3) encodes a polypeptide which binds an antibody
which is also bound by an MSH receptor, and/or 4) encodes a
polypeptide which is an MSH receptor or which has the same binding
capacity as an MSH receptor.
3. A DNA fragment which is a subsequence of the DNA fragment
according to claim 1 or 2 which comprises at least 15 nucleotides,
tides, preferably at least 18 nucleotides, more preferably at least
21 nucleotides, even more preferably at least 27 nucleotides and
most preferably at least 51 nucleotides.
4. A DNA fragment according to claim 1 encoding a polypeptide
comprising amino acids no. 1-317 shown in SEQ ID NO:2.
5. A DNA fragment according to claim 2 encoding a polypeptide
comprising amino acids no. 1-325 shown in SEQ ID NO:16.
6. A DNA fragment which shows at least 55% homology, preferably at
least 70%, more preferably at least 80% and most preferably at
least 95% homology, to the DNA sequence shown in SEQ ID NO:1 or SEQ
ID NO:15.
7. A DNA fragment which is a modified DNA fragment or a subsequence
thereof which differs from the DNA sequence SEQ ID NO:1, SEQ ID
NO:15, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 or the corresponding
subsequence in that at least one nucleotide has been substituted,
added, inserted, deleted and/or rearranged.
8. A DNA fragment which is a fusion DNA fragment comprising a DNA
fragment according to any of the claims 1-7 in frame with one or
more second DNA fragments different from or identical to the DNA
fragment according to any of claims 1-7, the second DNA fragment
preferably selected from the group consisting of DNA fragments
encoding, diphtheria toxin, a staphylococcus protein, a ricin
toxin, a pseudomonas endotoxin, abrin and fungal
ribosome-inactivation proteins (RIP), the resulting DNA fragment
encoding a fusion protein.
9. A DNA fragment having the nucleotide sequence shown in SEQ ID
NO:3 or an analogue thereof, wherein the nucleotides 13 and/or 15
and/or 23 optionally are substituted by C, or the nucleotide
sequence shown in SEQ ID NO:4, or an analogue thereof, wherein the
nucleotides 19 and/or 29 and/or 32 optionally are substituted by C
and wherein the nucleotides 20 and/or 31 optionally are substituted
by G.
10. A polypeptide having the amino acid sequence shown in SEQ ID
NO:2 or an analogue or subsequence thereof which 1) is an MSH
receptor or is capable of binding to MSH or an analogue thereof,
and/or 2) is encoded by a DNA fragment which is at least 50%
homologous with the DNA fragment shown in SEQ ID NO:1, and/or 3)
binds an antibody which is also bound by an MSH receptor.
11. A polypeptide having the amino acid sequence shown in SEQ ID
NO:16 or an analogue or subsequence thereof which 1) is an MSH
receptor or is capable of binding to MSH or an analogue thereof,
and/or 2) is encoded by a DNA fragment which is at least 50%
homologous with the DNA fragment shown in SEQ ID NO:15, and/or 3)
binds an antibody which is also bound by an MSH receptor.
12. A polypeptide which is a subsequence of the polypeptide
according to claim 10 comprising from 5 to 316 amino acids,
preferably at least 7 amino acids, more preferably at least 10
amino acids, even more preferably at least 15 amino acids and most
preferably at least 30 amino acids.
13. A polypeptide which is a subsequence of the polypeptide
according to claim 11 comprising from 5 to 324 amino acids,
preferably at least 7 amino acids, more preferably at least 10
amino acids, even more preferably at least 15 amino acids and most
preferably at least 30 amino acids.
14. A polypeptide which shows at least 55% homology, preferably at
least 70%, more preferably at least 80% and most preferably at
least 95% homology, to the polypeptide shown in SEQ ID NO:2 or the
polypeptide shown in SEQ ID NO:16.
15. A polypeptide consisting of or comprising a subsequence of the
polypeptide shown in SEQ ID NO:2, the subsequence being selected
from the group consisting of amino acids 1-40, 99-117, 181-189,
268-277, 62-76, 141-158, 212-244, 300-317, 39-63, 75-100, 116-141,
157-182, 188-213, 243-269 and 276-301, and analogues thereof, or a
subsequence of the polypeptide shown in SEQ ID NO:16 the
subsequence being selected from the group consisting of amino acids
1-38, 97-115, 179-187, 265-274, 61-74, 138-156, 211-240, 297-326,
37-62, 73-98, 114-139, 155-180, 186-212, 239-266 and 273-298, and
analogues thereof.
16. A polypeptide according to any of claims 10-15 which is
glycosylated and/or coupled to a carbohydrate or lipid moiety
and/or contains a palmitoyl anchor or a part thereof and/or
provided with a detectable label and/or coupled to a solid
support.
17. A polypeptide which is a fusion polypeptide comprising a
polypeptide according to any of claims 10-16 or a subsequence
thereof fused to a second polypeptide which may be different from
or identical to the polypeptide according to any of claims 10-16,
which fusion polypeptide preferably has retained the capability of
binding to MSH or an analogue thereof.
18. A polypeptide according to any of claims 10-17 in substantially
pure form.
19. A polypeptide according to any of claims 10-18 in lipid soluble
form.
20. A DNA fragment or a subsequence or analogue thereof which shows
a homology with any of the nucleotide sequences shown in SEQ ID
NO:5, SEQ ID NO:7 or SEQ ID NO:9 of at least 40%, preferably 50%,
more preferably at least 55%, even more preferably at least 70%,
still more preferably at least 80% and most preferably at least
95%, and/or which can be isolated by using the nucleotide sequence
shown in SEQ ID NO:13 and/or SEQ ID NO:14 as a primer, and/or which
has the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7 or
SEQ ID NO:9.
21. A DNA fragment which is a fusion DNA fragment comprising a DNA
fragment according to claim 20 in frame with one or more second DNA
fragments different from or identical to the DNA fragment according
to claim 20, the second DNA fragment preferably being selected from
the group consisting of DNA fragments encoding a melanotropic
hormone receptor, an MSH receptor and an ACTH receptor.
22. A polypeptide or a subsequence or analogue thereof, which shows
a homology of at least 40%, preferably at least 50%, more
preferably at least 55%, even more preferably at least 70%, still
more preferably at least 80% and most preferably at least 95% with
the polypeptide shown in SEQ ID NO:6, SEQ ID NO:8 or SEQ ID
NO:10.
23. A polypeptide which is a fusion polypeptide comprising a
polypeptide according to claim 22 or a subsequence thereof fused to
a second polypeptide which may be different from or identical to
the polypeptide according to claim 22, the second polypeptide
preferably being selected from the group consisting of a
melanotropic hormone receptor, an MSH receptor and an ACTH receptor
or an analogue or subsequence thereof.
24. A DNA fragment coding for a polypeptide as defined in any of
claims 10-19, 22 and 23.
25. A replicable expression vector carrying a DNA fragment
according to any of claims 1-9, 20, 21 and 24, which vector is
capable of replicating in a host organism or a cell line.
26. A vector according to claim 25 which is pB-11D deposited under
the deposition number DSM 7214 at Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, or pE-MC-2 deposited under
the deposition number DSM 8440 at Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH.
27. A cell which carries and is capable of replicating the DNA
fragment according to any of the claims 1-9, 20, 21 and 24.
28. A cell according to claim 27, which is selected from the group
consisting of a bacterium, a yeast and a protozoan, or the cell is
derived from a multicellular organism selected from the group
consisting of a fungus, an insect, a plant, and a mammal, and the
cell preferably being a bacterium selected from the group
consisting of the genus Bacillus, Escherichia and Salmonella.
29. A method of producing a polypeptide as defined in any of claims
10-19, 22 and 23, comprising the following steps of: (a) inserting
a DNA fragment as defined in any of the claims 1-9, 20, 21 and 24
into an expression vector, (b) transforming a suitable host cell
according to claim 27 or 28 with the vector produced in step (a),
(c) cultivating the host cell produced in step (b) under suitable
conditions for expressing the polypeptide, (d) harvesting the
polypeptide, and (e) optionally subjecting the polypeptide to
posttranslational modification, or comprising liquid and/or solid
phase peptide synthesis procedures.
30. A stable cell line which produces the polypeptide according to
claim 10 or 11 and which preferably contains and expresses cDNA
encoding the polypeptide of claim 10 or claim 11.
31. A method of preventing or stimulating the coupling of an MSH
receptor to its guanine nucleotide binding protein in an animal, in
particular a mammal, comprising administering a substance which in
advance has been found to bind to a polypeptide according to any of
claims 10-19 so as to occupy one or several of the cytoplasmic
loops and/or the C-terminal sequence.
32. A method of preventing or stimulating the binding of MSH and
similar peptides to an MSH receptor in an animal, in particular a
human, comprising administering, to the animal, a substance which
in advance has been found to bind to a polypeptide according to any
of claims 11-19 so as to occupy the binding site of the receptor
using an antagonist, a blocker or a compound such as a derivative
of MSH having a structure similar to MSH, and optionally thereby
preventing or stimulating the generation of second messenger
elements.
33. A method of increasing or decreasing the generation of second
messenger elements, and/or increasing or decreasing the production
of an MSH receptor and/or optionally increasing or decreasing the
binding affinity of MSH to an MSH receptor, comprising
administering to an animal, in particular a human, a medicament
which is or becomes bound to a substance which in advance has been
found to bind to a polypeptide according to any of claims
10-19.
34. A method of targeting, with a medicament, a cell that contains
an MSH receptor on its surface, comprising administering a
substance optionally linked to a medicament which substance in
advance has been found to bind to a polypeptide according to any of
claims 10-19 and which substance binds to the MSH receptor.
35. A method according to the claim 34, wherein the medicament is a
radionuclide or a toxin or any other molecule of natural or
synthetic origin.
36. A method according to any of claims 31-35 for the treatment of
an MSH receptor expressing disease condition selected from the
group consisting of melanoma, skin cancer, vitiligo, pyretic
condition, inflammatory condition, nociceptive condition, catatonic
condition, impaired memory condition, reduced or increased skin
tanning, pigmentation condition, epilepsy and nerve damage.
37. A method for treating conditions caused by MSH receptor
deficiency or impaired MSH receptor function in a mammal, such as a
human, comprising administering a polypeptide according to claim 19
which is an MSH receptor, or is capable of binding to MSH, or is an
analogue thereof, to the mammal.
38. A method for treating conditions caused by MSH receptor
deficiency or impaired MSH receptor function, such as
tyrosinase-positive albinism, in a mammal, such as a human,
comprising introducing a DNA fragment according to any of claims
1-9, 20, 21 and 24 encoding an active form of an MSH receptor.
39. A method for increasing or decreasing the melanin content of
the skin in a mammal, such as a human, comprising administering
substances as defined in any of claims 31-34 that are active
through an MSH receptor, preferably to increase the skin tanning
without or with reduced exposure to sunlight or to avoid
sunburns.
40. A method of activating the antipyretic and/or anti-inflammatory
and/or antinociceptive and/or memory improving and/or nerve
regenerating action effected via an MSH receptor, comprising
administering a substance that acts on the MSH receptor to bring
about the antipyretic and/or antiinflammatory and/or
antinociceptive and/or memory improving and/or nerve regenerating
actions.
41. A method of diagnosing an MSH receptor expressing disease
condition such as melanoma or skin cancer, comprising targeting a
cell containing an MSH receptor on its surface with diagnostic
agent capable of binding to the MSH receptor, which diagnostic
agent can be detected following binding to the receptor.
42. The method according to claim 41, as used in the assessment of
the prognosis and/or guidance for further treatment of melanoma or
skin cancer.
43. A method for detecting an MSH receptor in a biological sample,
such as a tissue sample, a cell culture or a cell suspension,
wherein the sample is treated with a optionally labelled substance
that binds to the MSH receptor, and detecting or visualizing the
presence of the bound substance.
44. An antibody optionally provided with a detectable label which
antibody is reactive with a polypeptide according to any of claims
10-19, 22 and 23 and which is preferably a monoclonal antibody.
45. A method for detection and/or quantitation of the MSH receptor
mRNA, comprising extracting RNA from a biological sample such as a
cell, a tissue sample, a cell culture or a cell suspension and
measuring the hybridization of said RNA to a labelled DNA fragment
according to any of claims 1-9, 20, 21 and 24 or a labelled RNA
fragment constructed from the DNA fragment according to any of
claims 1-9, 20, 21 and 24.
46. A method for detection and/or quantitation of the MSH receptor
mRNA, comprising extracting RNA from cells or tissues and
converting it into cDNA for subsequent use in the polymerase chain
reaction (PCR), preferably using PCR primer(s) which is/are
synthesized based on the DNA fragment claimed in any of claims 1-9,
20, 21 and 24.
47. The use of any of the methods of claims 43, 45 and 46 for
diagnosis of an MSH receptor expressing disease condition such as
melanoma and skin cancer.
48. The use of a DNA fragment according to any of claims 1-9, 20,
21 and 24 for the isolation of other similar DNA fragments using
techniques such as PCR or hybridization.
49. The use of a polypeptide according to any of claims 10-19, 22
and 23 for designing DNA probes for use in techniques such as PCR
and hybridization.
50. The use of a polypeptide according to any of claims 10-19, 22
and 23 for the deduction of the three-dimensional structure of an
MSH receptor or an analogue thereof having MSH binding capacity for
use in the design of a substance capable of binding to the MSH
receptor.
51. A method for selecting a substance which is capable of binding
to a melanotropic hormone receptor polypeptide such as an MSH
receptor and which substance may optionally be capable of
preventing or stimulating the generation of a second messenger
element in a cell such as a mammalian cell, in particular a human
cell, by its binding to the melanotro- pic hormone receptor
polypeptide the method comprising one or more of the following
steps: 1a) incubating a sample containing a melanotropic hormone
receptor polypeptide or an analogue thereof, the melanotropic
hormone receptor polypeptide preferably being a polypeptide
according to any of claims 10-19, 22 and 23, with radioactively
labelled melanotropic hormone or an analogue thereof and with the
substance to be tested, and 1b) measuring the binding affinity of
the substance to be tested for the melanotropic hormone receptor
polypeptide by separating bound from free labelled melanotropic
hormone or an analogue thereof using either filtration,
centrifugation, superflow or chromatography followed by measuring
the radioactivity retained in the sample by standard nuclear
counting, or 2a) incubating a sample containing a melanotropic
hormone receptor polypeptide or an analogue thereof, the
melanotropic hormone receptor polypeptide preferably being a
polypeptide according to any of claims 10-19, 22 and 23, with
melanotropic hormone or an analogue thereof and with the substance
to be tested, and 2b) measuring the binding affinity of the
substance to be tested for the melanotropic hormone receptor
polypeptide by separating free melanotropic hormone or the analogue
thereof from the bound melanotropic hormone or the analogue thereof
using either filtration, centrifugation, superflow or
chromatography followed by measuring the bound melanotropic hormone
or the analogue thereof by a detection system capable of detecting
melanotropic hormone or the analogue thereof, preferably using a
detection system such as radio immunoassay, immunefluorescense
assay, UV light absorption spectrometry or fluorescence emission
spectrometry.
52. A method for selecting a substance which is-capable of binding
to a melanotropic hormone receptor polypeptide such as an MSH
receptor and which substance may optionally be capable of
preventing or stimulating the generation of a second messenger
element in a cell such as a mammalian cell, in particular a human
cell, by its binding to the melanotropic hormone receptor
polypeptide the method comprising one or more of the following
steps: 1a) incubating a sample containing a melanotropic hormone
receptor polypeptide or an analogue thereof linked to a solid
support, the melanotropic hormone receptor polypeptide preferably
being a polypeptide according to any of claims 10-19, 22 and 23,
with melanotropic hormone or an analogue thereof- and with the
substance to be tested, and 1b) measuring the binding affinity of
the substance to be tested for the melanotropic hormone receptor
polypeptide or an analogue thereof by separating free melanotropic
hormone or an analogue thereof from the melanotropic hormone or an
analogue thereof bound to the melanotropic hormone receptor
polypeptide or analogue thereof by washing, followed by measuring
the bound melanotropic hormone or an analogue thereof by using a
ligand, preferably an antibody, capable of binding to the bound
melanotropic hormone or an analogue thereof which ligand is in
itself detectable, or which ligand is a first ligand which can be
rendered detectable using a second ligand, preferably an antibody
capable of binding to the said first ligand, or 2a) incubating a
sample containing, preferably in a soluble form or in a solid phase
being attached to a matrix, the melanotropic hormone receptor
polypeptide which preferably is a polypeptide according to any of
claims 10-19, 22 and 23 or an analogue thereof, with melanotropic
hormone and with the substance to be tested, and 2b) measuring the
alteration in the degree of interaction of the melanotropic hormone
receptor with a G-protein caused by the binding of the substance to
be tested to the melanotropic hormone receptor.
53. A method according to any of claims 31-34, 39, 40, 43, and
50-52 wherein the substance is an antibody or a part thereof or a
molecule of natural or synthetic origin having affinity for an MSH
receptor or the melanotropic hormone receptor polypeptide.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a DNA fragment encoding the
human melanocyte stimulating hormone receptor (MSH receptor) or an
analogue or subsequence thereof. The DNA fragment contains an open
reading frame of 951 bp which codes for a polypeptide of 317 amino
acids, said DNA fragment as well as its analogues, subsequences or
modifications constitute an important aspect of the invention. The
DNA fragment has been expressed in an eukaryotic cell line and the
expressed protein has been found to have properties identical to
that of a native MSH receptor. The invention also relates to a DNA
fragment encoding a subtype of the human MSH receptor (in this
application designated MC-2) which contains an open reading frame
of 975 bp which codes for a polypeptide of 325 amino acids, said
DNA fragment as well as its analogues, subsequences or
modifications also constitute an important aspect of the invention.
This DNA fragment has also been expressed in an eukaryotic cell
line and the expressed protein has been found to have properties to
that of an MSH receptor subtype.
[0002] The invention also relates to a polypeptide encoded by a DNA
fragment of the invention and to analogues and subsequences of said
polypeptide. Furthermore, the invention relates to the use of the
DNA fragments or analogues or subsequences thereof, and to the use
of polypeptides of the invention encoded by the DNA fragments of
the invention. Especially interesting is the use of the
polypeptides of the invention which have MSH receptor activity. The
use of the polypeptides of the invention or analogues or
subsequences thereof for generation of antibodies constitutes yet
another aspect of the invention. Also, the invention relates to
diagnostic and therapeutic methods and diagnostic and therapeutic
agents for use in the diagnosis and treatment of MSH receptor
expressing disease conditions such as vitiligo, melanoma, skin
cancer, pyretic conditions, inflammatory conditions and nociceptive
conditions, catatonic conditions and impaired memory conditions,
and to methods for detecting and quantitating the MSH receptor. In
addition, the invention provides methods for testing substances
capable of interfering with the activity of the MSH receptor and
methods for treatment of MSH receptor expressing disease
conditions. The patent application also relates to the use of the
MSH receptor coding fragments or the MSH receptor during
non-disease conditions for the control or diagnosis and/or
determination and/or production control of skin and/or hair and/or
fur color in man and/or animals. Moreover, the patent application
relates to the elucidation of the structure of the MSH receptor in
three dimensions by the utilization of computer modelling methods
and/or by application of structure analysis by crystallographic
approaches and/or NMR (Nuclear Magnetic Resonance) and to the use
of the knowledge of the receptor structure for the design of drugs
with binding affinity for the MSH receptor and/or its subtype
(MC-2).
[0003] The present invention which comprises a DNA fragment
encoding the MSH receptor or analogues thereof and the application
of these and in this connection methods for identifying products
which pertains to the MSH receptor and/or its biological functions
constitutes significant contributions which will become useful for
biotechnological, pharmaceutical, medical and veterinary practices.
As a background to the uses of a DNA fragment and analogues and
subsequences thereof and the application of these, some of the most
important facts regarding the MSH receptor and its biological
functions in man and animals are summarized below.
GENERAL BACKGROUND
[0004] Although information existed regarding the MSH receptor
(reviewed below), the structure of the MSH receptor gene as well as
the primary amino acid sequence of the MSH receptor has not been
known before the priority date of the present patent application.
As appears from the following, the MSH receptor is a very important
receptor with a number of different functions such as
anti-inflammatory and antipyretic function and involved in a number
of diseases such as melanoma and skin cancer and moreover, it is
having an important role in the control of skin, hair and fur color
in man and animals.
MSH Receptor and its Biological Functions
[0005] The MSH receptor belongs to a large class of receptors
showing functional and structural similarities. These receptors
mediate their cellular effects via coupling proteins termed guanine
nucleotide regulatory proteins (G-proteins), of which several types
are known (e.g. G.sub.s, G.sub.i, G.sub.k and others). The MSH
receptor is a cell membrane bound protein which serves as a
recognition site for .alpha.-MSH (melanocyte stimulating hormone).
