U.S. patent application number 09/991548 was filed with the patent office on 2002-10-31 for receptor derived peptides as modulators of receptor activity.
Invention is credited to Naranda, Tatjana, Olsson, Lennart.
Application Number | 20020160013 09/991548 |
Document ID | / |
Family ID | 27417098 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160013 |
Kind Code |
A1 |
Olsson, Lennart ; et
al. |
October 31, 2002 |
Receptor derived peptides as modulators of receptor activity
Abstract
Oligopeptides having an amino acid sequence corresponding to a
receptor's extracellular domain, and having sequence similarity to
regulatory peptides from MHC class I antigens, enhance or replace
the physiological response of ligand binding to the corresponding
receptor. The oligopeptides are used in diagnosis and therapy of
diseases that involve inadequate or inappropriate receptor response
as well as in the screening of drug candidates that affect surface
expression of receptors. Also useful for drug screening is a
modified receptor molecule, where the sequence corresponding to the
regulatory peptide is modified or deleted.
Inventors: |
Olsson, Lennart; (Mountain
View, CA) ; Naranda, Tatjana; (Mountain View,
CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
27417098 |
Appl. No.: |
09/991548 |
Filed: |
November 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09991548 |
Nov 20, 2001 |
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09028937 |
Feb 24, 1998 |
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6333031 |
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09028937 |
Feb 24, 1998 |
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08788820 |
Jan 23, 1997 |
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6346390 |
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08788820 |
Jan 23, 1997 |
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08701382 |
Aug 22, 1996 |
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6004758 |
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08701382 |
Aug 22, 1996 |
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08612999 |
Mar 8, 1996 |
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5952293 |
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Current U.S.
Class: |
424/185.1 ;
530/324; 530/325; 530/326; 530/327 |
Current CPC
Class: |
C07K 14/62 20130101;
C07K 14/71 20130101; C07K 14/72 20130101; C07K 2319/00 20130101;
C07K 14/705 20130101; C07K 14/70539 20130101; C07K 14/7155
20130101; C07K 14/7153 20130101; C07K 2319/75 20130101; C07K
2319/74 20130101 |
Class at
Publication: |
424/185.1 ;
530/324; 530/325; 530/326; 530/327 |
International
Class: |
A61K 039/00; C07K
014/74; C07K 007/08; C07K 007/06 |
Claims
What is claimed is:
1. An oligopeptide comprising at least about 8 amino acids and less
than about 40 amino acids which has an amino acid sequence
corresponding to the activation sequence of the extracellular
domain of a cell surface receptor.
2. An oligopeptide according to claim 1, wherein said oligopeptide
has at least about 35% sequence similarity with the sequence of an
.alpha.1-domain of an MHC Class I antigen.
3. An oligopeptide according to claim 2, wherein said sequence of
an a .alpha.1-domain of an MHC Class I antigen is SEQ ID NO:1.
4. An oligopeptide according to claim 1, wherein said cell surface
receptor is selected from the group consisting of insulin
responsive glucose transporter, insulin receptor, leptin receptor,
low density lipoprotein receptor, insulin like growth factor
receptor, granulocyte colony stimulating factor receptor,
interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15 and IL-17 receptors,
human growth hormone receptor, VEGF receptor, PDGF receptor, EPO
receptor, TPO receptor, transferrin receptor, prolactin receptor,
CNF receptor, T-ell receptor, and epidermal growth factor
receptor.
5. An oligopeptide according to claim 4, wherein said cell surface
receptor is human.
6. An oligopeptide selected from the group consisting of SEQ ID
NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID
NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID
NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; SEQ
ID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21;
SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID
NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ
ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; and SEQ ID
NO:35.
7. An oligopeptide at least about 90% homologous to a sequence
selected from the group consisting of SEQ ID NO:2; SEQ ID NO:3; SEQ
ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID
NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ
ID NO:14; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18;
SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID
NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ
ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32;
SEQ ID NO:33; SEQ ID NO:34; and SEQ ID NO:35.
8. A method of modulating the internalization of a cell-surface
receptor containing an activation sequence comprising binding an
exogeneous compound to said activation sequence.
9. A method according to claim 8 wherein said modulating is
inhibiting the internalization.
10. A method according to claim 9 wherein said exogeneous compound
comprises an oligopeptide comprising at least about 8 amino acids
and less than about 40 amino acids having an amino acid sequence
corresponding to an activation sequence of the extracellular domain
of a cell surface receptor; wherein when combined with a cell
expressing said cell surface receptor, said oligopeptide inhibits
receptor internalization upon ligand binding.
11. A method according to claim 10, wherein said oligopeptide has
at least about 35% sequence similarity with the sequence of an
.alpha.1-domain of an MHC Class I antigen.
12. An oligopeptide according to claim 11, wherein said sequence of
an .alpha.1-domain of an MHC Class I antigen is SEQ ID NO:1.