The term MSH relates to several peptides among which .alpha.-MSH,
.alpha.-MSH and .gamma.-MSH may be mentioned; the .alpha.-MSH
generally showing the largest activity. These hormones are
generally referred to as melanotropic hormones to which also the
ACTH (adrenocorticotropic hormone) belongs as well as a number of
related peptides, being present in man and animals. In the present
patent application, peptides which have binding affinity for MSH
receptors will collectively be referred to as MSH peptides or MSH
receptor ligands. Upon binding of MSH receptor ligands to the MSH
receptor, an activation of the receptor ensues which leads to
altered activity of the cell in which the receptor is located. MSH
receptors are known to be present in melanocytes which are pigment
cells and in humans give the skin a varying amount of dark
pigmentation and which have a role in protecting the skin from
UV-radiation. In animals, melanocytes also have a role in skin
pigmentation. In both animals and man changes in skin color are at
least partly mediated by melanocytes and these changes are also
partly regulated by the degree of activation of MSH receptors by
the peptide hormones that bind to the MSH receptor (Nordlund 1991;
Levine 1991). MSH receptors may also be localized in cell types
other than melanocytes (Tatro 1987) where they may have other types
of important physiological roles. .alpha.-MSH is known to be
produced in certain areas of the brain, such as the hypothalamus,
corpus amygdaloideum and cerebral cortex. Moreover,
proopiomelanocortin, which is the precursor molecule for
.alpha.-MSH, is found in lymphocytes of the thymus and spleen,
neutrophils, placenta, ovary as well as in the epidermis (Nordlund
1991). There is evidence that by acting on MSH receptors,
.alpha.-MSH may have roles in (i) mediating neurotransmitter
effects in the CNS, (ii) participating in endocrine regulation,
(iii) modulating immune-inflammatory responses, besides (iv)
regulating the skin pigmentation, as mentioned above (Nordlund
1991, Levine 1991). MSH receptors perform various functions in
neurochemical processes, such as the induction of antinociceptive
effects, the perturbation of grooming behaviour, the alteration of
stretch and yawn reflexes and the potentiation of catatonic states
(Hirsh and O'Donohue 1986). Moreover, MSH receptors are implicated
to have a function in the enhancement of visual and verbal learning
(Veith et al. 1978; Ward et al. 1979, Handelman et al. 1983). The
role for MSH receptors in endocrine function is indicated, for
example, observations that .alpha.-MSH may affect cortisol
secretion from the adrenal gland, and increase plasma levels of
growth hormone, luteinizing hormone and follicle-stimulating
hormone (Reid et al. 1984).
[0006] NSH receptors also seem to be mediating the powerful
antipyretic effect caused by .alpha.-MSH (Clark et al. 1985) as
well as the anti-inflammatory actions induced by .alpha.-MSH
(Rheins et al. 1989). Central MSH receptors are also involved in
the mediation of anti-convulsive effects since MSH peptides exert
anti-epileptic effects (De Wied 1993). Moreover, MSH receptors seem
to mediate the growth factor effect of MSH peptides which mediates
accelerated and enhanced nerve generation and muscle reinnervation
after peripheral nerve injury (Strand et al. 1993).
MSH Receptor on Melanoma Cells
[0007] Melanoma cell lines are derived from immortalized
melanocytes. Melanocytes are clinically the starting point of
malignant melanoma (reviewed below). MSH receptors are present on
many such melanoma cell lines, the reported frequency in different
cell lines being more than 70% (Tatro et al. 1990a). In
experimental melanoma, differentiation, tumorigenicity and
metastatic potential of the melanoma are influenced by MSH
(Kameyama et al. 1990). Moreover, .alpha.-MSH immunoreactivity has
been demonstrated to be present in human melanoma metastases
(Ghanem et al. 1989) indicating the possibility that locally formed
melanotropic activity has a role in the pathogenesis of
melanoma.
[0008] The presence of MSH receptors on melanoma cell lines
suggests that endogenous .alpha.-MSH, the major known form of
circulating melanotropin in mammals, may modulate melanoma cell
activity in vivo. The demonstration of specific binding sites in
melanoma tumours does not prove that these are linked to cellular
response systems in vivo, but this seems highly likely in view of
the close relationship between binding and biological responses in
cultured melanoma cells (Tatro et al. 1990b). Evidence suggests
that .alpha.-MSH may modulate proliferation and ability of melanoma
cells to establish metastatic colonies (Lerner et al. 1989;
Abdel-Malek et al. 1986).
[0009] It is well recognized that in mammalian melanocytes and
melanoma cells .alpha.-MSH acts through MSH receptor on an
intracellular pathway that involves the activation of adenylate
cyclase (Tatro et al. 1990b). This leads to an increase in the
production of cyclic AMP which in turn induces tyrosinase, a key
enzyme in the melanin biosynthesis. However, there is evidence that
melanotropins after binding to the MSH receptor increase the
intracellular calcium (Mac Neil et al. 1990). It is conceivable
that this effect is due to the fact that MSH/MSH receptor complex
activates phospholipase C (PLC), which then acts to produce
inositol 1,4,5-trisphosphate, which then in turn triggers
mobilization of intracellular calcium. This proposition is due to
the fact that receptor mediated activation of PLC is a G-protein
linked event, and that it has been shown that receptors may
simultaneously, e.g. in a promiscuous way, act via several of the
known G-protein linked metabolic pathways (Traiffort et al. 1992
and Gudermann et al. 1992). Activation of phospholipase C also
leads to the production of diacylglycerol, the activator of protein
kinase C. Indeed, it has recently been shown that MSH can activate
protein kinase C (Buffey et al. 1992). Two other G-protein coupled
receptors, namely the .alpha..sub.1b-adrenergic receptor and
serotonin receptor, which are also coupled through the above
mentioned second messenger system, are shown to be protooncogenic
(Allen et al. 1991; Julius et al. 1989), thus further indicating
the possibility that MSH receptor may have a pathogenic role in
melanoma. Moreover, melanotropins are shown to induce expression of
the growth associated oncogene c-fos (Hart et al. 1989) further
supporting this notion. Note also Sukhanov et al. (1991).
Malignant Melanoma
[0010] Malignant melanoma (melanocarcinoma) is a malignancy derived
from melanocytes. About 1 of all malignant tumours are malignant
melanomas. The incidence of malignant melanoma is increasing
rapidly. During the last decades the incidence has approximately
doubled every 10 years with both sexes being affected equally.
Malignant melanoma can develop at every site of the skin. There are
sites of predilection: feet followed by head and neck. Infrequent
sites are the genital organs, perineum, perianal region and mucous
membranes. The tumour has a high incidence of metastasis to
adjacent skin and regional lymph nodes. Haematogenous metastasis
may also occur.
[0011] The main factor for the development of malignant melanoma is
exposure to sunlight. The people who are mainly affected are those
who have fair skin that can be easily damaged by the sunlight.
Despite various therapeutic regimes the 5 year survival in melanoma
with distant metastasis is only 5% and with regional metastasis it
is 43% (Roses et al. 1991). Existing clinically approved therapies,
besides surgical removal of lesions, are non-specific and include
limb perfusion, chemotherapy, immunotherapy, radiotherapy and
hormonal therapy (Ho et al. 1990). Radiopharmaceuticals such as
iodoquinoline (Lambrecht et al. 1984), iodothiouracil (Coderre et
al. 1986) and N-(2-diethylaminoethyl)-4- -iodobenzamide (Michelot
et al. 1991) have been used for the diagnosis and therapy of
melanoma, albeit with very limited success. Another approach for
the diagnosis and therapy of melanoma is to use radiolabelled
monoclonal antibodies against melanoma associated antigens (Eary et
al. 1989; Larson 1991). This poses the problem of having a true
melanoma associated antigen. Also, different antigens are expressed
based on the developmental stage of the melanoma tumour, and
different tumour sites in the body may be expressing different
antigens. This would require the use of a mixture of monoclonal
antibodies, all of them with very high specificity. The composition
of such a mixture will vary between patients and between different
tumour stages of the same patient. All this would be very difficult
to achieve. Previous work has shown that MSH receptors are
detectable in melanoma metastases of about 80% of human patients
(see Tatro et al. 1992).
[0012] MSH receptors on the melanoma cells have been considered as
potential targets for novel drugs useful for treatment of the
disease. Diphtheria toxin and .alpha.-MSH fusion protein have been
constructed and shown to be selectively toxic for MSH receptor
bearing cells in vitro by a targeted delivery of the diphtheria
toxin (Murphy et al. 1986; Wen et al. 1991; Tatro et al. 1992). In
another approach MSH was coupled with an antibody directed towards
the CD3 receptor of cytotoxic T cells. The complex was shown to
mediate cell lysis of melanoma cells in vitro. The MSH moiety binds
to the MSH receptor of the melanoma cells whereas the antibody tags
CD3 bearing cytotoxic T-cells which mediate lysis of the melanoma
cell (Liu et al. 1988).
MSH Receptor and Skin Tanning and Control of Hair and Fur Color
[0013] Endogenous and exogenous melanotropins are suggested to
enhance human cutaneous pigmentation in vivo (Levine 1991; Mulligan
et al. 1982; Lerener et al. 1961). The mechanism of action by which
MSH and other melanotropins stimulate melanogenesis is well
studied. The melanotropins bind MSH receptors on melanocytes and
result in the activation of adenylate cyclase. Increased cAMP
activates tyrosinase enzyme which converts tyrosine to dopa and
dopa to dopaquinone, resulting in melanin formation. The melanin
thus formed is partly secreted from the melanocytes and taken up by
keratinocytes of the skin thus making the skin color become more
dark. Moreover, the pigment thus formed will constitute the color
of hair and fur in man and animal. Various colors will be produced
depending on the level of the presence of melanin pigment in the
hair, fur and skin.
MSH Receptor and Anti-pyretic and Anti-inflammatory Actions
[0014] The .alpha.-MSH is one of the most potent antipyretic agents
identified (Clark et al. 1985). Moreover, both afferent and
efferent inflammatory responses to chemicals and irritants, like
phorbol esters or contact allergens, are blocked by the topical
application of .alpha.-MSH. These anti-pyretic and
anti-inflammatory effects seem to reside in the carboxy terminal
region of the hormone supporting the notion of their mediation via
an action on MSH receptors.
MSH Receptor and Vitiligo
[0015] In vitiligo areas of loss of skin pigmentation is a
characteristic feature. Such loss of skin color is due to loss
and/or malfunction of pigment cells. Due to localization of MSH
receptor on skin pigment cells it is considered that MSH receptor
has a role in vitiligo. One of the causes of the vitiligo can be
autoimmune reactions of the host against the MSH receptor protein
and/or polypeptides. Thus, the MSH receptor constitute an
interesting target in the cure and/or amelioration of the vitiligo
condition.
DISCLOSURE OF THE INVENTION
[0016] The above summarized activity and involvement of the MSH
receptor in a number of biological functions of various cells
clearly shows the importance of the present invention which relates
to a DNA fragment encoding a polypeptide having MSH receptor
activity. Despite considerable efforts to elucidate the sequence of
such a DNA fragment, nobody had prior to the present invention
succeeded in doing this.
[0017] Accordingly, the present invention relates to a novel DNA
fragment having the nucleotide sequence shown in SEQ ID NO:1 or an
analogue or subsequence thereof which
[0018] 1) has a homology with the DNA sequence shown in SEQ ID NO:1
of at least 50%, and/or
[0019] 2) encodes a polypeptide, the amino acid sequence of which
is at least 50% homologous with the amino acid sequence shown in
SEQ ID NO:2, and/or
[0020] 3) encodes a polypeptide which binds an antibody which is
also bound by an MSH receptor, and/or
[0021] 4) encodes a polypeptide which is an MSH receptor or which
has the same binding capacity as an MSH receptor.
[0022] The DNA fragment with the nucleotide sequence shown in SEQ
ID NO: 1 is derived from a human cDNA library and has been found to
contain an open reading frame of 951 bp which codes for a
previously unknown polypeptide of 317 amino acids which is shown in
SEQ ID NO:2. This polypeptide constitutes the entire polypeptide of
an MSH receptor.
[0023] A detailed description of the molecular cloning and
nucleotide sequencing of the cDNA on the basis of the carefully
constructed primers is given in Example 1. The cDNA of the MSH
receptor represents a rather rare clone, based on the fact that its
messenger RNA was found only in the melanoma cells and not in the
other tissues examined like brain, thymus, parathyroid gland,
parotid gland, salivary gland, adrenal gland, testis, liver, lung,
heart, spleen, skeletal muscle, intestine and colon, cf. Example
1.
[0024] Transmembrane segments of the above-mentioned polypeptide
(corresponding to nucleotides 286-351,-394-465, 517-588, 640-711,
733-804, 898-972 and 997-1068 in SEQ ID NO:1, respectively) were
determined by hydropathy analysis (Kyte et al. 1982).
[0025] Glycosylation sites are found at amino acid residues 15 and
29 in SEQ ID NO:2, possible phosphorylation sites are found at
amino acid residues 42-45, 151-154 and 306-308 SEQ ID NO: 2, and a
possible palmitylation site is found at amino acid residue 316 in
SEQ ID NO:2.
[0026] The abbreviations of the amino acids used herein are the
following:
1 Three-letter One-letter Amino acid abbreviation symbol Alanine
Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine
Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine
His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0027] Each of the nucleotides shown herein is represented by the
abbreviations generally used, i.e.
[0028] A represents deoxyadenine
[0029] T represents deoxythymidine
[0030] G represents deoxyguanine
[0031] C represents deoxycytosine
[0032] N represents deoxyinosine
[0033] Using the DNA fragment G-8 described below (with the
nucleotide sequence shown in SEQ ID NO:7) as a hybridization probe,
another novel DNA fragment has been isolated from a human genomic
library. This DNA fragment is in the present application numbered
as SEQ ID NO:15. This fragment constitutes another interesting
aspect of the invention as it has been shown to code for a
previously unknown polypeptide which is also an MSH receptor and/or
an MSH receptor subtype. The polypeptide encoded by the fragment is
in the present context numbered as SEQ ID NO:16. It is believed
that the polypeptide is melanotropic hormone receptor such as an
.alpha.-MSH receptor and/or a .beta.-MSH receptor and/or a
.gamma.-MSH receptor and/or an ACTH receptor and is interchangeably
referred to as MC-2 and MC-2 receptor herein. The MC-2 receptor is
in particular known to be located in the central nervous system and
also in peripheral organs such as gut, lung, heart, liver, spleen,
smooth and skeletal muscle tissues and the immune system.
[0034] The novel DNA fragment with the nucleotide sequence SEQ ID
NO: 15 comprises 1650 nucleotides and was sequenced as described
herein. The nucleotides from 1 to 615 form the 5' untranslated
region while the nucleotides 1591 to 1650 form the 3' untranslated
region. The coding fragment from nucleotide 616-1590 encodes a
polypeptide of 325 amino acids which is shown in SEQ ID NO:16. The
DNA-fragment was isolated from a human genomic library as described
in Example 6.
[0035] Thus, an aspect of the invention relates to a DNA fragment
having the nucleotide sequence shown in SEQ ID NO:15 or an analogue
or subsequence thereof which
[0036] 1) has a homology with the DNA sequence shown in SEQ ID NO:
15 of at least 50%, and/or
[0037] 2) encodes a polypeptide, the amino acid of which is at
least 50% homologous with the amino acid sequence shown in SEQ ID
NO: 16, and/or
[0038] 3) encodes a polypeptide which binds an antibody which is
also bound by an MSH receptor, and/or
[0039] 4) encodes a polypeptide which is an MSH receptor or which
has the same binding capacity as an MSH receptor.
[0040] While one particular aspect of the invention relates to DNA
fragments having the nucleotide sequence shown in SEQ ID NO: 1 or
in SEQ ID NO:15 and encoding a polypeptide of the invention, an
analogue or subsequence thereof comprising at least 15 nucleotides
is another important aspect of the invention. The invention relates
to the coding part of the described nucleotide sequence shown in
SEQ ID NO:1 or SEQ ID NO: 15 as well as the non-coding part. A DNA
fragment which shows at least 55% homology, preferably at least
70%, more preferably at least 80% and most preferably at least 95%
sequence homology with a DNA fragment of the same length obtained
from the DNA sequence shown in SEQ ID NO:1 or SEQ ID NO:15 is also
an interesting aspect of the invention as such fragments and
subsequences may encode polypeptides capable of acting as epitopes
and thus capable of eliciting an antibody response directed
thereto. Such antibodies can also bind to a polypeptide
constituting an MSH receptor and thereby being important in
diagnosis and treatments of MSH receptor related diseases and
conditions, as will appear from the following. In addition, such
fragments and subsequences may among other utilities be used as
probes in the identification of other DNA fragments as will appear
from the following. In this respect a fragment and/or subsequence
of the non-coding part of the DNA fragments shown in SEQ ID NO:1
and 15 is equally important as the fragments and/or subsequences of
the coding parts of these DNA fragments.
[0041] When used in the present context with regard to nucleotide
sequences, the term "subsequence" indicates a nucleotide sequence
which is derived from a DNA fragment of the invention and which has
retained a characteristic nucleotide sequence thereof as evidenced
by its conforming to at least one of the criteria 1)-4) above.
Typically, the subsequence is a part of a nucleotide sequence shown
in SEQ ID NO:1 or SEQ ID NO:15, the subsequence being either a
consecutive stretch of nucleotides taken from a nucleotide sequence
shown in SEQ ID NO:1 or SEQ ID NO:15 or being composed of one or
more separate nucleotides or nucleotide sequences of a nucleotide
sequence shown in SEQ ID NO:1 and/or SEQ ID NO:15.
[0042] It is important to note that a "characteristic nucleotide
sequence" in the present context is meant to indicate a nucleotide
sequence of a DNA fragment of the invention which is identifying
the DNA fragment according to one or more of the following
criteria:
[0043] it encodes a peptide with binding properties of an MSH
receptor, and/or
[0044] it encodes a peptide which is bound (with high specificity)
by an antibody which also binds (with high specificity) to the
polypeptide encoded by the original DNA fragment from which it is
derived (the binding for instance being assessed as described in
example 10), and/or
[0045] it will be useful as a hybridization probe for identifying
the original DNA fragment from which it is derived.
[0046] When a compound "X", such as a e.g. receptor, "binds" to a
polypeptide, an antibody or another substance "Y" it is in the
present application defined as a substantial specific binding of
"X" to "Y" as assessed by the ability of "X" to distinguish between
"Y" and other substances under physiological conditions (e.g. in a
Ringer solution at 37.degree. C. or e.g. using the binding buffer
and conditions essentially as described in Example 3). It is
preferred that "X" binds "Y" with known affinity. Preferably, the
dissociation constant (defined as 1 K = A X A Y A XY ,
[0047] wherein A.sub.X, A.sub.Y and A.sub.XY are the activities of
"X", "Y" and "XY" in the system XY=X+Y) of the complex "XY" is less
than 100 .mu.M, more preferably less than 1 .mu.M, even more
preferably less than 100 nM and most preferably the dissociation
constant of the complex "XY" is less than 10 nM. Moreover, in a
still further meaning, when "X" is said to bind to "Y", the latter
which is also bound to a compound "Z" with a known high affinity
(it has been established that "Y" binds to "Z" with such high
affinity that it can be regarded as a test-tool), the dissociation
constant of the complex "XY" formed is not more than 100,000-fold
higher than that for "YZ", preferably not more than 10,000-fold,
more preferably not more than 1,000-fold and most preferably not
more than 100-fold higher than that for "YZ".
[0048] One way of determining such binding characteristics is to
employ the method of example 3, wherein a panel of melanotropic
hormones are tested with regard to their ability to inhibit binding
of .sup.125I-NDP-MSH to a suspected MSH receptor.
[0049] According to the above, a degree of homology of-as little as
30% between a DNA fragment and one of the DNA fragments shown in
SEQ ID NO:1 or 15 or a subsequence thereof may in some instances
ensure that a characteristic DNA sequence is retained in the first
DNA fragment. Thus, the invention also relates to DNA fragments
which have retained a characteristic DNA sequence of the DNA
sequences in SEQ ID NO:1 and 15, said characteristic DNA sequences
having as little as 30% homology with any of the sequences shown in
SEQ ID NO:1 and 15. This will most likely be the case when the
characteristic sequence is a part of a functional important part of
the polypeptide and therefore has little resemblance with other
polypeptides. However, it is preferred that the degree of homology
is at least 40% when the characteristic sequence codes a less
functionally important part of the molecule. The degree of homology
may in some instances be so high as 95%; this might be the case
when the characteristic DNA fragment encodes a intramembraneous
part of the polypeptide, wherein the amino acid sequence might be
much like other sequences of transmembraneous polypeptides.
[0050] In the present specification and claims, the term
"subsequence" thus designates a nucleotide sequence which
preferably has a size of at least 15 nucleotides, more preferably
at least 18 nucleotides, still more preferably at least 21
nucleotides, even more preferably at least 27 nucleotides and most
preferably at least 51 nucleotides. It is well known that small
fragments are useful as epitopes, DNA-probes for hybridization with
DNA or RNA, in PCR techniques as is described herein, or useful in
that they encode peptides comprising epitopes capable of eliciting
the production of antibodies.
[0051] The term "analogue" with regard to the DNA fragments of the
invention is intended to indicate a nucleotide sequence which
encodes a polypeptide identical or substantially identical to a
polypeptide encoded by a DNA fragment of the invention shown in SEQ
ID NO:1 or 15.
[0052] It is well known that the same amino acid may be encoded by
various codons, the codon usage being related, inter alia, to the
preference of the organisms in question expressing the nucleotide
sequence. Thus, one or more nucleotides or codons of a DNA fragment
of the invention may be exchanged by others which, when expressed,
result in a polypeptide identical or substantially identical to the
polypeptide encoded by the DNA fragment in question.
[0053] Thus, a DNA fragment encoding a polypeptide comprising the
amino acids 1-317 of SEQ ID NO:2, or a DNA fragment encoding a
polypeptide comprising the amino acids 1-325 SEQ ID NO:16 are very
important embodiments of the invention.
[0054] Also, the term "analogue" is used in the present context to
indicate a DNA fragment or a DNA sequence of a similar nucleotide
composition or sequence as the DNA sequence encoding the amino acid
sequence constituting an MSH receptor, allowing for minor
variations which do not have an adverse effect on the ligand
binding properties and/or biological function and/or immunogenicity
as compared to the MSH receptor, or which give interesting and
useful novel binding properties or biological functions and
immunogenicities etc. of the analogue. The analogous DNA fragment
or DNA sequence may be derived from an animal or a human or may be
partially or completely of synthetic origin as described above. The
analogue may also be derived through the use of recombinant DNA
techniques.
[0055] Furthermore, the terms "analogue" and "subsequence" are
intended to allow for variations in the sequence such as
substitution, insertion (including introns), addition, deletion and
rearrangement of one or more nucleotides, which variations do not
have any substantial effect on the polypeptide encoded by a DNA
fragment or a subsequence thereof. The term "substitution" is
intended to mean the replacement of one or more nucleotides in the
full nucleotide sequence with one or more different nucleotides,
"addition" is understood to mean the addition of one or more
nucleotides at either end of the full nucleotide sequence,
"insertion" is intended to mean the introduction of one or more
nucleotides within the full nucleotide sequence, "deletion" is
intended to indicate that one or more nucleotides have been deleted
from the full nucleotide sequence whether at either end of the
sequence or at any suitable point within it, and "rearrangement" is
intended to mean that two or more nucleotide residues have been
exchanged with each other.
[0056] When using the term "any substantial effect on the
polypeptide" is understood that the DNA fragment encodes a
polypeptide which has retained its antigenicity and/or MSH binding
properties compared to the MSH receptor polypeptide encoded by the
DNA fragment from which the analogue/subsequence is derived.
[0057] The terms "fragment", "sequence", "subsequence" and
"analogue", as used in the present specification and claims with
respect to fragments, sequences, subsequences and analogues
according to the invention should of course be understood as not
comprising these phenomena in their natural environment, but
rather, e.g., in isolated, purified, in vitro or recombinant
form.
[0058] The terms "homology" and "homologous" are, with respect to
DNA fragments, intended to mean a homology between the nucleotides
in question between which the homology is to be established, in the
match with respect to identity and position of the nucleotides of
the DNA fragments. With respect to polypeptides and fragments
thereof described herein, the terms are intended to mean a homology
between the amino acids in question between which the homology is
to be established, in the match with respect to identity and
position of the amino acids of the polypeptides.
[0059] "Binding capacity of an MSH receptor" is in this context
meant as the binding properties of an MSH receptor assessed by a
test wherein the binding between the receptor and various possible
ligands is determined with respect to their ability of inhibiting
binding of .sup.125I-NDP-MSH
(.sup.125I-(Nle.sup.4,D-Phe.sup.7)-MSH) as described herein.
[0060] "Same binding capacity as an MSH receptor" is defined herein
as a binding profile which shows that a substance binds
125I-NDP-MSH with a higher affinity than other compounds although
the substance may be unable to elicit the effects exerted by the
binding of MSH to an MSH receptor.
[0061] When reference is being made to "an analogue of MSH" or "an
analogue of a melanotropic hormone" it is intended to mean a
substance which shows binding capacity for an MSH receptor as
defined above. Thus, examples of analogues of MSH and analogues of
melanotropic hormones are .alpha.-MSH, .beta.-MSH, .gamma.-MSH and
NDP-MSH.
[0062] The term "melanotropic hormone" is intended to refer both to
a natural peptide being derived from proopiomelanocortin (POMC),
the natural peptide typically having a biological activity of that
of MSH or ACTH, and a synthetic peptide, the synthetic peptide
having the ability to induce at least one of the biological effects
which may be induced by the natural melanotropic hormones. Examples
of melanotropic hormones are .alpha.-MSH, .beta.MSH, .gamma.-MSH,
ACTH and NDP-MSH.
[0063] The term "melanotropic hormone receptor" is intended to mean
a receptor which can be activated by a melanotropic hormone so as
to induce a second messenger response (or any other typical
receptor response) or a biological effect generally being referred
to as a melanotropic hormone response. Stimulation of melanin
formation in melanocytes and the stimulation of corticosteroid
synthesis in the adrenal gland by the melanotropic hormones are
typical examples of melanotropic hormone responses. Examples of
melanotropic hormone receptors are the MSH receptor, the ACTH
receptor and the MC-2 receptor.
[0064] The present invention is based on the construction of the
primers shown as SEQ ID NO:3 and SEQ ID NO:4 and the analogues
thereof which are defined below. As appears from the above, great
interest and many efforts have been exerted in order to examine the
function of the MSH receptor and thus, there has been obvious
interest in isolating the DNA encoding the MSH receptor. The very
careful work performed by the inventors of the present invention
when designing these primers such as described in details in
Example-1 rendered the present invention possible.
[0065] Thus, the DNA fragments of the invention used as primers
constitute another interesting aspect of the invention and have
various important utilities such as detection and isolation of
other DNA fragments encoding polypeptides having similar functions
and/or,binding capacity as an MSH receptor. In particular, the
primers can be used in the detection of other G-protein coupled or
binding receptors. The invention therefore also relates to a DNA
fragment having the nucleotide sequence SEQ ID NO:3 (from segment
3) or analogues thereof, wherein the nucleotides 13 and/or 15
and/or 23 optionally are substituted by c and to a DNA fragment
having the nucleotide sequence SEQ ID NO:4 (from segment 6) or
analogues thereof wherein the nucleotides 19 and/or 29 and/or 32
optionally are substituted by C, and wherein the nucleotides 20
and/or 31 are optionally substituted by G.
[0066] In order to examine a DNA fragment of the invention or an
analogue or subsequence thereof or an RNA fragment transcribed
therefrom, such as to examine the relatedness to other foreign DNA
fragments, hybridization is a useful method. Hybridization may be
performed as follows: A DNA fragment or an analogue or a
subsequence thereof of the invention is labelled with any of the
labelling principles available (radioactive system, color reaction
system, light based system, or variations of these) so as to
constitute a probe. The foreign DNA/RNA to be examined is coupled
to a matrix. The matrix is subjected to a suitable treatment so as
to couple the DNA/RNA to the matrix. The matrix is exposed to a
prehybridization solution of a composition, at a temperature and
for a period of time suited to the matrix and the foreign DNA/RNA
in question. The matrix is then placed in a hybridization solution
containing labelled denatured DNA probe. Hybridization is carried
out at a suitable temperature and the period of time. The matrix is
then washed with a solution of a composition, at a temperature and
for a period of time suited to the matrix and the foreign DNA/RNA
in question. The matrix is then subjected to a suitable detection
system based on the nature of the label in the DNA probe. The
results are then analyzed. Any hybridization of the foreign DNA/RNA
and the DNA probe is an indication of similarity of the two
species, and may be used to examine whether the foreign DNA/RNA is
a part of the invention. In the above hybridization procedure a RNA
probe corresponding to a polypeptide or an analogue or a
subsequence thereof of the invention can also be used in place of a
DNA probe. Another approach of determining similarity between DNA
sequences is by determining the nucleotide sequence of the DNA
fragment to be compared with a DNA fragment or an analogue or
subsequence thereof of the invention by conventional DNA sequencing
analysis, and comparing the degree of homology with the DNA
fragment or an analogue or subsequence thereof of the
invention.
[0067] Polymerase chain reaction (PCR) primers can be synthesized
based on the nucleotide sequence of the cloned MSH receptor, or on
the basis of other known similar sequences. These primers can then
be used to amplify the whole or a part of an MSH receptor sequence
or the sequences of its analogues. Primers as shown in SEQ ID NO:3
and SEQ ID NO:4 which constitute part of the invention may be used
in this aspect. Polymerase chain reaction enzyme, a type of heat
stable DNA polymerase, generally incorporates wrong nucleotides at
a frequency of 1 in 10000 (Tindall et al. 1988) during
amplification. Because of the iterative nature of the amplification
this frequently attributes a new altered sequence to the amplified
MSH receptor.
[0068] The DNA fragment described above and constituting an
important aspect of the invention may be obtained directly from the
genomic DNA or by isolating mRNA and converting it into the
corresponding DNA sequence by using reverse transcriptase, thereby
producing a cDNA. When obtaining the DNA fragment from genomic DNA,
it is derived directly by screening for genomic sequences such as
is described in Example 1. It can be accomplished by hybridization
to a DNA probe designed on the basis of knowledge of an MSH
receptor sequence, or the sequence information obtained by amino
acid sequencing of the purified MSH receptor. When the DNA is of
complementary DNA (cDNA) origin, it may be obtained by preparing a
cDNA library with mRNA from cells containing MSH receptor or parts
thereof. Hybridization can be accomplished by a DNA probe designed
on the basis of knowledge of an MSH receptor sequence, or the
sequence information obtained by amino acid sequencing of the
purified MSH receptor.
[0069] The DNA fragments of the invention or analogues or
subsequences thereof can also be obtained using other methods
(Wright et al. 1992) like expression cloning in cell line (Xie et
al. 1992) or the expression cloning in the oocyte (Julius et al.
1988; Masu et al. 1987).
[0070] A DNA fragment of the invention or an analogue or
subsequence thereof can be obtained from other animals, such as
mammals. The DNA obtained in this way could be exactly similar to
the one shown in SEQ ID NO:1 or SEQ ID NO:15 or could have
differences in structure attributed to well known interspecies
variations.
[0071] A DNA fragment of the invention or an analogue or
subsequence thereof can be replicated by fusing it with a vector
and inserting the complex into a suitable microorganism or a
mammalian cell line. Alternatively, the DNA fragment can be
manufactured using chemical synthesis.
[0072] As mentioned above the polypeptides encoded by the DNA
fragments of the invention shown in SEQ ID NO:1 and 15 have been
shown to contain coding regions encoding an MSH receptor and an MSH
receptor/MSH receptor subtype, respectively.
[0073] Thus, in another particular important aspect, the invention
relates to a polypeptide having the amino acid sequence shown in
SEQ ID NO:2 or an analogue or subsequence thereof which
[0074] 1) is an MSH receptor or which is capable of binding to MSH
or an analogue thereof, and/or
[0075] 2) is encoded by a DNA fragment which is at least 50%
homologous with the DNA fragment shown in SEQ ID NO:1, and/or
[0076] 3) binds an antibody which is also bound by an MSH
receptor.
[0077] Another most important aspect of the invention thus relates
to a polypeptide having the amino acid sequence shown in SEQ ID
NO:16 or an analogue or subsequence thereof which 1) is an MSH
receptor or which is capable of binding to MSH or an analogue
thereof, and/or
[0078] 2) is encoded by a DNA fragment which is at least 50%
homologous with the DNA fragment shown in SEQ ID NO:1, and/or
[0079] 3) binds an antibody which is also bound by an MSH
receptor.
[0080] By the use of the term "MSH receptor" is meant a polypeptide
being capable of binding MSH and by the term "analogue thereof" is
meant any polypeptide having the same binding capacity as an MSH
receptor in that the polypeptide is capable of binding MSH. Thus,
included is also a polypeptide from different sources, such as
different animals, such as mammals, in particular a human, which
vary for example in the carbohydrate part, or the phosphorylation
and/or in tissue distribution. In this context the term MSH
receptor also refers to both the above-mentioned polypeptides of
the invention.
[0081] The term analogue also includes polypeptides being capable
of binding antibodies which also bind to an MSH receptor. Such
analogues may be capable of eliciting or stimulating an immune
response which is also directed against the MSH receptor or which
can also be elicited by the MSH receptor. These and other analogues
are encoded by a DNA fragment or analogue or subsequence thereof of
the invention which with respect to analogues have been defined
above. The analogues may in a particular aspect be of synthetic
origin as discussed herein.
[0082] The term "analogue" with regard to a polypeptide is also
used in the present context to indicate a protein or polypeptide of
a similar amino acid composition or sequence as the characteristic
amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:16 (or
another polypeptide of the invention), allowing for minor
variations which do not have an adverse effect on the ligand
binding properties and/or biological function and/or
immunogenicity, or which may give interesting and useful novel
binding properties or biological functions and immunogenicities
etc. of the analogue. The analogous polypeptide or protein may be
derived from an animal or a human or may be partially or completely
of synthetic origin. The analogue may also be derived through the
use of recombinant DNA techniques.
[0083] It is being widely recognized that the same or similar gene
may be present in two or several copies in the genome of the same
animal. Because gene mutations will always tend to induce
divergence of a DNA sequence the structure of the protein coded by
the same and/or similar genes will tend to diverge during
evolution. Thus, in the present context it is obvious that the MSH
receptor, because of the existence of the DNA according to SEQ ID
NO:1 and SEQ ID NO:15 in fact exist in at least two copies in the
genome. Thus SEQ ID NO:1 and 15 are both coding for proteins which
have MSH receptor properties, albeit the binding properties of the
two proteins for melanotropic hormones differ. It is therefore
predicted that even more copies of the MSH receptor are present in
the genome of an animal, for instance in homo sapiens. Because of
the similarity between the amino acid composition of such proteins
and the MSH receptors described herein, theses proteins are in the
present application being regarded as being analogues of the MSH
receptor. Thus such proteins are also part of the invention.
[0084] In the present context the term "characteristic amino acid
sequence derived from an "MSH receptor" is intended to mean an
amino acid sequence which comprises amino acids constituting a
substantially consecutive stretch (in terms of linear or spatial
conformation) of the polypeptide shown in SEQ ID NO: 2 or the amino
acid sequence shown in SEQ ID NO:16 and encoding an MSH receptor.
Such secondary or tertiary conformation may have interesting and
useful properties and may constitute epitopes.
[0085] In the present context, the term "epitope" refers to any
polypeptide of the invention or an analogue thereof capable of
stimulating or interacting with immunocompetent cells and capable
of stimulating the production of antibodies which also bind to a
polypeptide constituting an MSH receptor. Especially epitopes
showing desirable properties with regard to diagnosis and therapy
constitute important aspects of the present invention.
[0086] In the present context, the term "epitope" also refers to
any polypeptide of the invention or a characteristic amino acid
sequence or an analogue thereof capable of interacting or binding
existing or novel substances which are also bound by a polypeptide
constituting an MSH receptor. The said substances can be organic
molecules, small peptides or large polypeptides or derivatives of
any of the above. Such an approach can find use in the drug
screening programme.
[0087] The term "receptor subtype" is intended to mean a receptor
which is capable of binding the same ligand and/or ligands as
another receptor, albeit the affinities of the ligands for the
receptors may be different for the compared receptors.
[0088] Thus, when referring to MC-2 as an "MSH receptor subtype" it
is indicated that MC-2 is an MSH receptor, but that the pattern of
binding to various ligands/substances is different from that of
another MSH receptor, such as e.g. the MSH receptor with the amino
acid sequence shown in SEQ ID NO:2.
[0089] The term "subsequence" with regard to a polypeptide
designates a polypeptide sequence which comprises a part of the
polypeptide sequence shown in SEQ ID NO:2 or SEQ ID NO:16 or other
polypeptide sequences of the invention which may optionally have
retained its capability of binding MSH. Included are also
polypeptide subsequences which have been analogized by
modifications as explained herein. Polypeptides constituting
interesting epitopes or encoded by a nucleotide subsequence of the
invention as defined above are also included.
[0090] "A derivative of an MSH receptor" is meant to indicate both
an analogue, subsequence or subtype of an MSH receptors.
[0091] In a most important aspect, the invention relates to a
polypeptide encoded by the DNA fragment shown in SEQ ID NO:1,
preferably the polypeptide shown in SEQ ID NO:2, and to a
polypeptide encoded by the DNA fragment with the nucleotide
sequence shown in SEQ ID NO:15, preferably the polypeptide shown in
SEQ ID NO:16.
[0092] The polypeptides of the invention also comprises
polypeptides which show a degree of homology of at least 55%,
preferably at least 70%, more preferably at least 80% and most
preferably at least 95% homology to a polypeptide of the same
length which has an amino acid sequence which is a part of the
sequences shown in SEQ ID NO:2 and SEQ ID NO:16.
[0093] The invention also relates to a characteristic amino acid
sequence being a subsequence comprising from at least 5 amino acids
to 316 acids of SEQ ID NO:2 and to a subsequence comprising from 5
to 324 amino acids of SEQ ID NO:16, and any analogue to such
polypeptides. Preferably the subsequence comprises at least 7 amino
acids, more preferably at least 10 amino acids, even more
preferably at least 15 amino acids and most preferably at least 30
amino acids. The polypeptide may be coupled to any other
moiety.
[0094] The present invention also relates to a substantially pure
polypeptide which has the same binding capacity as an MSH receptor
or which is recognized by an antibody raised against or reactive
with a polypeptide of the invention. Furthermore the invention
relates to any polypeptide of the invention in substantially pure
form.
[0095] Furthermore, the invention relates to a polypeptide as
defined herein which is glycosylated or which is linked to a
carbohydrate or lipid moiety. Also a polypeptide containing a
palmitoyl anchor or a part thereof constitutes an interesting
aspect as well as any polypeptide of the invention in lipid soluble
form which may, in one interesting aspect of the invention as
described herein, be used in the treatment of an animal, in
particular a human, having impaired function of the receptor.
[0096] The lipid soluble form of polypeptide of the invention may
be a form comprising components such as liposomes, micelles and
phospholipid so as to allow the polypeptide to be incorporated in
the cell membrane of the recipient. It is important that the lipid
soluble form is a form which ensures the stability of the
polypeptide and preferably in a form which is pharmaceutically
acceptable so as to allow the administration of the lipid soluble
form to an animal, in particular a human. The lipid soluble form
may also be in a form comprising components such a detergent, oil,
such as mineral oil or vegetable oil or water, and which may be a
suspension of one or more of the above mentioned components.
[0097] In the present context, the term "substantially pure" is
understood to mean that the polypeptide in question is
substantially free from other components, e.g. other polypeptides
or carbohydrates, which may result from the production and/or
recovery of the polypeptide or otherwise be found together with the
polypeptide. The high purity of a polypeptide of the invention is
advantageous when the polypeptide is to be used for, e.g., the
production of antibodies. Also due to its high purity, the
substantially pure polypeptide may be used in a lower amount than a
polypeptide of a conventional lower purity for most purposes. The
purification of a polypeptide of the invention may be performed by
methods known to a person skilled in the art.
[0098] The polypeptides of the invention having the amino acid
sequences shown in SEQ ID NO:2 and 16 and which are MSH receptors
bear similarity with other G-protein coupled receptors. They have
the most common feature of passing through the cell membrane 7
times, like all other G-protein coupled receptors. Based on the
observations of homology between the transmembrane segments of
different G-protein coupled receptors, it has been hypothesized
that the extracellular loops and the transmembrane segments are
involved in the ligand binding, such as e.g. the binding of MSH or
an analogue of MSH or of a synthetic organic molecule serving as
ligand for the receptor. The intracellular loops have been assigned
the role of coupling to the G-proteins and possible involvement in
other intracellular activities. In the present context the above
mentioned extra and intracellular loops as well the transmembrane
segments that are intended to be involved in the binding of the
ligand and/or the coupling of G-proteins are one or two or several
or all of the mentioned segments and loops of the receptor.
[0099] Intense efforts to solve the 3-dimensional (3D) structure of
G-protein coupled receptors and some related proteins (e.g.
bacteriorhodopsin and opsins) are ongoing in several laboratories
world wide. It is expected that once the 3D structure of one of
these proteins is solved, the 3D structure of other G-protein
coupled receptors will become easily solvable using computational
methods, provided that their primary amino acid structure is known.
This is due to the fact that all G-protein coupled receptors are
likely to show similar gross 3D structure (Sankara-Ramakrishnan
& Vishveshwara 1989; Findlay & Eliopoulos 1990; Hibert M.
F. et al. 1991).
[0100] Thus, in certain embodiments of the invention are considered
especially important amino acids 1-39, 100-116, 182-188 and 269-276
which are considered to constitute the extracellular loops of the
MSH receptor. These regions will be of particular importance as
epitope targets for antibodies intended for clinical use in e.g.
targeted drug delivery. or for drug design.
[0101] In other embodiments of the invention are considered
especially important the amino acids 63-75, 142-157, 213-243 and
301-317 which are considered to constitute the intracellular loops
of the MSH receptor. These regions will be particularly important
in the elucidation of the mechanisms for the coupling of the MSH
receptor to G-proteins. These regions may serve as targets for
drugs aiming for the modulation of the interaction of the receptor
with G-proteins. It may also in particular be desired to alter the
amino acid sequences, by e.g. deletions, site directed mutations,
insertions of extra amino acids, or combinations thereof, to
generate novel MSH receptor analogues showing altered properties.
Such altered receptors may be desired to further the understanding
of the molecular mechanisms in the coupling of the receptor with
G-proteins. Moreover, yet another aspect pertaining to this
particular part of the invention is the DNA sequences coding for
the intracellular segments, in particular base pairs 636-726, but
also the other segments, as these regions are considered to be less
homologous with other G-protein coupled receptor coding fragments.
Thus, such regions of the sequence may serve to generate DNA probes
which in hybridization studies, as is described in detail below,
are selective for the MSH receptor DNA or mRNA.
[0102] In yet other embodiments of the invention are considered
particularly important the amino acids 40-62, 76-99, 117-140,
158-181, 189-212, 244-268 and 277-300 which are considered to
constitute the transmembrane segments of the MSH receptor. It may
here be desired to alter one or several specific amino acids to
generate MSH receptor analogues showing altered properties. Such
altered receptors may be desired to further the understanding of
the molecular mechanisms in the binding of MSH and MSH analogues to
the receptor. Moreover, yet another aspect pertaining to this
particular part of the invention is the DNA sequences coding for
the transmembrane segments as -these regions are considered to be
highly homologous with other G-protein coupled receptor coding
fragments which are natural variants of the MSH receptor. Such
receptor coding fragments may exist in other species which code for
species variants of the MSH receptor. Such receptor coding
fragments may also exist in humans as well as animals which encode
homologous receptors which are subtypes of the MSH receptor or
which are closely related receptor types possibly belonging to the
same class of melanotropic hormone receptor family. By using
homology screening methods utilizing DNA sequences derived from the
transmembrane segments it may be possible to obtain the DNA
sequences of these homologous receptor coding fragments.
[0103] Because of the difference in sequence of SEQ ID NO:16 from
SEQ ID NO:2 the following fragments of the MC-2 receptor are
considered especially important: Amino acids 1-37, 98-114, 180-186
and 266-273 of SEQ ID NO:16 which are considered to constitute the
extracellular loops of the MC-2 receptor. These regions will be of
particular importance as targets for antibodies intended for
clinical use in e.g. targeted drug delivery or for drug design.
[0104] In other embodiments of the invention are considered
especially important the amino acids 62-73, 139-155, 212-239 and
298-325 of SEQ ID NO:16 which are considered to constitute the
intracellular loops of the MC-2 receptor. These regions will be
particularly important in the elucidation of the mechanisms for the
coupling of the MC-2 receptor to G-proteins. These regions may
serve as targets for drugs aiming for the modulation of the
interaction of the receptor with G-proteins. It may also in
particular be desired to alter the amino acid sequences, by e.g.
deletions, site directed mutations, insertions of extra amino
acids, or combinations thereof, to generate novel MC-2 receptor
analogues showing altered properties. Such altered receptors may be
desired to further the understanding of the molecular mechanisms in
the coupling of the receptor with G-proteins. Moreover, yet another
aspect pertaining to this particular part of the invention is the
DNA sequences coding for the intracellular segments, in particular
base pairs 633-717 of SEQ ID NO:15, but also the other segments, as
these regions are considered to be less homologous with other
G-protein coupled receptor coding fragments. Thus, such regions of
the sequence may serve to generate DNA probes which in
hybridization studies, as is described in detail below, are
selective for the MC-2 receptor DNA or mRNA.
[0105] In yet other embodiments of the invention are considered
particularly important the amino acids 38-61, 74-97, 115-138,
156-179,187-211, 240-265 and 274-297 of SEQ ID NO:16 which are
considered to constitute the transmembrane segments of the MC-2
receptor. It may here be desired to alter one or several specific
amino acids to generate MC-2 receptor analogues showing altered
properties. Such altered receptors may be desired to further the
understanding of the molecular mechanisms in the binding of MSH and
MSH analogues to the receptor. Moreover, yet another aspect
pertaining to this particular part of the invention is the DNA
sequences coding for the transmembrane segments as these regions
are considered to be highly homologous with other G-protein coupled
receptor coding fragments which are natural variants of the MC-2
receptor. Such receptor coding fragments may exist in other species
which code for species variants of the MC-2 receptor. Such receptor
coding fragments may also exist in humans as well as animals which
encode homologous receptors which are subtypes of the MC-2 receptor
or which are closely related receptor types possibly belonging to
the same class of melanotropic hormone receptor family. By using
homology screening methods utilizing DNA-sequences derived from the
transmembrane segments it may be possible to obtain the DNA
sequences of these homologous receptor coding fragments.
[0106] The above mentioned specific amino acid sequences are
prominent examples of subsequences according to the invention. It
is to be understood that the other important subsequences according
to the invention are subsequences which are modifications of the
above mentioned subsequences in that, and with respect to
particular subsequences of which they are modifications, they
fulfil any one of the criteria 1)-3) for the polypeptide as stated
above. Also included in this aspect of the invention is a DNA
fragment encoding any such amino acid sequence.
[0107] Using the primers of the invention, three DNA fragments have
been isolated and sequenced. These DNA fragments termed G-6 shown
in SEQ ID NO:5, G-8, shown in SEQ ID NO:7 and G-10, shown in SEQ ID
NO:9, share homologies with the DNA sequence shown in SEQ ID
NO:1.
[0108] Thus, the present invention also relates to a DNA fragment
or a subsequence or an analogue thereof which shows a homology with
any of the nucleotide sequences shown in SEQ ID NO:5, SEQ ID NO:7
or SEQ ID NO:9 of at least 40%, or which can be isolated by using
the nucleotide sequence shown in SEQ ID NO: 13 and/or SEQ ID NO:14
as a primer, or which has any of the nucleotide sequences shown in
SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9. The homology may in some
aspects be at least 50%, preferably at least 55%, more preferably
at least 70%, even more preferably at least 80% and most preferably
at least 95% with any of the DNA sequences shown in SEQ ID NO: 5,
SEQ ID NO:7 or SEQ ID NO:9, respectively.
[0109] Polypeptide sequences or subsequences or analogues thereof
which show a homology of at least 40% with any of the polypeptides
shown in SEQ ID NO:6, 8 or 10 encoded for by the DNA sequences
shown in SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9, respectively,
constitute yet another embodiment of the invention. The homology
may in some aspects be at least 50%, preferably at least 55%, more
preferably at least 70%, even more preferably at least 80% and most
preferably at least 95% with any of the polypeptides shown in SEQ
ID NO:6, SEQ ID NO:8 and SEQ ID NO:10, respectively.
[0110] Any of the DNA fragments of the invention may in a
particular embodiment of the invention be used for the preparation
of a DNA probe which may be labelled or unlabelled and which is
used to isolate full length coding fragments and/or to detect or
quantitate RNA. The information of any of these DNA fragments may
also be used to make PCR primers and for making a polypeptide.
[0111] As mentioned above and described in the examples, the G-8
fragment has been used to isolate a new MSH receptor subtype, MC-2.
It is therefore contemplated to perform similar experiments using
the two other fragments G-6 and G-10.
[0112] The DNA fragments G-6 and G-10 represent two potential
G-protein coupled receptors, and can be used to isolate full length
coding fragments for them using for example the same methodology as
described in Example 1.
[0113] In particular, because of substantial homology of G-6 and
G-10 with the primers shown in SEQ ID NO:3 and SEQ ID NO:4 of the
invention, it is considered that one or both of G-6 and G-10
represent coding fragments encoding receptors for peptides sharing
similar origin as the MSH receptor; that is the POMC
(proopiomelanocortin) receptors, such as e.g. the ACTH receptor,
the met-enkephalin receptor and the .beta.-endorphin receptor. In
particular, it is considered that one or both of G-6 and G-10 are
the .mu., .delta. and .sigma. opioid receptors because of their
dissimilarities to the .kappa. opioid receptor.
[0114] However, G-6 and G-10 each has unique sequence
characteristics starting in the intracellular segment 5, that is
the amino acids "L-Y-V/I-H-M", and it may be contemplated that one
or both of G-6 and G-10 represent parts of DNA sequences encoding
receptors for a novel class of peptide hormone receptors; e.g. the
corticotropin releasing hormone receptor, the growth hormone
releasing hormone receptor, the gonadotropin releasing hormone
receptor, the thyrotropin releasing hormone receptor, the
luteinizing hormone releasing hormone receptor, the follicle
stimulating hormone releasing hormone receptor, the chorionic
gonadotropin hormone receptor and/or the glucagon receptor.
[0115] However, G-6 and G-10 also has short extracellular loops,
indicating that the receptors encoded by these fragments constitute
the small peptide hormone type of G-protein coupled receptors.
Thus, one or both of G-6 and G-10 may represent the neuropeptide Y
receptor, the tumour necrosis factor receptor, the colony
stimulating factor receptor, the interleukin 1 receptor, the neurot
sin receptor, the atrial natriuretic factor receptor, the kallidin
receptor, the bulbogastrin receptor, the motilin receptor, the
pancreatic polypeptide receptor, the olfactory receptor subtype,
the spermatozoon feromone receptor subtype, the insulin like growth
factor receptor, the taste receptor subtype, the gustuducin coupled
receptor subtype, the inhibin receptor subtype and/or the kyotropin
receptor.
[0116] The dissimilarity of the G-6 and G-10 with other known
nucleotide fragments coding for G-protein coupled receptors, the
latter of which include a substantial amount of well characterized
receptors for amines, may indicate that one or both of G-6 and G-10
by contrast encode genes for lipids, e.g. more specifically
receptors for prostanoids, that is, more specifically the
prostaglandin E.sub.1 receptor, the prostaglandin E.sub.2 receptor,
the prostaglandin F.sub.2a or receptors for prostacyclins, e.g. the
PGI.sub.2 receptor, and/or receptors for leukotrienes, e.g. the
leukotriene D.sub.4 receptor and/or the leukotriene C.sub.4
receptor and/or a receptor for a non-lipid, e.g. the phosphatidic
acid receptor and/or the thromboxane A2 receptor and/or the
platelet activating factor receptor.
[0117] Due to general characteristics of G-6 and G-10 being
G-protein coupled receptors, it is considered that one or both of
G-6 and G-10 by contrast encode genes for subtype of the substance
P receptor, substance K receptor, endothelin receptor, angiotensin
receptor, chemoattractant peptide receptor, bombesin receptor,
oxytocin receptor, vasopressin receptor, antidiuretic hormone
receptor, gastrin receptor, cholecystokinin receptor, canabinoid
receptor, follicle stimulating hormone receptor, luteinizing
hormone receptor, growth hormone receptor, thyrotropin receptor,
calcitonin receptor, calcitonin gene related peptide receptor
and/or parathyroid hormone receptor.
[0118] Based on the knowledge of the DNA fragments of the invention
(or the knowledge of the analogues or RNA fragments) the described
DNA fragments of the invention can be produced containing one or
more modified nucleotides to improve resistance against nucleases
or to improve transport across cell membranes.
[0119] Suitable polypeptides can be produced using recombinant DNA
technology. More specifically, the polypeptides may be produced by
a method which comprises cultivating or breeding an organism
carrying a DNA fragment or an analogue or a subsequence thereof of
the invention under conditions leading to expression of said DNA
fragment, and subsequently recovering the expressed polypeptide
from the said organism.
[0120] The organism which can be used for the production of such a
polypeptide may be a higher organism e.g. an animal, or a lower
organism e.g. a microorganism. Irrespective of the type of organism
used, a DNA fragment of the invention or an analogue or a
subsequence thereof (described above) should be introduced in the
organism either directly or with the help of a suitable vector.
Alternatively, the polypeptides may be produced in the mammalian
cell lines by introducing a DNA fragment of the invention either
directly or with the help of an expression vector.
[0121] The DNA fragments or analogues or subsequences thereof of
the invention can also be cloned in a suitable stable expression
vector and then put into a suitable cell line. The cells expressing
the desired polypeptides are then selected using the conditions
suitable for the vector and the cell line used. The selected cells
are then grown further and form a very important and continuous
source of the desired polypeptides.
[0122] A polypeptide of the invention can also be made by in vitro
translation of the RNA complementary to a DNA fragment of the
invention. This can be achieved for the whole molecule, or a part
or parts of the molecule, in free form or in fusion with one or
several proteins. The methods which can be used are described
(Sambrook et al. 1989; Spirin et al. 1988). The polypeptides of the
invention can also be expressed in vitro as functional proteins in
the fused or the unfused form (Zozulya et al. 1990).
[0123] In line with the above, the invention relates to a vector
containing a recombinant DNA insert coding for an MSH receptor
polypeptide of the invention or a fusion polypeptide as defined
herein. In one particular important embodiment, a DNA fragment or
an analogue or subsequence thereof of the invention or a fusion DNA
fragment of the invention as defined herein may be carried by a
replicable expression vector which is capable of replicating in a
host organism or a cell line.
[0124] The vector may in particular be a plasmid, phage, cosmid,
mini-chromosome or virus. In an interesting embodiment of the
invention, the vector may be a vector which, when introduced in a
host cell, is integrated in the host cell genome. Included as an
important aspect of the invention is also an organism which carries
and is capable of expressing a DNA fragment of the invention. Such
a plasmid vector has been constructed and is designated pE-11D
herein. This vector constitutes yet another aspect of the
invention.
[0125] Also, the invention relates to an organism which carries and
is capable of replicating a DNA fragment of the invention and also
such a plasmid vector designated pB-11D has been constructed and
constitutes a part of the invention. This vector was deposited on
Aug. 24, 1992 under the number DSM 7214 in the Deutsche Sammlung
von Mikroorganismen under the terms and conditions of the Budapest
Treaty.
[0126] Further, yet another plasmid vector, designated pB-MC-2, has
been constructed, which is capable of replicating a DNA of the
present invention. pB-MC-2 thus constitutes yet a very important
aspect of the invention. The construction of pB-MC-2 is described
in detail in Example 6.
[0127] Moreover, still yet another plasmid vector, designated
pE-MC-2, which is also a very important embodiment of the
invention, has been constructed. The pE-MC-2 plasmid vector is
capable of expressing the MC-2 receptor of the present invention.
The construction of pE-MC-2 is detailed in Example 7 and the use of
pE-MC-2 for the expression of the MC-2 receptor is exemplified in
Example 8. This vector has been deposited on Aug. 9, 1993 under the
number DSM 8440 in the Deutsche Sammlung von Mikroorganismen under
the terms and conditions of the Budapest Treaty.
[0128] Organisms which may be used in this aspect of the invention
of producing the peptides of the invention comprise a cell which is
a microorganism such as a bacterium, a yeast, a protozoan, or which
is derived from a multicellular organism such as a fungus, an
insect, a plant, a mammal or it may be a cell line. If the organism
is a bacterium, it is preferred that the bacterium is of the genus
Bacillus, e.g. B. subtilis, Escherichia, e.g. E. coli, or
Salmonella.
[0129] If a higher organism is used, transgenic techniques may be
employed for the production of the polypeptides. Examples of
suitable animals are sheep, cattle, pigs etc. A DNA fragment
encoding a polypeptide of the invention is expressed together with
a polypeptide which is inherently expressed by the animal, e.g. a
milk protein or the like. The resulting fusion protein may then be
subjected to post-translational modifications so as to obtain a
polypeptide of the invention.
[0130] In another aspect of the invention a MSH receptor may be
obtained from a suitable cell type found to naturally express MSH
receptor from DNA encoding an MSH receptor. Such cells may be e.g.
a melanoma cell line, as is shown in Example 3 for WM 266-4 cells,
or they may be obtained from any tissue containing cells expressing
a DNA fragment of the invention.
[0131] A stable cell line capable of producing a polypeptide of the
invention having MSH binding properties, has been established. The
cell lines of COS-7 cells constitute other important aspects of the
invention. The cell line harbours the DNA fragment with the
nucleotide sequence SEQ ID NO:1 and steadily produces polypeptides
having binding properties substantially identical to the binding
properties described below. The production of this stable cell line
is described in detail in example 5.
[0132] Thus, the invention also relates to a stable cell line
producing a polypeptide of the invention which optionally binds
NDP-MSH with high affinity; the establishment of such a cell line
may be performed according to the technique described in Example 5,
or to any other method known to the person skilled in the art.
[0133] In one particular aspect of the invention, a DNA fragment of
the invention may comprise one or more second nucleotide
sequence(s) encoding one or more polypeptide(s) different from or
identical to a polypeptide of the invention fused in frame to a DNA
fragment of the invention or an analogue thereof encoding a
polypeptide or an analogue or subsequence thereof of the invention
with the purpose of producing a fused polypeptide which polypeptide
constitutes yet another interesting aspect of the invention. When
using recombinant DNA technology the fused DNA sequences may be
inserted into a suitable vector or genome. Alternatively, one of
the nucleotide sequences is inserted into the vector or genome
already containing the other nucleotide sequence(s). A fusion
polypeptide can also be made by inserting the nucleotide sequences
separately and allowing the expression to occur. The host organism,
which may be of eukaryotic or prokaryotic origin is grown under
conditions ensuring expression of fused sequences. The fused
polypeptide is then purified and a polypeptide of the invention
separated from its fusion partner using a suitable method. The
fusion polypeptide may in a particular embodiment of the invention
still be capable of binding to MSH or an analogue thereof.
[0134] The second polypeptide to which a polypeptide of the
invention is fused may in one particular embodiment of the
invention be a DNA fragment encoding a diphtheria toxin, a
staphylococcus protein, a ricin toxin, Pseudomonas endotoxin, abrin
or fungal ribosome-inactivation proteins (RIP). In other
embodiments of the invention the second DNA fragment may encode a
subsequence of a melanotropic hormone receptor, an MSH receptor or
an ACTH receptor.
[0135] The fusion polypeptides of the invention may be modified as
well as other polypeptides of the invention, e.g. they may be
glycosylated, coupled to a carbohydrate or lipid moiety, contain a
palmitoyl anchor or a part thereof bound to a solid support and be
provided with a detectable label.
[0136] The present invention also relates to a polypeptide of the
invention in substantially pure form and to a method of producing
the polypeptide. The method of producing a polypeptide of the
invention comprises the following steps:
[0137] (a) inserting a DNA fragment of the invention in an
expression vector,
[0138] (b) transforming a suitable host organism with the vector
produced in step (a),
[0139] (c) cultivating the host organism produced in step (b) under
suitable conditions for expressing the polypeptide,
[0140] (d) harvesting the polypeptide, and
[0141] (e) optionally subjecting the polypeptide to
posttranslational modifications.
[0142] A DNA fragment or an analogue or subsequence thereof of the
invention encoding a polypeptide of invention can be modified
before or after it has been inserted into the vector or organism
for expression. The polypeptide product may also be subjected to
modification. The modification may comprise substitution, addition,
insertion, deletion or rearrangement of one or more nucleotides and
amino acids in the DNA and polypeptide, respectively. The term
"substitution" is intended to mean the replacement of one or more
nucleotides or amino acids in a DNA fragment or polypeptide of the
invention. The term "addition" means addition of one or more
nucleotides and amino acids at either end of a DNA
fragment/polypeptide of the invention or a part of them. Insertion
is intended to mean the introduction of one or more nucleotides and
amino acids in a DNA fragment or polypeptide of the invention or a
part of them. Deletion is intended to mean the removal of one or
more nucleotides and amino acids from a DNA fragment or polypeptide
of the invention or from a part of them. Rearrangement is intended
to indicate that one or more nucleotides or amino acids have been
exchanged within the DNA or polypeptide sequence, respectively. The
DNA fragment may, however, also be modified by mutagenesis either
before or after inserting it in the organism. A DNA or protein
sequence of the invention may be modified in such a way that it
does not lose any of its biophysical, biochemical or biological
properties, or part of such properties (one and/or all) or all of
such properties (one and/or all).
[0143] The polypeptide produced as described above may be subjected
to posttranslational modifications such as thermal treatment,
chemical treatment (formaldehyde, glutaraldehyde etc.) or enzyme
treatment (peptidases, proteinases and protein modification
enzymes). The polypeptide may be processed in a different way when
produced in an organism as compared to its natural production
environment. It may or may not be advantageous to remove or alter
the processing characteristics caused by the host organism in
question.
[0144] When a polypeptide according to the invention is produced in
a prokaryotic organism such as a bacterium, a useful
post-translational modification may be refolding of the peptide in
order to obtain the peptide in a native and functional form due to
the fact that peptides produced this way are often found as
insoluble non-functional inclusion bodies inside the microorganism.
The refolding of such peptides of such inclusion bodies are
traditionally refolded by denaturing the peptide followed by a
gradual continuous renaturation.
[0145] The term "truncated" polypeptide refers to a polypeptide
deleted of one or more amino acids eventually resulting in changing
of the properties of the polypeptide, such as e.g. solubility. In a
further meaning, the term "truncated" polypeptide refers to a
mixture of polypeptides all derived from one polypeptide or
expressed from the coding fragment(s) encoding said
polypeptide.
[0146] Subsequent to the expression according to the invention of
the polypeptide in an organism or a cell line, the polypeptide can
either be used as such or it can first be purified from the
organism or cell line. If the polypeptide is expressed as a
secreted product, it can be purified directly. If the polypeptide
is expressed as an associated product, it may require the partial
or complete disruption of the host before purification. Examples of
the procedures employed for the purification of polypeptides
are:
[0147] (i) immunoprecipitation or affinity chromatography with
antibodies,
[0148] (ii) affinity chromatography with a suitable ligand,
[0149] (iii) other chromatography procedures such as gel
filtration, ion exchange or high performance liquid chromatography
or derivatives of any of the above,
[0150] (iv) electrophoretic procedures like polyacrylamide gel
electrophoresis, denaturating polyacrylamide gel electrophoresis,
agarose gel electrophoresis and isoelectric focusing,
[0151] (v) any other specific solubilization and/or purification
techniques.
[0152] Also, preparation of polypeptides of the invention may be
performed by the well known methods of liquid or solid phase
peptide synthesis utilizing the successive coupling of the
individual amino acids of the polypeptide sequence. Alternatively,
the polypeptide can be synthesized by the coupling of individual
amino acids forming fragments of the polypeptide sequence which are
later coupled so as to result in the desired polypeptide. These
methods thus constitute another interesting aspect of the
invention.
[0153] Very important and thus constituting important aspects of
the invention are various methods of regulating the activity
exerted by an MSH receptor. This activity which has been described
above in details may have important implications for the various
disease conditions connected to the MSH receptor and for the
various other biological functions. Thus, methods for preventing or
stimulating the binding of the MSH receptor to various molecules
constitute important aspects of the invention.
[0154] One of such aspects of the invention relates to a method of
preventing or stimulating the coupling of the described MSH
receptor to its guanine nucleotide binding protein comprising using
a method wherein a ligand is bound to an epitope of the receptor
which normally interacts with the G-protein, in particular one or
several of the extracellular loops and/or the transmembrane
sequence and/or the cytoplasmic loops and/or the C-terminal
sequence, thereby inhibiting or stimulating the coupling between
the G-protein and the receptor. Thus, in one aspect this method
comprises administering a substance to an animal, in particular a
human, which substance in advance has been found to bind to a
polypeptide having the amino acid sequence shown in SEQ ID NO:2 or
16 or a subsequence comprising any of the subsequences encoding one
or several of the extracellular loops and/or the transmembrane
sequence and/or the cytoplasmic loops and/or the C-terminal
sequence or a combination thereof.
[0155] In this context a "ligand" will refer to a substance, which
may be natural or synthetic, and which will bind in a preferably
reversible, but also possibly irreversible manner to the MSH
receptor.
[0156] Another method according to the invention of preventing or
stimulating the binding of MSH or similar peptides or a G-protein
to the described MSH receptor comprises administering a substance
to an animal, in particular a human, which substance in advance has
been found to bind to a polypeptide having the amino acid sequence
shown in SEQ ID NO:2 or 16 or an analogue or subsequence thereof
comprising any of the subsequences encoding one or several of the
extracellular loops and/or the transmembrane sequence and/or the
cytoplasmic loops and/or the C-terminal sequence or a combination
thereof, so as to occupy the binding site of the receptor using an
agonist, an antagonist, a blocker or a substance such as a
derivative of MSH having a structure similar to MSH, and optionally
thereby preventing or stimulating the generation of second
messenger elements. The substance may be a synthetic ligand such as
a peptide, an organic compound or an antibody capable of binding to
the receptor or a part thereof. The antibody may be a monoclonal or
polyclonal antibody.
[0157] A method of reducing or increasing the binding affinity of
the MSH receptor is an interesting aspect of the invention and may
be obtained by the use of allosteric modulation. Further a way of
preventing the coupling of the MSH receptor to its guanine
nucleotide binding protein according to the invention is to reduce
the production of the MSH receptor. This may be obtained by using
antisense oligotherapy wherein a DNA or RNA fragment complementary
to at least part of the mRNA corresponding to a polypeptide of the
invention or an analogue thereof may be effective in arresting the
translation of the polypeptide in human cells and thereby
inhibiting the synthesis of MSH receptor polypeptide.
[0158] A method for increasing the production of the MSH receptor
may be receptor upregulation. Also a method of decreasing or
increasing the generation of second messenger elements and/or
increasing the production of the MSH receptor and/or optionally
increasing or decreasing the binding affinity of MSH to the MSH
receptor is part of the invention. The method comprises
administering to an animal, in particular a human, a medicament
which is or becomes bound to a substance, which substance in
advance has been found to bind to a polypeptide having the amino
acid sequence shown in SEQ ID NO:2 or 16 or an analogue or
subsequence comprising any of the subsequences encoding one or
several of the extracellular loops and/or the transmembrane
sequence and/or the cytoplasmic loops and/or the C-terminal
sequence or a combination thereof.
[0159] A substance as the above indicated could be chosen by
employing the methods described below for identifying substances
which can prevent or stimulate the effect exerted by MSH
receptors.
[0160] A method for internalization of an MSH receptor, thereby
making it unavailable for the binding of the hormone, constitutes
another aspect of the invention. The method comprises using a
substance which in advance has been found to bind to a polypeptide
having the amino acid sequence shown in SEQ ID NO: 2 or 16 or a
subsequence comprising any of the subsequences encoding one or
several of the extracellular loops and/or the transmembrane
sequence and/or the cytoplasmic loops and/or the C-terminal
sequence or a combination thereof, such as a substance or a
modified form of the MSH which is either able or unable to initiate
the normal processes activated by the hormone but which substance
causes internalization of the receptor. This method may also be
used to regulate the effect exerted by the MSH receptor.
[0161] By using radioligand binding techniques and expressed MSH
receptor protein and peptides the binding affinities of substances
(ligands) for the MSH receptors may be measured. Such measurements
are typically performed in the screening of novel drugs (synthetic
or natural) with potential activity on MSH receptors. In
pharmacological terms such drugs may act as agonists, partial
agonists or as antagonists at the MSH receptor. All types of such
substances are testable using the method according to the
invention. The substances/ligands acting on the MSH receptor (or
its derivative) can also be coupled to toxic agents (toxins,
radionuclides) aiming to destroy the MSH receptor (or the MSH
receptor derivative) bearing organisms or cells.
[0162] A very important aspect of the invention relates to methods
for identifying substances which may be used for preventing or
stimulating the effect exerted by an MSH receptor such as the
generation of a second messenger element in a cell, such as a
mammalian cell, in particular a human cell. Inhibiting the binding
of MSH to the MSH receptor is one way of achieving this, and
therefore methods for identifying substances which are capable of
binding to the MSH receptors are very important aspects of the
invention. The methods involve various methods of assessing the
capability of the substance in question to compete with the binding
of MSH to the MSH receptor. The substance may prevent this binding
by blocking the MSH binding site on the MSH receptor resulting in
blocking of the effects exerted by the MSH receptor upon binding of
MSH. In another embodiment, the substance may be a substance with
optionally increased binding capacity to the MSH receptor (compared
to MSH) and which is in addition capable of activating the effects
exerted by the MSH receptor.
[0163] One embodiment of this aspect of the invention is to
incubate an MSH receptor protein or its analogue, obtained as
described in example 3, with radioactively labelled MSH or MSH
analogue together with the test substance. Depending on the binding
activity of the test substance the amount of labelled MSH or MSH
analogue becoming bound to the MSH receptor will vary. Test
substances with high binding affinity for the MSH receptor will
exclude the binding of the labelled MSH or MSH analogue at lower
concentration than test substances with lower binding affinity.
Separation of bound versus free labelled MSH or MSH analogue is
accomplished using techniques such as filtration, centrifugation,
superflow or chromatography. Measurement of radioactivity either
retained on the receptor or being present in the solution separated
from the receptors is made using standard nuclear counting. In
another variant of this embodiment of the invention, the amount of
MSH or MSH analogue being bound to the receptor or being present in
the solution separated from the receptors is detected by using any
other suitable detection system capable of detecting MSH or MSH
analogue. Examples of such detection systems are immune assays such
as radio immune assay and ELISA (Enzyme linked immune sorbent
assay), immune fluorescence assay, UV light absorption spectrometry
or fluorescence emission spectrometry.
[0164] In another embodiment of this aspect of the invention, the
amount of test substance bound to the MSH receptor is indirectly
and/or approximately estimated by measuring the alteration in the
degree of interaction of the MSH receptor with a G-protein caused
by the binding of test substance to the MSH receptor. In a variant
of such a test system, the effect of the test substance is studied
alone. In another variant of such an assay, the ability of the test
substance to compete for MSH or MSA analogue is studied by the
simultaneous addition of test substance and MSH or MSH analogue.
The degree of activation of the G-protein by the MSH receptor
caused by the test substance, the MSH or the MSH analogue can be
measured directly by e.g. measuring the GTPase activity of the
system using previously described methods (Aktories and Jakobs
1981; Vachon et al. 1986), or by using other suitable methods.
Alternatively the degree of activation of the system may be studied
indirectly by measuring other biochemical or physiological
parameters which may become altered as a consequence of the primary
interaction of the ligand(s) with the MSH receptor. Examples of
measurable entities in this context are adenylate cyclase activity,
cAMP-levels, skin pigmentation, tyrosinase activity and
[.sup.35S]methionine incorporation (Burchill et al. 1990).
[0165] In yet another embodiment of the invention, which is
particularly useful if the ligand is a macromolecule, such as when
the ligand is an antibody, the detection of the binding of the
ligand to the receptor may be done using other approaches. In one
variant of this embodiment, the ligand is incubated with a
substantially pure preparation of the MSH receptor or its analogue,
the latter which has been tagged with a suitable molecule which,
after separation of bound versus free MSH receptor, will allow the
detection of the MSH receptor ligand complex by e.g. nuclear
counting, color, fluorescence or enzymatic activity. Separation of
bound and free ligand may, for example, be accomplished by adding a
second antibody which is directed towards the ligand thereby
forming a precipitate of the ligand-receptor complex. In other
variants of the invention, a substantially pure preparation of the
MSH receptor is attached to a solid support. The ligand is then
incubated with the solidified receptor whereafter detection of the
amount of ligand bound to the receptor may be done using
conventional ELISA or using any similar suitable approach.
[0166] It will be understood that the ligands described herein may
be provided with a detectable label. The ligands themselves can be
macromolecules, such as monoclonal or polyclonal antibodies or they
may be substances of natural or synthetic origin which are able to
bind to MSH receptor.
[0167] Using the above and similar approaches substances can be
identified which can block the binding of the MSH receptor (or its
derivative) by the receptor ligands. In the present context the
term "blocking of the MSH receptor (or its derivative)" means that
the MSH receptor (or its derivative) is occupied by the substance
so that the receptor ligands cannot bind the MSH receptor (or its
derivative) or that the MSH receptor ligands are capable of binding
to the MSH receptor but unable to activate the MSH receptor.
[0168] It will be understood that methods similar to those
mentioned above for identifying substances which bind to an MSH
receptor can be used for identifying substances which bind to other
melanotropic hormone receptors. Since the DNA fragments of the
invention which have the nucleotide sequences shown in SEQ-ID NO:5
and 9, coding for the polypeptides of the invention shown in SEQ ID
NO:6 and 10 may be derived from other melanotropic hormone
receptors, the binding properties of these melanotropic hormone
receptors may be of great importance. Thus, important aspects of
the invention are methods as the above-mentioned for identifying
substances which are capable of binding to melanotropic hormone
receptors.
[0169] It is of course also important to note, that the DNA
fragments having the nucleotide sequence shown in SEQ. ID NO:5, 7
and 9 can be modified in the same manner as other DNA fragments of
the invention, and thus, all disclosure in the present
specification relating to the modifications of the DNA fragments
having the nucleotide sequence shown in SEQ. ID NO: 1 and 15
applies analogously or mutatis mutandis to modifications of the DNA
fragments having the nucleotide sequence shown in SEQ ID NO:5, 7
and 9. Also, as aspects of the invention, the DNA fragments having
the nucleotide sequences shown in SEQ ID NO:5, 7 and 9 can be used
in the same manners as described in the present specification for
the DNA fragments having the nucleotide sequences shown in SEQ ID
NO:1 and 15. Likewise, the polypeptides having the amino acid
sequences SEQ ID NO:6, 8 and 10 can be modified in the same manner
as the polypeptides shown in SEQ ID NO:1 and 15, and thus, all
disclosure in the present specification relating to the
modification of the polypeptides having the sequences shown in SEQ
ID NO:6, 8 and 10 applies analogously or mutatis mutandis to
modifications of the polypeptides shown in SEQ ID NO:1 and 15.
Also, as aspects of the invention, the polypeptides having the
amino acid sequences shown in SEQ ID NO:6, 8 and 10 can be used in
the same manners as described in the present specification for the
polypeptide having the amino acid sequence shown in SEQ ID NO:1 and
15.
[0170] The polypeptide with the amino acid sequence SEQ ID NO:2 has
been identified as an MSH receptor, based on the results of such
binding experiments. A series of POMC (pro-opiomelanocortin)
derived peptides showed differential potencies in inhibiting
.sup.125I-NDP-MSH binding to MSH receptor coding fragment
transfected COS-7 cells. The potency order was NDP-MSH
(K.sub.i=23.+-.0.5 pM)>.alpha.-MSH (K.sub.i=92.+-.19 pM)>ACTH
(1-39) (K.sub.i=170.+-.37 pM)>.beta.-MSH (K.sub.i=449.+-.74
pM)>.gamma.-MSH (K.sub.i=1010.+-.200 pM). ACTH (4-10) showed
very low binding affinity (K.sub.i=22,400.+-.7200 pM), whereas the
non-melanotropic POMC peptide .beta.-endorphin showed no affinity
for the expressed MSH receptor.
[0171] Moreover, in addition the polypeptide with the amino acid
sequence shown in SEQ ID NO:16, referred to as the MC-2 receptor,
has been identified as an MSH receptor and/or MSH receptor subtype
based on the result of such binding experiments. A series of POMC
derived peptides showed the following differential potencies in
inhibiting 125I-NDP-MSH binding to MC-2 receptor coding fragment
transfected COS-7 cells: NDP-MSH (Ki=5.18.+-.0.54
nM)>.alpha.-MSH (Ki=928.+-.314 nM)=ACTH (1-39) (Ki=929.+-.389
nM)>.beta.-MSH (Ki=1.75.+-.0.67 .mu.M) >.gamma.-MSH
(Ki=3.45.+-.0.88 .mu.M). The non-melanotropic POMC peptide
.beta.-endorphin showed no affinity for the expressed MC-2
receptor.
[0172] The above mentioned binding experiments can be done using
whole animal systems, human clinical trials, a tissue specimen, a
microorganism and/or a cell, in particular a cell line expressing
the said receptor protein or its analogue. It can also be achieved
using the purified protein of the invention. The purified protein
can be used in a soluble form or in the solid phase being attached
to a suitable matrix.
[0173] Drugs can be designed so as to act on very specific parts of
a polypeptide of the invention. Drugs can be acting on either only
the regions of or within the extracellular loops or transmembrane
segments. In either case it may be affecting the binding of the
natural ligands to the MSH receptor or its derivatives. Specific
drugs can also be directed towards the regions of intracellular
loops. Such drugs could be affecting the coupling of the MSH
receptor or its derivatives to the intracellular systems like the
G-proteins. Such drugs could also be affecting the G-proteins so
that they cannot couple to the MSH receptor or its derivatives.
[0174] Various ways of treating the disease conditions wherein an
MSH receptor is involved are provided by the present invention.
These diseases include MSH receptor expressing disease condition
such as melanoma, skin cancer, pyretic condition, inflammatory
condition, nociceptive condition, catatonic condition, impaired
memory condition, reduced or increased skin tanning and/or
pigmentation conditions, epilepsy. The invention also includes a
method to improve nerve repair, muscle reinnervation and/or neuron
growth.
[0175] Due to its central nervous system localization the MC-2
receptor is in particular a target for drugs used in treating
conditions such as pain, pyretic, catatonic and impaired memory
conditions. Moreover, due to peripheral localization of the MC-2
receptor it is an interesting target for the anti-inflammatory
drugs. The MC-2 receptor is also an interesting target for drugs
improving growth and/or regeneration and/or repair of neurons being
damaged due to disease and/or toxic influence and/or age and/or by
other condition being associated with or leading to neuron damage.
Moreover, due its ability to improve muscular reinnervation the
MC-2 receptor is an interesting target for drugs treating condition
of impaired muscle innervation. In addition due to its central
nervous system localization the MC-2 receptor is also an important
target for drugs used in the treatment of epilepsy.
[0176] Thus, the invention relates to a method of targeting a cell
that contains an MSH receptor on the surface with a medicament
comprising administering the medicament in the form of a substance
that binds to the MSH receptor. In a particular embodiment of the
invention, the medicament may be attached to a substance such as an
antibody or a part thereof or be a molecule of natural or synthetic
origin having affinity for the MSH receptor. The medicament may be
a radionuclide or a toxin or any other molecule of natural or
synthetic origin. The use of an antibody such as a monoclonal
antibody as a substance to which a medicament is bound comprises an
important aspect of the invention. The antibody could be tagged
with toxin or radioactivity for diagnostic or therapeutic purposes.
Such an approach is expected to be superior to the MSH toxin
conjugates mentioned above, because of the expected high avidity
and specificity of such MSH receptor antibodies. Moreover, the MSH
toxin conjugates might induce untoward effects by virtue of their
potential hormonal activity. The use of toxin or radiation coupled
monoclonal antibodies against the MSH receptor may prove to be a
very attractive approach as the MSH receptor is the most common and
most specific component of the melanoma cells.
[0177] In another embodiment of the invention the substance is a
natural or synthetic organic compound, or a peptide or derivative
thereof, that binds to the receptor or an epitope thereof and which
optionally may become discovered by using methodology described in
the present application. Such a substance may in particular be a
synthetic and/or a natural compound which have or do not have any
structural resemblance to MSH. Such substances are typically
composed of one or two or several aromatic and/or non-aromatic
rings and/or heterocyclic rings, with side chains appropriately
attached and may in addition have chains interconnecting the ring
structures. Such substances vary considerably in their structure
and there exist several different classes of such substances which
is due to the fact that they either bind to the same or the
differing epitopes of the MSH receptor. Some of these substances
share partly or totally the same binding epitope on the MSH
receptor as the MSH peptide, whereas other substances bind to other
and/or partially other epitopes of the MSH receptor. Some of these
substances have the ability to mimic the action of MSH in that when
they bind to the receptor they cause the same effects in a cell
and/or organ and/or tissue as when MSH binds to the MSH receptor.
Other substances by contrast have the ability to prevent the action
of MSH on the receptor by their binding to the MSH receptor.
[0178] In a very important aspect of the invention, a lipid soluble
form of an MSH receptor may be used in the treatment of an animal,
in particular a human, by administering this form to the
animal.
[0179] Also in a very important aspect of the invention, conditions
caused by MSH receptor deficiency or impaired MSH receptor function
in an animal, in particular a human may be treated by introducing a
DNA fragment encoding an active form of an MSH receptor. One such
condition which may be treated by the present invention is
tyrosinase-positive albinism.
[0180] As stated previously, endogenous and exogenous melanotropins
are suggested to enhance human cutaneous pigmentation in vivo
(Levine 1991; Mulligan et al. 1982; Lerener et al. 1961). A
treatment which produces tanning without sun exposure will be
helpful to people who tan poorly and sunburn easily. Increased
melanin in the skin might afford these people protection against
ultraviolet light and thus put them at low risk for skin
cancer.
[0181] The use of MSH and other melanotropins may be an effective
and safe means of achieving skin darkening without harmful excess
sun exposure. In addition, the resultant increased skin
pigmentation might provide protection against the effects of
subsequent sun exposure. In patients with tyrosinase-positive
albinism, the molecular machinery to make melanin is present, but
functions suboptimally (King et al. 1988). Perhaps, by acting on
MSH receptor melanotropins could in these patients stimulate
tyrosinase resulting in an increase in pigmentation. This may
afford these individuals protection from ultraviolet light while
improving their appearance and social acceptance.
[0182] In the present context melanotropin is intended to be a
substance that is binding to an epitope of an MSH receptor and
thereby e.g. induces a similar skin tanning effect as MSH or any
other desirable effect similar to that of MSH. Such a substance may
be found using the methodology described in the present application
and may in particular be a synthetic substance which have or do not
have any structural resemblance to MSH.
[0183] Also, the invention relates to a method for increasing the
melanin content of the skin in an animal, in particular a human,
comprising using substances that are active through an MSH
receptor. Thus, the skin tanning may be obtained without or with
reduced exposure to sunlight which will make it possible to avoid
sunburns, which is most desirable as already explained above.
[0184] Detection of the MSH receptors of the invention is important
in various diagnostic aspects of the invention and may facilitate
the diagnosis of various of the disease conditions associated with
a content of MSH receptors in the tissue that is higher than
normally found in said tissue and improve the prognosis of some of
the diseases such as melanoma and skin cancer. Especially important
aspects of the invention are the use of the detection of MSH
receptor in MSH receptor expressing diseases, such as melanoma or
skin cancer, in assessing the prognosis and/or guidance for further
treatment.
[0185] Thus, the invention relates to a method of diagnosing an MSH
receptor expressing disease condition such as melanoma or skin
cancer comprising targeting a cell containing an MSH receptor on
the surface with a diagnostic agent capable of binding to the MSH
receptor, which diagnostic agent can be detected following binding
to the receptor. The diagnostic agent may be administered bound to
a substance that binds to MSH receptor.
[0186] The diagnostic agent may be a radioactive substance, or may
be linked to a radioactive substance. In other embodiments, the
diagnostic agent may be a colored or color generating substance or
linked to a color or color generating agent.
[0187] One diagnostic method of the invention is detecting an MSH
receptor in a biological sample, wherein the sample is treated with
a substance that binds to the MSH receptor, and detecting or
visualizing the presence of the bound substance. In a particular
interesting embodiment of the invention, the substance is an
antibody or a part thereof. The antibody may be an antibody that
distinguishes between possible different forms of the MSH receptor.
The antibody may be labelled with radionuclide, or biotinylated or
may be unlabelled and later detected by immunostaining. An
important method in connection with this part of the invention is
detection and/or measurement of the bound antibody by a method of
the ELISA type or by a method of the radioimmunoassay type.
[0188] The terms "a sample" or "a biological sample" as used herein
are defined as a cell, a subcellular fraction, a cell fraction, a
tissue sample, a cell culture, or a cell suspension.
[0189] In connection with the above, the invention also relates to
polyclonal and monoclonal antibodies which are reactive with a
polypeptide or an analogue or subsequence thereof of the invention.
A detailed description of the various aspect of the invention
involving antibodies and which constitutes parts of the invention
is given herein.
[0190] In the present context the term antibody is understood as
the whole antibody molecule or any fragments thereof. An antibody
can be fragmented during and/or after the production. It can also
be made in the fragmented form to begin with and used as such or
used after joining different fragments.
[0191] The animal used for the preparation of antibodies to a
polypeptide of the invention is preferably selected from the group
consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse
and guinea pigs. The cells producing the antibodies may be spleen
cells or peripheral blood lymphocytes. The antibody or fragments
thereof may be of a monospecific (polyclonal) kind. The
monospecific antibody may be prepared by injecting a suitable
animal with a substantially pure preparation of a polypeptide of
the invention. This can be followed by one or more booster
injections at suitable intervals before the first bleeding. The
animals are bled about 5-7 days after each immunization. Antibodies
may optionally be isolated from the serum using standard antibody
purification techniques (Sambrook et al. 1989).
[0192] Using the sequence of SEQ ID NO:2 polyclonal antibodies have
been prepared by chemically synthesizing two peptides which had the
amino acid sequences identical to amino acids 4-19 and 25-35 of SEQ
ID NO:2, respectively.
[0193] These two peptides were (separately) coupled to
thyroglobulin and separately injected into rabbits in Freund's
adjuvant. After four booster injection both the conjugates were
found to have induced formation of sera in the rabbits which were
highly reactive against MSH receptor containing cells. The details
of the manufacturing of the anti MSH rabbit sera is given in
Example 10.
[0194] A monoclonal antibody or fragments thereof may be raised
against an essential component of an MSH receptor, i.e. an epitope.
The monoclonal antibody may be produced using conventional
techniques (Kohler et al. 1975) by use of a hybridoma cell line, or
by clones or subclones thereof or by cells carrying genetic
information from the hybridoma cell line producing said monoclonal
antibody. The monoclonal antibody may be produced by fusing cells
producing said monoclonal antibody with cells of a suitable cell
line, and cloning the resulting hybridoma cells producing said
monoclonal antibody. Alternatively, the monoclonal antibody may be
produced by immortalizing an unfused cell line producing said
monoclonal antibody. The monoclonal antibodies are ultimately
harvested from the cell growth medium. Hybridoma cells used to make
monoclonal antibody may be grown in vitro or in the body cavity of
an animal. The monoclonal antibody or fragments thereof may also be
made using the recombinant DNA techniques (Huse et al. 1989).
[0195] Monoclonal antibodies may also be made by immunizing the
suitable animals with a unpurified preparation of an MSH receptor
protein. The resulting hybridoma clones secreting monoclonal
antibodies can be screened for their ability to block the binding
of MSH or its analogue to the MSH receptor e.g. using the approach
described in example 3.
[0196] The idiotypic (antigen binding) structure of the antibody is
antigenic and can thus give rise to specific antibodies directed
against the idiotypic structure. The antibodies raised against the
idiotype are called the anti-idiotypic antibodies. Such antibodies
may mimic the structure of the original antigen and therefore may
function as the original antigen. Such antibodies may be able to
substitute the original antigen (MSH receptor protein, polypeptides
or their analogues) for a part or all of the functions, usability
and properties of the original polypeptide of the invention.
[0197] Preferably the monoclonal antibodies or fragments thereof
will be used in most cases but polyclonal antibodies or fragments
thereof may also be used. Typical uses of MSH receptor antibodies
are as follows:
[0198] For purification of proteins: The antibodies can be used to
purify an MSH receptor or its derivatives from the biological
samples, using the affinity chromatography or the
immunoprecipitation techniques.
[0199] For diagnosis and therapy: The monoclonal antibodies against
an MSH receptor or its derivatives can be used in the diagnosis and
therapy of disease conditions in animals and humans. The diagnostic
and therapeutic antibodies may be valuable for the disorders of
skin, like skin cancer generally described as melanoma. The finding
that MSH receptor is consistently found on melanoma tissues
supports this notion. The diagnostic agent may be an antibody with
the specificity for a polypeptide of the invention. The antibody
can be coupled to another protein or a solid support and/or can be
used in the agglutination tests or the color developing tests. Such
antibodies can also be used to quantitate the MSH receptor or its
derivatives in biological samples using the standard histochemistry
or immunochemistry techniques.
[0200] For toxin therapy: The specific monoclonal antibodies can be
coupled to different toxins like ricin or diphtheria toxin.
Generally the A-chain of the plant toxin ricin or the A-chain of
the diphtheria toxin is conjugated to the monoclonal antibody in
order to assemble hybrid proteins which have a targeted
cytotoxicity. Moreover, the toxin used may alternatively be
selected from Pseudomonas endotoxin, abrin or fungal
ribosome-inactivation proteins (RIP). In the present context, a
hybrid between the monoclonal antibody against a polypeptide of the
invention and a toxin moiety can be used to bring about the killing
of the MSH receptor bearing cells in an organism. Moreover, in the
present context the toxin is intended to mean any toxin that is
suitable for the purpose of killing and/or damaging the cell
wherein the MSH receptor is located. The toxic effect of the toxin
may be brought about when the toxin is still conjugated with the
antibody. However, more likely the toxin will be processed once the
antibody-toxin conjugate has become attached to the MSH receptor
bearing cell. Such processing may involve e.g. internalisation of
the antibody-toxin complex, cleavage of toxin from antibody and
transportation of the toxin within the cell to its site of action.
The processing is being done by the natural machinery of the MSH
receptor bearing cell and careful engineering of the properties of
the antibody-toxin complex will maximise its toxicity by affording
the most favourable processing pathway for the complex.
[0201] In order to improve the clinical efficiency of the
antibody-toxin complex the monoclonal antibody can be designed so
as to reduce its size by e.g. utilising F(ab').sub.2 or Fab'
fragments instead of the whole intact antibody. The reduction in
the size of the molecule will increase the ability of the
antibody-toxin complex to diffuse from the blood to the site of the
tumour cell. Moreover, selecting a small sized toxin for the
conjugation to the antibody, such as e.g. fungal RIP, will afford
the same effect. Moreover, elimination of the Fc fragment, as is
achieved with the use of F(ab')2 or Fab' fragments for toxin
conjugation, will eliminate the possibility that the antibody-toxin
conjugate will bind to cells containing Fc receptors thus
minimising non-specific binding of the toxin complex to other cells
than MSH receptor bearing cells. Thus, this measure will increase
the selectivity of the antibody-toxin complex and increase its
cytotoxic effect since a higher dose will be possible to
administer. The problem of inducing a humoral immune response in
the patent to whom the antibody-toxin conjugate is administered may
be minimised by prior and/or concomitant administration of a drug
which suppress the immune response. Such drugs may e.g. be selected
from cyclophosphamide, prednisone, azathioprine and/or cyclosporin.
Moreover, another approach for the same purpose is to administer a
monoclonal antibody directed towards CD4 antigen. Yet, another
approach for the same purpose is to carefully engineer the
antibody-toxin complex to minimise its immunogenicity. Such
engineering is afforded by eliminating the most immunogenic
epitopes of the complex while still retaining its ability to bind
to the MSH receptor with high affinity and retaining its desired
toxic effect.
[0202] Moreover, the engineering will also have the purpose to
increase crease the stability of the complex after it has been
administered to the patient. Increased stability is essential to
afford a good therapeutic effect.
[0203] Increased clinical efficiency of the antibody-toxin complex
may also be afforded by concomitant administration to the patient
of another agent which will potentiate the toxicity of the complex.
The other agent may e.g. be cyclophosphamide, daunorubicin and/or
interferon. Moreover, increased toxicity may be afforded by
simultaneously utilising antibody-toxin complexes where two and/or
several toxins with different mechanism for their toxicity has been
included. As an alternative the simultaneous administration of
another antibody-toxin complex which is directed for yet another
melanoma protein, with the anti MSH receptor antibody-toxin complex
of the present invention, will afford increased clinical
efficiency. Yet another measure to afford increased clinical
efficiency of the antibody-toxin complex will be afforded by the
additional coupling to the MSH receptor antibody-toxin complex a
suitable radionuclide which by virtue of RIT induces a cytotoxic
effect. The approach of RIT for therapy of melanoma is described
further below.
[0204] For radiodiagnosis and radiotherapy: The monoclonal
antibodies against an MSH receptor or its derivatives can be used
in the diagnosis and therapy of disease conditions in animals and
humans. The diagnostic and therapeutic antibodies may be valuable
for the disorders like skin cancer generally described as melanoma.
The finding that MSH receptor is consistently found on melanoma
tissues supports this notion.
[0205] The specific monoclonal antibodies can also be coupled to
different radionuclides like, .sup.123I, .sup.125I, .sup.131I,
.sup.99mTC, .sup.111In, .sup.186Re, .sup.188Re, .sup.211At,
.sup.212Bi, .sup.90Y, .sup.67Cu and .sup.67Ga. Such radiolabelled
antibodies can be used for diagnosis (Radioimmunoscintigraphy) or
therapy (Radioimmunotherapy).
[0206] In the present context a radionuclide is intended to mean
any radionuclide that is suitable for the purpose of detecting the
cell and/or cell cluster that is hosting an MSH receptor. Coupling
of radionuclides to anti MSH receptor antibodies may be afforded by
a number of techniques. For .sup.123I, .sup.125I and/or .sup.131I
coupling is afforded by well established chloramine-T, iodogen,
lactoperoxidase and/or hydroxyperoxidase methods. Moreover,
radioiodination may as an alternative be afforded using
Bolton-Hunter reagent. Radiolabelling with e.g. .sup.111In and/or
.sup.90Y and/or 67Cu may be afforded by the coupling of a
bifunctional chelating agent to the antibody and then adding the
radionuclide to the complex. By virtue of the chelating ability of
the complex the radionuclide will become attached to the complex.
Suitable chelators may be selected from eg. isothiocyanatobenzyl
EDTA (CITC), diethylenetriaminepentaacetic acid (DTPA) and be
coupled via the mixed anhydride or the cyclic anhydride (Hnatowich
1990). However, since such complexes may provide somewhat unstable
chelation and moreover during their manufacture intra and
intermolecular cross linking of antibodies, other chelators such as
e.g. GYK-DTPA or SCN-BZ-DTPA may be used as an alternative
(Hnatowich 1990). Radiolabelling of .sup.99mTc to the antibody may
be afforded by using direct labelling techniques such as by
reducing disulphide bonds on the antibody thereby providing sites
for stable attachment of .sup.99mTc. One method for this end is to
use tin which will afford reduction of disulphide bonds and adding
[.sup.99mTc]pertechnetate which also will provide .sup.99mTc by
reduction with the tin (Hnatowich 1990). In the present context tin
may be provided in the form of e.g. stannous tartrate or any other
form suitable for the purpose. As an alternative to tin another
suitable reducing agent may be used such as e.g. dithiotreitol
and/or 2-mercaptoethanol. Moreover, [.sup.99mTc]-glucartate and/or
[.sup.99mTc]-phosphonate may substitute for
[.sup.99mTc]pertechnetate as source for .sup.99mTc. Yet another
approach for .sup.99mTc labelling of anti MSH antibodies is to use
chelators as was described above. For the purpose of .sup.99mTc
chelation a promising concept is to couple metallothioneins to the
anti MSH receptor antibody since the proteins afford strong
chelation of .sup.99mTc. Still another chelator that may be
employed is the diamide demercaptide chelator (Fritzberg et al.
1986).
[0207] Radioimmunoscintigraphy procedure is based on the fact that
the labelled antibody will recognize the MSH receptor (or its
derivative) on the cells, normal or diseased, and that the antibody
will not bind to the cells devoid of the MSH receptor (or its
derivative). The ultimate quality of the scintigraphic examination
is dependent on the absolute quantity of the MSH receptor (or its
derivative) in the specimen under examination and the background
activity. It is possible to detect tumours using this technique
when the tumour to non-tumour signal ratios are 1.5/1 or higher.
Imaging is initially generally performed as planar scintigraphic
examination. Anatomical landmarks are indicated with a point
source, separately recorded, and afterwards added with computer
assistance. A Single Photon Emission Computed Tomography (SPECT)
can be performed, acquiring data-by a 360 degree rotation of the
gamma camera around the object under examination. Transverse,
coronal, sagittal or oblique sections are then reconstructed using
mathematical calculations. SPECT appears to improve sensitivity and
requires low tumour/non-tumour signal ratio. In the present context
the above mentioned approach can be used in the diagnosis of skin
cancers and other disease conditions where MSH receptor (or its
derivative) is expressed and can be approached by the monoclonal
antibody against a polypeptide of the invention.
[0208] Radioimmunotherapy (RIT) for killing diseased cells by a
toxic agent bound to a specific monoclonal antibody is a promising
concept. In order to be efficient RIT has requirements besides the
tumour/non-tumour signal ratio. The amount of radioactivity has to
be sufficient to eradicate tumour without giving a high radiation
dose to the surrounding normal tissue. Furthermore, distribution of
the labelled monoclonal antibody in the tumour has to be
homogeneous, allowing radiation of all tumour cells. The biological
half life of the monoclonal antibody in the tumour has to be long
enough to allow the radionuclide to exert maximal radiation
effects. In the present context, the above mentioned approach can
be used in the therapy of skin cancers and other disease conditions
where MSH receptor (or its derivative) is expressed and can be
approached by the monoclonal antibody against a polypeptide of the
invention.
[0209] As ligand binding blockers: The prevention of the binding of
an MSH receptor (or its derivative) by the receptor ligands can be
suitably performed by the antibodies with the specificity for a
polypeptide of the invention. In the present context the term
"blocking of an MSH receptor (or its derivative)" means that the
MSH receptor (or its derivative) is occupied by the antibodies so
that the receptor ligands cannot activate the MSH receptor (or its
derivative).
[0210] In accordance with the above, the invention also relates to
an antibody capable of binding to a polypeptide of the invention
provided with a detectable label, and to a polypeptide of the
invention provided with a detectable label. The polypeptide or the
antibody may in some embodiments be coupled to a solid support. The
support may be selected from the group consisting of plates,
strips, beads, particles, films and paper, and the solid support
may be of latex, polystyrene, polyvinyl chloride, polyolefin,
nylon, polyvinylidene difluoride, cellulose, silicone or
silica.
[0211] Other methods for detection and/or quantitation of an MSH
receptor comprise detection of the DNA or RNA and such methods are
preferably based on the principles of hybridization which have been
described in details above. Thus, in one such aspect the invention
relates to a method for detection and/or quantitation of the mRNA
of an MSH receptor comprising extracting RNA from a biological
sample such as a subcellular fraction, a cell, a tissue sample, a
cell culture or a cell suspension and measuring the hybridization
of said RNA to a labelled DNA fragment of the invention or a
labelled RNA fragment which can be constructed from a DNA fragment
of the invention. Also, methods for measuring RNA such as northern
blot or dot blot may be employed. The hybridization may be
performed in situ or a labelled antisense mRNA probe may be used.
In another embodiment detection and/or quantitation of the MSH
receptor mRNA may be obtained by extracting RNA from cells or
tissues and converting it into cDNA for subsequent use in the
polymerase chain reaction (PCR). The PCR primer(s) may be
synthesized based on a DNA fragment of the invention such as the
DNA fragments shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4 or
any other DNA fragment of the invention. This method for detection
and/or quantification may be used as a diagnostic method for
diagnosing an MSH receptor expressing disease condition such as
melanoma, skin cancer, pyretic condition, inflammatory condition,
nociceptive condition, catatonic condition, impaired memory
condition, reduced or increased skin tanning condition and/or
pigmentation condition.
[0212] In another specific embodiment of the invention this method
for the detection of MSH receptor RNA and/or DNA is used as
production control in the breeding of animals for obtaining a
desired fur and/or skin color in the animal. Animals for which such
a control of fur and/or skin color is desired may be selected from
e.g. mammals and reptiles and may in particular e.g. be a snake,
alligator, crocodile, mink, fox, hamster or chinchilla.
[0213] In yet a further embodiment of the invention the MSH
receptor coding fragment(s) or a subsequence thereof is being
analyzed in an animal by using e.g. cloning or PCR as described
above. The thus obtained DNA and/or cDNA is subjected to sequence
analysis using known methodology with the purpose of detecting a
specific variant of an MSH receptor. The detection of such variants
of MSH receptor may be desired e.g. in production control for the
breeding of animals in order to obtain a desired skin and/or fur
color. Animals for which such a control of fur and/or skin color is
desired may be selected from e.g. mammals and reptiles and may in
particular e.g. be a snake, alligator, crocodile, mink, fox,
hamster or chinchilla.
[0214] In still a further embodiment of the invention a desired
skin and/or fur color of the animal is being obtained by
introducing into the animal the desired variant of the MSH receptor
by e.g. manufacturing a transgenic animal which will appropriately
produce the MSH receptor variant. As an alternative the desired MSH
receptor variant is being obtained by mutating a natural MSH
receptor coding fragment in situ in the animal. Animals where the
introduction of MSH receptor variants are desired may be selected
from e.g. mammals and reptiles and may in particular e.g. be a
snake, alligator, crocodile, mink, fox, hamster or chinchilla.
[0215] In the present context an MSH receptor variant is intended
to mean a homologue and/or analogue of SEQ ID NO:1, SEQ ID NO: 2,
SEQ ID NO:15 or SEQ. ID NO:16.
[0216] The lack of detailed structural information at the atomic
level about the tertiary structure of the MSH receptor (a member of
the G-protein coupled receptor family) continues to hamper efforts
to understand receptor function at the molecular level. A
polypeptide of the invention can be produced in large quantities
using protein purification methods, and/or recombinant DNA
techniques and/or synthetic chemistry approach. The polypeptide of
the invention can then be crystallized. Crystallization, the
ordered packing of molecules in three dimensions, relies on
achieving the right balance of attractive and dispersive or
repulsive forces between protein molecules in solution. To
solubilize an integral membrane protein, like an MSH receptor or
its derivatives, it is necessary to replace the natural
phospholipid environment by the detergent. The resultant shell of
detergent around the protein is considered to be the most important
factor impeding the formation of ordered crystals. Removal of the
detergent leads to the precipitation of the protein and renders it
useless for the crystallographic studies.
[0217] The main problem in crystallisation of an MSH receptor is to
find the ideal detergents which forms stable or semi-stable
complexes with the membrane section of the MSH receptor, where
there are 7 transmembrane sections. It is also a pre-requisite that
the overall structure of the receptor is not affected too much,
something which can be validated using functional assays such as
e.g. radioligand binding as has been described in the present
application. Several classes of detergents, which are useful for
crystallisation, are presently known and may be applied. It is a
good approach for each of these detergents to obtain individual
phase diagram for the solubility of the MSH receptor and analyze
these diagrams in detail in order to find optimal conditions for
crystallisation. Using such approach it will be possible to find
and refine a buffered solution or a mixture of detergents and salts
to force the protein unit to arrange intermolecular contacts for
crystal growth. By salts are in the present context intended mono
and/or divalent ions which may support the interaction of
individual MSH receptor molecules to arrange in a crystal lattice.
Moreover, crystallisation may be afforded by alternatively or in
addition adding MSH or an MSH analogue which will bind to the
receptor and increase the possible surface(s) for intramolecular
contact. In addition the solubility of the MSH receptor may be
analyzed using temperature gradients. The initial aggregation of
nucleus, indicating crystallisation, can be analyzed in combination
with e.g. laser light scattering. To obtain crystals the approaches
such as e.g. sitting and hanging drop as well as micro-batch
applications may be utilised. Improved crystals may be obtained
using microgravity conditions. As a final approach heavy atom
cluster can also be applied.
[0218] Crystal aggregates may be analyzed by subjecting them to
e.g. synchrotron radiation at a suitable wave length, such as e.g.
an 1 .ANG. wave length or a wave length more or less than 1 .ANG.,
and collecting data for radiation diffraction. The application of
anomalous scattering to solve the phase problem in crystallography
can be applied. Moreover, cooling of crystals a suitable
temperatures such as e.g. -10.degree. C. and 4.degree. C. or the
flash freezing of MSH receptor crystals, which are very radiation
sensitive, may be applied. The solution of 3D structure of the MSH
receptor from X-ray diffraction pattern may be afforded using well
known computational techniques.
[0219] Although no G-protein coupled receptor (family to which the
MSH receptor belongs) has yet formed crystals, two other integral
membrane proteins, the photosynthetic reaction center (Allen et al.
1987) and bacteriorhodopsin (Henderson et al. 1990), have been
successfully studied. Similar techniques as singly or in
combination can be applied to the crystallization and/or atomic
structure determination of the polypeptides of the invention.
Moreover, other techniques aiming at the elucidation of the 3D
structure of proteins are being rapidly developed. One such
technique, which is already far advanced, is two-dimensional NMR
(Wright 1989), as well as modern multidimensional NMR-techniques.
In order to utilize such a technique for elucidation of MSH
receptor 3D structure it is required to have sufficient amounts of
pure MSH protein and then obtain the appropriate two-dimensional or
multidimensional NMR data which is used along with the known
primary amino acid sequence of the receptor applying appropriate
computational methods. In addition computational methods are also
being developed aiming to elucidate the 3D structure of proteins in
the computer. These methods are generally and collectively referred
to as molecular modelling. It is predicted that once the 3D
structure of one member of the G-protein coupled receptor family is
solved, it will be possible to rapidly solve the 3D structure of
the other members provided that their primary amino acid sequences
are known, by using one, two or several of above mentioned methods.
This is due to the predicted high similarities in the 3D structure
of these receptors. Successful elucidation of the 3D structure of
other classes of proteins, such as the elucidation of the 3D
structure of renin based on its homology to trypsin (Radung 1988),
has already been successfully achieved, and an analogous approach
may be used to obtain the 3D structure of an MSH receptor. In such
an approach the backbone of the MSH receptor protein is aligned
with the backbone of another G-protein coupled receptor using the
most homologous parts of the amino acid sequences (subsequences) of
the two proteins. In general only the transmembrane segments of the
receptors are considered in this alignment. After that the MSH
receptor has been aligned with the backbone of the other G-protein
coupled receptors refinement of the structure of the MSH receptor
is being made by careful positioning of the transmembrane segments
of the MSH receptor, e.g. involving the rotation and tilting of
transmembrane segments, as well as the positioning of amino acid
side chains, until eventual Wan der Waals overlaps has been
eliminated. Moreover, further refinement of the structure is being
made by finding positions of the amino acid side chains which will
form suitable bonds, such as e.g. hydrogen bonds, in between
different amino acids of the same and/or the adjacent transmembrane
segments. Finally further refinement of the structure is being made
by minimizing the energy of the system using well known
computational techniques. The energy of the system is usually
calculated by approximate methods e.g. by using the Amber force
field but also the more exact methods of quantum mechanical
calculations may be applied. Such computations are readily being
made using commercially available computer programmes such as e.g.
Hyperchem, Sybyl etc.
[0220] It is understood that once the atomic structure of one of
the G-protein coupled receptors is known, it will be relatively
easy to do the same for other members, including an MSH receptor,
of this very important receptor family.
[0221] Knowledge of the atomic structure on the one hand will help
to understand the receptors function in minute details and on the
other hand will facilitate the improvement of the specific drug
developments through computational and/or other suitable methods.
Among the methods that can be applied are 3D graphical analysis of
epitopes, the docking of ligands to potential epitopes of the MSH
receptor and de novo design of substances in the computer.
[0222] Thus, the present invention also relates to the use of a
polypeptide sequence of the invention for the deduction of three
dimensional structure of an MSH receptor or an analogue thereof
having MSH binding capacity for use in the design of a substance
capable of binding to the MSH receptor.
[0223] It will be understood that the above disclosed DNA fragments
can be used for finding and isolating other similar DNA fragments,
i.e. DNA fragments of the invention, by employing techniques like
PCR and hybridization. Also, it is possible to use the polypeptides
of the invention for designing DNA probes to be used for such
finding and isolation. Thus, using this approach oligonucleotide
primers are deduced from the polypeptide sequences of the present
invention using the universal genetic code. Such primers can be
used to perform PCR to find and isolate other similar DNA fragments
as described in Examples 1 and 4. Thus, such uses of DNA fragment
and polypeptides of the invention also constitute important aspects
of the invention.
FIGURE LEGENDS
[0224] FIG. 1. Agarose gel electrophoresis analysis. Lane
1--molecular weight markers. Lane 2--PCR reaction as described in
Example 1. Three DNA products can be seen at 705 bp, 501 bp and 372
bp. The band at 372 bp position was later used to clone the full
length coding fragment for human MSH receptor.
[0225] FIG. 2. Northern blot analysis of the tissue distribution of
the GE4 mRNA. Ten .mu.g of poly(A).sup.+RNA from different tissues,
namely brain (lane 2), thymus (lane 3), parathyroid gland (lane 4),
Parotid gland (lane 5), salivary gland (lane 6), adrenal gland
(lane 7), testis (lane 8), liver (lane 9), lung (lane 10), heart
(lane 11), spleen (lane 12), skeletal muscle (lane 13), intestine
(lane 14), colon (lane 15) and WM 266-4 human melanoma cells (lane
16), was separated by electrophoresis and blotted onto a membrane.
It was then hybridized to .sup.32P-labelled GE4 probe. As can be
seen a positive band was seen only in lane 16 corresponding to the
WM 266-4 human melanoma cells. The GE4 DNA was later found to be a
part of the MSH receptor coding fragment. See Example 1 for
details.
[0226] FIG. 3. Plasmid map of pB-11D.
[0227] FIG. 4. Plasmid map of pE-11D.
[0228] FIG. 5. .sup.125I-NDP-MSH binding to the WM266-4 human
melanoma cells.
[0229] FIG. 6. Relative potencies of melanotropins for inhibiting
.sup.125I-NDP-MSH binding to COS-7 cells (available from ATCC)
transfected with MSH receptor cDNA. Competition curves are shown
for non-labelled NDP-MSH (.box-solid.), .alpha.-MSH
(.circle-solid.), ACTH (1-39) (.quadrature.), .beta.-MSH
(.tangle-solidup.), .gamma.-MSH (.tangle-soliddn.), ACTH (4-10) (o)
and .beta.-endorphin (.DELTA.). Experimental procedures are as
described in Examples 2 and 3. Each point represents the mean of
quadruplicate determinations. Non-specific binding was less than 8
percent of the total binding.
[0230] FIG. 7. Plasmid map of pB-MC-2.
[0231] FIG. 8. Plasmid map of pE-MC-2.
[0232] FIG. 9. Relative potencies of melanotropins for inhibiting
125I-NDP-MSH binding to COS-7 cells (available from ATCC)
transfected with pE-MC-2 plasmid DNA. Competition curves are shown
for non-labelled NDP-MSH (.box-solid.), .alpha.-MSH
(.circle-solid.), ACTH (1-39) (.quadrature.), .beta.-MSH
(.tangle-solidup.), .gamma.-MSH (.tangle-soliddn.), and
.beta.-endorphin (o). Experimental procedures are as described in
Examples 7 and 8. Each point represents the mean of quadruplicate
determinations. Non-specific binding was less than 8 percent of the
total binding.
[0233] FIG. 10. Agarose gel electrophoresis analysis of the PCR
products generated from the human brain and melanoma cells mRNA.
Lane 1-molecular weight markers, Lane 2-human brain mRNA without
reverse transcription, Lane 3-human brain mRNA after reverse
transcription, Lane 4-human melanoma cells mRNA without reverse
transcription, Lane 5-human melanoma cells mRNA after reverse
transcription. A specific product at the expected position of 380
bp is seen only in the human brain sample after reverse
transcription.
EXAMPLES
Example 1
Molecular Cloning and Nucleotide Sequencing of the MSH Receptor
cDNA
Design of Polymerase Chain Reaction (PCR) Primers
[0234] The primers were designed by careful examination of the
sequence homologies in the membrane spanning segments 3 and 6 of
the earlier cloned G-protein coupled receptors. The design of the
primers was not inclined towards any one receptor or any one class
of the receptors. The primers were degenerate (redundancy=8 for
primer from segment 3 and redundancy=32 for primer from segment 6)
and also had an inert nucleotide called deoxyinosine at places
which otherwise would have become highly degenerate. The primers
were chemically synthesized on a custom order basis by Symbicom AB
(Tvistevgen 48, Ume.ang., Sweden). Primers were synthesized with
EcoRI (segment 3) and BamHI (segment 6) linkers at the 5'-ends to
facilitate the cloning. The primer sequences are shown in SEQ ID
NO:3 and SEQ ID NO: 4.
Polymerase Chain Reaction (PCR) on Human Genomic DNA
[0235] One .mu.g of human genomic DNA (Purchased from Clontech
Laboratories Incorporated 4030 Fabian Way, Palo Alto Calif. 94303
USA) was subjected to PCR using the above described primers. The
PCR was done in a final volume of 50 .mu.l. It contained one .mu.g
of human genomic DNA, 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM
MgCl.sub.2, 0.001% gelatin, 200 .mu.M of each deoxynucleotide, 1
.mu.M of each primer and 1 unit of the enzyme Taq DNA polymerase
(enzyme was obtained from Perkin Elmer Cetus, 761 Main Av.,
Noewalk, Conn. 06859, USA).
[0236] The PCR thermal profile used was 93.degree. C. for 60
seconds, 55.degree. C. for 40 seconds and 72.degree. C. for 60
seconds for a total of 25 cycles, using a thermal Cycler (Hybaid,
111-113 Waldegrave road, Teddington, Middlesex, TWIL 8LL, UK).
[0237] Ten percent of the reaction was analyzed by agarose gel
electrophoresis, using the standard methods (Sambrook et al. 1989)
(see FIG. 1). Three products were identified at approximately 705
bp, 501 bp and 372 bp. The 705 and 501 bp products were later
identified as DNA for two of the already cloned receptors, but the
372 bp product was identified as a novel G-protein coupled
receptor, and was the only one processed further.
Sequencing of the 372 bD Product
[0238] The 372 bp product was cloned into the pGEM7zf(+) vector
(Promega Corp. Madison, Wis., USA) using the standard techniques
(Sambrook et al. 1989) and one of the resulting plasmid termed as
GE4 was sequenced using the chain termination method (Sanger et al.
1977). It was found to contain the sequence shown in SEQ ID
NO:11.
Tissue Distribution of the GE4 mRNA
[0239] Poly(A)+ RNA was prepared using the oligo-dT purification
scheme as described in standard protocols (Sambrook et al. 1989)
from the following tissues: brain, thymus, parathyroid gland,
parotid gland, salivary gland, adrenal gland, testis, liver, lung,
heart, spleen, skeletal muscle, intestine, colon and WM 266-4 human
melanoma cells (ATCC # CRL 1676). Ten .mu.g of Poly(A).sup.+RNA was
subjected to electrophoresis through a 0.8% agarose-formaldehyde
gel as described (Sambrook et al. 1989). The RNA was then blotted
on to a Genescreen membrane (New England Nuclear, USA), and cross
linked to the membrane with UV light. The membrane was then placed
in a sealed plastic bag containing 10 ml of prehybridization
solution (50% formamide, 5.times.SSC, 5.times.Denhardt's solution,
0.1% SDS, 10 mM Sodium phosphate pH 7.0, 10 mM EDTA and 100
.mu.gg/ml denatured calf thymus DNA) at 42 .degree. C. for 4 hours.
The prehybridization solution was then replaced with the 10 ml of
hybridization solution (10 ml prehybridization solution+.sup.32P
labelled GE4 DNA probe). The GE4 DNA was labelled with .sup.32P
using a commercial multiprime kit (Amersham, Aylesbury,
Buckinghamshire, HP20 1BR, U.K.). The membrane was left in the
hybridization solution for 12 hours at 42.degree. C. The membrane
was then washed in a solution of 0.1.times.SSC and 0.1% SDS at
60.degree. C. for 30 minutes, air dried and then exposed to
autoradiographic film for 16 hours. See FIG. 2. A positive signal
can only be seen in the lane #16, which is for human melanoma WM
266-4 cells.
Construction and Screening of the cDNA Library from WM 266-4
Cells
[0240] The WM 266-4 cells were obtained from ATCC, Bethesda, Md.,
USA. The cells were grown in the medium as advised by the ATCC.
Poly(A).sup.+ RNA from these cells was made using the fast track
mRNA isolation kit (InVitrogen corp., 3985 Sorrento Valley Blvd.
#B, San Diego, Calif. 92121, USA). Five .mu.g of this RNA was then
used to make a random primed cDNA library in the lambda gt11 vector
using the materials and the conditions described by the
manufacturer of the kit used (Amersham, Aylesbury, Buckinghamshire,
HP20 1BR, U.K.).
[0241] Approximately 7.times.10.sup.5 plaque forming units from the
unamplified library were plated on the agar-LB plates (Sambrook et
al. 1989), grown for 8 hours and were then transferred to Hybond-C
filter discs (Amersham, Aylesbury, Buckinghamshire, HP20 1BR,
U.K.). The DNA on the filter discs was then denatured and fixed as
described (Sambrook 1989). The filter discs were then placed in
sealed bags (4 filters/bag) containing the prehybridization
solution (6.times.SSC, 5.times.Denhardt's solution, 10 mM sodium
phosphate pH 7.0, 1 mM EDTA, 0.5% SDS and 0.1 mg/ml of denatured
Salmon testis DNA) for 6 hours at 60.degree. C. The filters were
then placed in the hybridization solution (prehybridization
solution+.sup.32P-labelled GE4 DNA probe), for 12 hours at
60.degree. C. The GE4 DNA was labelled with .sup.32P using a
commercial multiprime kit (Amersham, Aylesbury, Buckinghamshire,
HP20 1BR, U.K.). The filters were then washed at 65.degree. C. in a
solution of 0.1.times.SSC and 0.1% SDS for 20 minutes, air dried
and then exposed to the autoradiographic film for 24 hours. The
positive plaques were picked and after repeating the screening for
two more times a positive plaque designated 11D was isolated.
Subcloning and Sequencing of the 11D cDNA
[0242] A large scale lambda DNA preparation was made for the 11D
clone as described (Sambrook et al. 1989). The insert was excised
out with-the EcoRI and HindIII enzymes. This took out all of the
coding and 5'-untranslated sequences with part of the
3'-untranslated sequence. The EcoRI-HindIII fragment was cloned
between the EcoRI and Hind III sites of the pGEM7Zf(+) vector
(Promega Corp., Madison, Wis., USA) using the standard methods
described (Sambrook 1989). The resulting plasmid DNA (See FIG. 3;
pB-11D) was then transfected into competent DH5alfa E.Coli (BRL,
8400 Helgerman court, Gaithersburg, Md. 20877, USA). Bacterial
colonies were grown on agar plates containing ampicillin.
Individual colonies were picked in ampicillin containing 5 ml LB
medium (Sambrook 1989) and grown overnight. Plasmid DNA was
prepared as described (Sambrook 1989). Prepared plasmid DNAs were
checked for the presence of 11D cDNA in correct position. The
plasmid constructs with 11D cDNA in correct position were then
amplified further in 500 ml cultures. Plasmid DNA from such large
scale preparations were prepared with Qiagen Kits (Qiagen Inc.,
11712 Moorpark Street, Studio City, Calif. 91604, USA). Both the
strands of DNA in the entire coding sequence and the
5'-untranslated region and a small portion of the 3'-untranslated
region were sequenced by making the overlapping fragments. The
method of sequencing was the chain termination method (Sanger et
al. 1977). The cloned 11D cDNA was found to have the nucleotide
sequence shown in SEQ ID NO: 1 and was shown to contain 7
hydrophobic segments (corresponding to nucleotides 286-351,
394-465, 517-588, 640-711, 733-804, 898-972 and 997-1068 in SEQ ID
NO:1, respectively).
Example 2
[0243] Cloning of the 11D cDNA into an Expression Vector
[0244] The expression vector pcDNAI (Invitrogen Corp., 3985
Sorrento Valley Blvd. #B, San Diego, Calif. 92121, USA), is a 4.2
kb multifunctional eukaryotic expression vector. It has the human
CMV promoter and enhancer for high level expression.
[0245] The 111D cDNA was excised from pGEM7Zf(+) vector (described
above in 5.1.6) with EcoRI and NsiI enzymes, and ligated into the
same sites of pcDNAI vector. The resulting plasmid DNA (See FIG. 4;
pE-11D) was then transfected into competent MC1061/P3 E.Coli.
Bacterial colonies were grown on agar plates containing ampicillin.
Individual colonies were picked in 5 ml LB-ampicillin medium
(Sambrook 1989) and grown over-night. Plasmid DNA was prepared as
described (Sambrook 1989). Prepared plasmid DNAs were checked for
the presence of11D cDNA in correct position. The plasmid constructs
with 11D cDNA in correct position were then amplified further in
500 ml cultures. Plasmid DNA from such large scale preparations
were prepared with Qiagen Kits (Qiagen Inc., 11712 Moorpark street,
Studio city, Calif. 91604, USA).
Example 3
Expression of the 11D cDNA and Establishment of its Identity
[0246] The WM266-4 human melanoma cells (from which the MSH
receptor cDNA has been cloned) were grown under conditions
described by ATCC. These cells were subjected to radioligand
binding as described below for the transfected COS-7 cells. The
WM-266-4 cells were shown to bind the .sup.125I-labelled NDP-MSH in
a specific manner. (FIG. 5)
[0247] COS-7 cells were grown in Dulbecco's modified Eagle medium
with 8% foetal calf serum and non-essential amino acids (Gibco/BRL,
8400 Helgerman Court, Gaithersburg, Md. 20877, USA). Eighty percent
confluent cultures were transfected with 1 .mu.g of pE-11D plasmid
DNA and 40 .mu.g lipofectin (BRL, 8400 Helgerman Court,
Gaithersburg, Md. 20877, USA) in serum free medium. Five hours
after transfection, serum containing medium was replaced, and cells
were cultivated for 20 hours. Cells were then scraped off,
centrifuged, resuspended in serum containing medium, plated on 48
well plates, and allowed to grow for 24 hours. The cells were then
washed with 0.3 ml of binding buffer (minimum essential medium with
Earle's salts, 25 mM HEPES pH 7.0, 0.2% bovine serum albumin, 1 mM
1,10-phenanthroline, 0.5 mg per liter leupeptine and 200 mg per
liter bacitracin) and then incubated at 37.degree. C. for 2 hours
with 0.3 ml binding buffer containing 24,000 cpm of
.sup.125I-NDP-MSH and appropriate concentration of unlabelled
peptides. NDP-MSH was labelled with .sup.125Iodine (see below for
details) to the specific activity of 8.6.times.10.sup.4 cpm per
Mol. The plates were then put on ice, cells washed with 0.3 ml of
ice cold binding buffer and detached from plates with 0.3 ml of 0.1
N NaOH. Radioactivity was counted and data analyzed by iterative,
non-linear curve fitting programme suitable for radioligand binding
analysis (see FIG. 6). A series of POMC (pro-opiomelanocortin)
derived peptides (purchased from Saxon Biochemicals GmbH, Hannover,
Germany) showed differential potencies in inhibiting
.sup.125I-NDP-MSH binding to pE-111D transfected COS-7 cells. The
potencies and reciprocals of binding affinities (K.sub.is) were
determined by testing several (e.g. 11-12) concentrations of every
tested peptide and fitting the data for the counts found to be
bound to the cells to the four parameter logistic function using
non-linear regression analysis using previously described methods
(Bergstrom and Wikberg 1986). The K.sub.i-values were then
calculated from the IC-50 values estimated thus estimated by using
the Cheng and Prusoff equation, as previously described (Cheng and
Prusoff 1973). The potency order and K.sub.i values found from the
analysis were NDP-MSH (K.sub.i=23.+-.0.5 pM)>.alpha.-MSH
(K.sub.i=92.+-.19 pM)>ACTH (1-39) (K.sub.i=170.+-.37
pM)>.beta.-MSH (K.sub.i=449.+-.74 pM)>.gamma.-MSH
(K.sub.i=1010.+-.200 pM). ACTH (4-10) showed very low binding
affinity (K.sub.i=22,400.+-.7200 pM), whereas the non-melanotropic
POMC peptide .beta.-endorphin showed no affinity for the expressed
MSH receptor. These results conclusively prove that the cloned DNA
of the invention is the MSH receptor cDNA.
Iodination of NDP-MSH
[0248] Four .mu.g of the peptide NbP-MSH was iodinated with 1 mCi
of 125-iodine using the Iodobeads (Pierce, Rockford, Ill., USA) in
100 mM sodium phosphate buffer (pH 6.5) for 10 minutes. The
Iodobead was then removed from the solution which was applied to
the C-18 reverse phase chromatography cartridge pre-equilibrated
with 15% acetonitrile/0.05 M ammonium acetate pH 5.8. The cartridge
was washed with 5 ml of the pre-equilibration buffer and then
eluted at a flow rate of 1 ml/minute using a peristaltic pump. The
elution gradient was 15% to 35% of acetonitrile containing 0.05 M
ammonium acetate pH 5.8. Fractions of 1 ml were collected and the
radioactivity determined by counting 2.5 .mu.l from each fraction
on to a gamma counter. Fractions 25 to 29 were pooled, dried under
vacuum and redissolved in 1 ml water. The radioactivity was counted
and the specific activity was calculated.
Example 4
Identification of DNA Sequences Related to the Cloned MSH Receptor
cDNA
[0249] Two PCR primers were designed based on the sequence of the
cloned MSH receptor cDNA. Their nucleotide sequences are shown in
SEQ ID NO:13 and in SEQ ID NO:14, respectively.
[0250] These primers were used to perform PCR on human genomic DNA
in exactly the same way as described in Example 2 except for the
thermal profile, which was 94.degree. C. for 30 sec, 45.degree. C.
for 20 sec, 72.degree. C. for 20 sec for 5 cycles and then
94.degree. C. for 30 sec, 60.degree. C. for 20 sec, 72.degree. C.
for 20 sec for 25 cycles. Ten percent of the reaction was analyzed
by agarose gel electrophoresis, using the standard methods
(Sambrook et al. 1989). The products obtained were cloned into the
pGEM7zf(+) vector and sequenced to completion. They were shown to
have the nucleotide sequences shown in SEQ ID NO:5, SEQ ID NO:7 and
SEQ ID NO:9, respectively.
Example 5
Cloning of the 11D cDNA into a Stable Expression Vector
Cloning of the11D cDNA
[0251] The expression vector pRC/CMV (Invitrogen corp., 3985
Sorrento Valley Blvd. #B, San Diego, Calif. 92121, USA), is a 4.5
kb multifunctional eukaryotic expression vector. It has the cMV
promoter and enhancer for high level expression, and neomycin gene
for selection of stable transfectants.
[0252] The 11D cDNA was excised from pcDNA I vector as described in
example 2 with Hind III enzyme and ligated into the same site of
pRC/CMV vector. The resulting plasmid DNA was then transfected into
competent INV.alpha.F' E. coli. Bacterial colonies were grown on
agar containing ampicillin. Individual colonies were picked in 5 ml
LB-ampicillin medium (Sambrook 1989) and grown overnight. Plasmid
DNA was prepared as described (Sambrook 1989). Prepared plasmid
DNAs were checked for the presence of 11D cDNA in correct position.
The plasmid constructs with 11 D cDNA in correct were then
amplified further in 500 ml cultures. Plasmid DNA from such large
scale preparations were prepared with Qiagen Kits (Qiagen Inc.,
11712 Moorpark Street, Studio City, Calif., 91604, USA).
Stable Expression of the 11D cDNA
[0253] COS-7 cells (available from ATCC) were grown in Dulbecco's
modified Eagle medium with 8% fetal calf serum and non-essential
amino acids (Gibco/BRL, 8400 Helgerman Court, Gaithersburg, Md.
20877, USA) in serum free medium, five hours after transfection,
serum containing medium was replaced, and cells were cultivated for
48 hours. At this time selection for the calls stably harbouring
the pRC/CMV-11D plasmid was begun by growing the cells in neomycin
(0.5 mg/ml) containing medium (selection medium). Selection
procedure was continued for two weeks, replacing the selection
medium every 4th day. surviving cells were collected and maintained
in the selection medium. These cells constitute the stable cell
line.
[0254] To investigate the binding properties cells were scraped off
from the culture flask, centrifuged, resuspended in selection
medium, plated on 48 well, and allowed to grow for 24 hours. The
cells were then washed with 0.3 ml of binding buffer (minimum
essential medium with Earle's salts, 25 mM HEPES pH 7.0, 0.2%
bovine serum albumin, 1 mM 1,10-phenanthroline, 0.5 mg per liter
leupeptine and 200 mg per liter bacitracin) and then incubated at
37.degree. C. for 2 hours with 0.3 ml binding buffer containing
24,000 cpm of .sup.125I-NDP-MSH and appropriate concentration of
unlabelled peptides. NDP-MSH was labelled with .sup.125Iodine as
explained in example 3 to the specific activity of
8.6.times.10.sup.4 cpm per Mol. The plates were then put on ice,
cells washed with 0.3 ml of ice cold binding buffer and detached
form plated with 0.3 ml of 0.1 N NaOH. Radioactivity was counted
and data analyzed by an iterative, non-linear curve fitting
programme suitable for radioligand binding analysis. A series of
POMC (pro-opiomelanocortin) derived peptides (purchased from Saxon
Biochemicals GMBH, Hannover, Germany) showed differential potencies
in inhibiting .sup.123I-NDP-MSH binding to the stable cell line.
The potency order found from the analysis were
NDP-MSH>.alpha.-MSH>ACTH(1-39)>.beta.-MSH>.gamma.-MSH.
ACTH(4-10) showed very low binding affinity, whereas the
non-melanotropic POMC peptide .beta.-endorphin showed no affinity
for the expressed MSH receptor. These results conclusively prove
that the cloned MSH receptor cDNA is produced and expressed in a
stable cell line.
Example 6
Molecular Cloning and Nucleotide Sequencing of the Full Length
Clone of G8 DNA
Screening of a Human Placental Genomic Library
[0255] A human genomic DNA library was purchased from Stratagene,
USA. Approximately 7.times.10.sup.5. plaque forming units from this
library were plated on the agar-LB plates (Sambrook et al. 1989),
grown for 8 hours and were then transferred to Hybond-N filter
discs (Amersham, Aylesbury, Buckinghamshire, HP20 1BR, U.K.). The
DNA on the filter discs was then denatured and fixed as described
(Sambrook et al. 1989). The filter discs were then placed in sealed
bags (4 filters/bag) containing the prehybridization solution
(6.times.SSC, 5.times.Denhardt's solution, 10 mM sodium phosphate
pH 7.0, 1 mM EDTA, 0.5% SDS and 0.1 mg/ml of denatured Salmon
testis DNA) for 6 hours at 60.degree. C. The filters were then
placed in the hybridization solution (prehybridization
solution+.sup.32P-labelled G-8 DNA probe, as in SEQ ID NO:7), for
12 hours at 60.degree. C. The G-8 DNA was labelled with .sup.32P
using a commercial multiprime kit (Amersham, Aylesbury,
Buckinghamshire, HP20 1BR, U.K.). The filters were then washed at
65.degree. C. in a solution of 0.1.times.SSC and 0.1% SDS for 20
minutes, air dried and then exposed to the autoradiographic film
for 24 hours. The positive plaques were picked and after repeating
the screening for two more times a positive plaque containing a
gene designated MC-2 was isolated.
Subcloning and Sequencing of the Full Length MC-2 DNA
[0256] A large scale lambda DNA preparation was made for the MC-2
clone as described (Sambrook et al. 1989). The insert was excised
out with the Sac I enzyme. This took out a 2.4 kb fragment
containing all of the coding and a parts of 5'- and 3'-untranslated
sequence. The Sac I fragment was cloned in the Sac I site of the
pGEM5Zf(+) vector (Promega Corp., Madison, Wis., USA) using the
standard methods described (Sambrook 1989). The resulting plasmid
DNA pB-MC-2 (FIG. 7) was then transfected into competent DH5alfa E.
Coli (BRL, 8400 Helgerman court, Gaithersburg, Md. 20877, USA).
Bacterial colonies were grown on agar plates containing ampicillin.
Individual colonies were picked in ampicillin containing 5 ml LB
medium (Sambrook et al. 1989) and grown overnight. Plasmid DNA was
prepared as described (Sambrook et al. 1989). Prepared plasmid DNAs
were checked for the presence of MC-2 DNA in correct position. The
plasmid constructs with MC-2 DNA in correct position were then
amplified further in 500 ml cultures. Plasmid DNA from such large
scale preparations were prepared with Qiagen Kits (Qiagen Inc.,
11712 Moorpark Street, Studio City, Calif. 91604, USA). Both the
strands of DNA in the entire coding sequence and the
5'-untranslated region and a small portion of the 3'-untranslated
region were sequenced by making the overlapping fragments and
primer walking. The method of sequencing was the chain termination
method (Sanger et al. 1977). The cloned MC-2 DNA was found to
contain the sequence shown in SEQ ID NO:15.
Example 7
Cloning of the MC-2 DNA into an Expression Vector
[0257] The expression vector pRC/CMV (Invitrogen Corp., 3985
Sorrento Valley Blvd. #B, San Diego, Calif. 92121, USA), is a 4.5
kb multifunctional eukaryotic expression vector. It has the human
CMv promoter and enhancer for high level expression.
[0258] The MC-2 DNA (from nucleotide no. 520 to nucleotide no. 1620
from the seq. I.D. no. 10) was ligated between the HindIII and the
XbaI sites of the pRC/CMV vector. The resulting plasmid DNA pE-MC-2
(FIG. 8) was then transfected into competent INV.alpha.F' E. Coli.
Bacterial colonies were grown on agar plates containing ampicillin.
Individual colonies were picked in 5 ml LB-ampicillin medium
(Sambrook et al. 1989) and grown overnight. Plasmid DNA was
prepared as described (Sambrook et al. 1989). Prepared plasmid DNAs
were checked for the presence of MC-2 DNA in correct position. The
plasmid constructs with MC-2 DNA in correct position were then
amplified further in 500 ml cultures. Plasmid DNA from such large
scale preparations were prepared with Qiagen Kits (Qiagen Inc.,
11712 Moorpark street, Studio city, Calif. 91604, USA).
Example 8
Expression of the MC-2 DNA and Establishment of its Identity
[0259] COS-7 cells were grown in Dulbecco's modified Eagle medium
with 8% foetal calf serum and non-essential amino acids (Gibco/BRL,
8400 Helgerman Court, Gaithersburg, Md. 20877, USA). Eighty percent
confluent cultures were transfected with 1 .mu.g of pE-MC-2 plasmid
DNA and 40 .mu.g lipofectin (BRL, 8400 Helgerman Court,
Gaithersburg, Md. 20877, USA) in serum free medium. Five hours
after transfection, serum containing medium was replaced, and cells
were cultivated for 20 hours. Cells were then scraped off,
centrifuged, resuspended in serum containing medium, plated on 48
well plates, and allowed to grow for 24 hours. The cells were then
washed with 0.3 ml of binding buffer (minimum essential medium with
Earle's salts, 25 MM HEPES pH 7.0, 0.2% bovine serum albumin, 1 mM
1,10-phenanthroline, 0.5 mg per liter leupeptine and 200 mg per
liter bacitracin) and then incubated at 37.degree. C. for 2 hours
with 0.3 ml of binding buffer containing 24,000 cpm of
.sup.1251-NDP-MSH and appropriate concentration of unlabelled
peptides. NDP-MSH was labelled with .sup.125Iodine (see below for
details) to the specific activity of 8.6.times.104 cpm per Mol. The
plates were then put on ice, cells washed with 0.3 ml of ice cold
binding buffer and detached from plates with 0.3 ml of 0.1 N NaOH.
Radioactivity was counted and data analyzed by iterative,
non-linear curve fitting programme suitable for radioligand binding
analysis (see FIG. 9). A series of POMC (pro-opiomelanocortin)
derived peptides (purchased from Saxon Biochemicals GmbH, Hannover,
Germany) showed differential potencies in inhibiting
.sup.125I-NDP-MSH binding to pE-MC-2 transfected COS-7 cells. The
potencies and reciprocals of binding affinities (K.sub.is) were
determined by testing several (e.g. 10-12) concentrations of every
tested peptide and fitting the data for the counts found to be
bound to the cells to the four parameter logistic function using
non-linear regression analysis using previously described methods
(Bergstrom and Wikberg 1986). The K.sub.i-values were then
calculated from the IC-50 values estimated thus estimated by using
the Cheng and Prusoff equation, as previously described (Cheng and
Prusoff 1973). The potency order and K.sub.i values found from the
analysis were NDP-MSH (K.sub.i=5.18.+-.0.54 nM)>.alpha.-MSH
(K.sub.i=928.+-.314 nM)=ACTH (1-39) (K.sub.i=929.+-.389
nM)>.beta.-MSH (K.sub.i=1.75.+-.0.67 .mu.M)>.gamma.-MSH
(K.sub.i=3.45.+-.0.88 .mu.M). The non-melanotropic POMC peptide
.beta.-endorphin showed no affinity for the expressed MC-2
receptor. These results conclusively prove that the cloned MC-2 DNA
of the invention is a new member of the melanotropic receptor
family.
[0260] Iodination of NDP-MSH: Four mg of the peptide NDP-MSH was
iodinated with 1 .mu.Ci of .sup.125Iodine using the Iodobeads
(Pierce, Rockford, Ill., USA) in 100 mM sodium phosphate buffer (pH
6.5) for 10 minutes. The Iodobead was then removed from the
solution which was applied to the C-18 reverse phase chromatography
cartridge preequilibrated with 15% acetonitrile/0.05 M ammonium
acetate pH 5.8. The cartridge was washed with 5 ml of the
pre-equilibration buffer and then eluted at a flow rate of 1
ml/minute using a peristaltic pump. The elution gradient was 15% to
35% of acetonitrile containing 0.05 M ammonium acetate pH 5.8.
Fractions of 1 ml were collected and the radioactivity determined
by counting 2.5 .mu.l from each fraction on to a gamma counter.
Fractions 25 to 29 were pooled, dried under vacuum and redissolved
in 1 ml of water. The radioactivity was counted and the specific
activity was calculated.
Example 9
MC-2 RNA Detection by PCR Analysis
[0261] RNA from human brain tissue (purchased from Clontech, USA)
and WM266-4 melanoma cells (made by Fast Track kit from Invitrogen
Corp. USA) were reverse transcribed with Superscript RNase H.sup.-
reverse transcriptase (BRL, USA). PCR was performed on samples
before and after reverse transcription to rule out the possibility
of genomic DNA contamination in RNA preparations. Five .mu.g of RNA
was used for reverse transcription and then all of it was used as
template in the first PCR. The first PCR was performed with primers
(described below as number 1 & 2) specific for the 5'- and
3'-untranslated regions of the MC-2 DNA. Ten percent of the first
PCR reaction was then subjected to a second PCR with primers
(described below as number 3 & 4) specific for the coding
region of the clone MC-2.
2 Primer 1: 5'-GGAAGCTTTCTTTGGTAGGCTG (SEQ ID NO:17) Primer 2:
5'-GGTCTAGAGCCACAGAGAGGAG (SEQ ID NO:18) Primer 3:
5'-CTGCATTTCTTGGATCT (SEQ ID NO:19) Primer 4: 5'-AAGCTGCACATGGATGC
(SEQ ID NO:20)
[0262] Both the PCRs were performed with Gene amplification kit
(Perkin Elmer Cetus, USA). The PCR thermal profile used was
93.degree. C. for 60 seconds, 55.degree. C. for 40 seconds and
72.degree. C. for 60 seconds for a total of 40 cycles. Fifty
percent of the reaction was analyzed by agarose gel
electrophoresis. The product was seen at the expected 380 bp
position (FIG. 10).
Example 10
Development of Polyclonal Antibodies Against the MSE Receptor with
Polypeptide Sequence According to SEQ ID NO:2
[0263] The following two peptides, which were based on the
polypeptide in SEQ ID NO:2, were synthesized:
[0264] Peptide M1-Y, amino acids 4-19 of SEQ ID NO:2:
[0265] Gln Gly Ser Gln Arg Arg Leu Leu Gly Ser Leu Asn Ser Thr Pro
Cys
[0266] Peptide M2-Y, amino acids 25-35 of SEQ ID NO:2:
[0267] Gly Leu Ala Ala Asn Gln Thr Gly Ala Arg cys
[0268] The peptides were synthesized commercially by the multiple
peptide system, U.S.A. The synthetic peptides were conjugated to
thyroglobulin (THY) by use of the MBS method. This method allows
coupling of the free sulfhydryl group of the cysteine-containing
peptide onto the carrier protein via the bifunctional crosslinker
MBS.
Immunization of Rabbits
[0269] Each of the THY-peptide (0.5 mg) conjugates were separately
emulsified in Freund's complete adjuvant(1:1) and separately
injected into SWL rabbits. After three weeks, the rabbits were
given an additional booster injections with 0.5 mg of conjugate in
incomplete adjuvant. Booster injection were then given with 4-weeks
intervals, using the same procedure, up until totally four booster
injections had been given. Sera were collected 12-14 days after the
last injection. A specimen of normal rabbits serum (pre-immune
serum) was taken from each rabbit before immunization. All sera
were aliquoted for storage at -80.degree. C. before being used.
Antibody Screening
[0270] Cultured cells which were, respectively, expressing and not
expressing the MSH receptor, were attached to poly-L-lysine coated
slides for 24 hours, and the slides were then gently washed in PBS.
(As MSH receptor expressing cells, COS-7 cells transfected with
pE-11D, using the method described in Example 3 were used. As
controls, which were not expressing MSH receptors, non-transfected
COS-7 cells were used). The cells, being attached to the slides,
were fixed in 4% paraformaldehyde for 10 minutes at 22.degree. C.
whereafter the slides were washed twice in PBS. Cells were then
permeabilized by incubating in 0.2% Triton X-100 in PBS for 4
minutes at 22.degree. C. and the slides were then again washed
gently in PBS with 3 changes in 5 minutes intervals. Slides were
then pre-incubated in 10% foetal calf serum for 30 minutes at
22.degree. C. whereafter they were incubated with either the
pre-immune sera diluted (1:100) or the antisera diluted (1:100) in
10% foetal calf serum, for 60 minutes at 22.degree. C. After this
procedure the slides were gently washed in PBS with 3 changes in 5
minute intervals. Slides were then incubated with TRITC-labelled
anti-rabbit secondary antibody (diluted 1:40) for 60 minutes at
22.degree. C. and then again washed gently in PBS with 3 changes in
5 minute intervals. The cells were then observed under a
fluorescent microscope using appropriate filters for the correct
wavelengths.
Results
[0271] The cells expressing the MSH receptor showed very little
fluorescence when tested with pre-immunesera (reaction can be
categorised as +).
[0272] The cells expressing the MSH receptor showed high
fluorescence both when tested with antisera developed against
Peptide M1-Y and when tested with antisera developed against
Peptide M2-Y (The reaction can in both cases be categorised as
++++).
[0273] The control cells not expressing the MSH receptor showed
very little fluorescence when tested with antisera developed
against Peptide M1-Y or against Peptide M2-y (the reaction can, in
both cases, be categorised as +).
[0274] 6. References
[0275] Clark, W G, M Holdeman & J M Lipton: Analysis of the
antipyretic action of .alpha.-melanocyte stimulating hormone in
rabbits. J. Physiol. 1985, 359, 459-465.
[0276] Findlay, J & E Eliopoulos: Three-dimensional modelling
of G protein-linked receptors. TiPS. 1990, 11, 492-499.
[0277] Ghanem, G. J Verstegen, A Libert, R Arnould & F Lejeune:
Alpha-melanocyte-stimulating hormone immunoreactivity in human
melanoma metastases extracts. Pigment Cell Res. 1989, 2,
519-523.
[0278] Handelman, G E, T L O'Donohue, D Forrested & Cook:
Alpha-melanocyte stimulating hormone facilitates learning of visual
but not of auditory discriminations. Peptides. 1983, 4,
145-148.
[0279] Hirsch, M D & T L O'Donohue: Structural modifications of
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Sequence CWU 1
1
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