13. A method according to claim 8, wherein said cell surface
receptor is selected from the group consisting of insulin
responsive glucose transporter, insulin receptor, leptin receptor,
low density lipoprotein receptor, insulin like growth factor
receptor, granulocyte colony stimulating factor receptor,
interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15 and IL-17 receptors,
human growth hormone receptor, VEGF receptor, PDGF receptor, EPO
receptor, TPO receptor, transferrin receptor, prolactin receptor,
T-cell receptor, CNF receptor, and epidermal growth factor
receptor.
14. A method according to claim 13, wherein said cell surface
receptor is human.
15. A method according to claim 13, wherein said oligopeptide is
selected from the group consisting of SEQ ID NO:2; SEQ ID NO:3; SEQ
ID NO:4; SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID
NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO: 12; SEQ ID NO:13; SEQ
ID NO:14; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18;
SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO.:22; SEQ ID
NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ
ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32;
SEQ ID NO:33; SEQ ID NO:34; and SEQ ID NO:35.
16. A mammalian cell comprising a modified cell surface receptor,
wherein said modification comprises an amino acid sequence
substitution, insertion or deletion in an activation sequence of
the region of the extracellular domain, and wherein said modified
sequence is of at least about 8 amino acids and less than about 40
amino acids.
17. A cell according to claim 16, wherein said cell surface
receptor is selected from the group consisting of insulin
responsive glucose transporter, insulin receptor, leptin receptor,
low density lipoprotein receptor, insulin like growth factor
receptor, granulocyte colony stimulating factor receptor,
interleukin receptors including IL-1, IL-2, IL-3, IL4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15 and IL-17 receptors,
human growth hormone receptor, VEGF receptor, PDGF receptor, EPO
receptor, TPO receptor, transferrin receptor, prolactin receptor,
CNF receptor, T-cell receptor, and epidermal growth factor
receptor.
18. A cell according to claim 17, wherein said cell surface
receptor is human.
19. A cell according to claim 16, wherein said modification
comprises the deletion of all or part of a sequence selected from
the group consisting of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ
ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID
NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ
ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19;
SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID
NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ
ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33;
SEQ ID NO:34; and SEQ ID NO:35.
20. A method of determining an activation sequence of a cell
surface receptor, said method comprising searching for a region of
sequence similarity between said cell surface receptor and the
sequence of an .alpha.1-domain of an MHC Class I antigen.
21. A method according to claim 20, wherein said oligopeptide has
at least about 35% sequence similarity with the sequence of an
.alpha.1-domain of an MHC Class I antigen.
22. An oligopeptide according to claim 21, wherein said sequence of
an .alpha.1-domain of an MHC Class I antigen is SEQ ID NO:1.
23. A method for screening for a bioactive agent capable of binding
to the activation sequence of a cell surface receptor, said method
comprising combining a cell surface receptor and a candidate
bioactive agent, and determining the binding of said candidate
agent to the the activation sequence of said cell surface
receptor.
24. A method according to claim 23, wherein said determination
comprises competitive binding of an oligopeptide according to claim
1.
25. A method according to claim 24, wherein either the candidate
bioactive agent or the oligopeptide is labelled.
26. A method according to claim 23, wherein said cell surface
receptor comprises the full length cell surface receptor.
27. A method for screening for a bioactive agent capable of binding
to the activation sequence of a cell surface receptor, said method
comprising the steps of: a) combining i) said cell surface
receptor; ii) a ligand bound by said cell surface receptor; and
iii) an oligopeptide according to claim 1, wherein said
oligopeptide binds to the activation sequence of said cell surface
receptor; to form a test mixture; b) adding to said text mixture a
candidate bioactive agent; and c) determining the binding of said
candidate bioactive agent to said activation sequence.
28. A method according to claim 27 wherein said oligopeptide is
labelled.
29. A method according to claim 27 wherein said candidate bioactive
agent is labelled.
30. A method according to claim 27, wherein said cell surface
receptor comprises the full length cell surface receptor.
31. A method for screening for an bioactive agent capable of
binding to the activation sequence of a cell surface receptor, said
method comprising the steps of: a) combining in a first sample said
cell surface receptor, a ligand bound by said cell surface
receptor, and an oligopeptide according to claim 1; b) combining in
a second sample a candidate bioactive agent, said cell surface
receptor, a ligand bound by said cell surface receptor, and an
oligopeptide according to claim 1; and c) determining the binding
of said oligopeptide to said cell surface receptor in said first
and said second samples; wherein a change in binding of said
oligopeptide in said second sample relative to said first sample
indicates that said agent is capable of binding to said activation
sequence.
32. A method for screening for an bioactive agent capable of
binding to the activation sequence of a cell surface receptor, said
method comprising the steps of: a) combining in a first sample a
receptor-derived oligopeptide according to claim 1, and a bioactive
peptide having the sequence of an .alpha.1-domain of an MHC Class I
antigen; b) combining in a second sample a candidate bioactive
agent, a receptor derived oligopeptide according to claim 1, and a
bioactive peptide having the sequence of an .alpha.1-domain of an
MHC Class I antigen; and c) determining the association of said
receptor-derived oligopeptide with said bioactive peptide having
the sequence of an .alpha.1-domain of an MHC Class I antigen in
said first and said second samples; wherein a change in said
association in said second sample relative to said first sample
indicates that said agent is capable of binding said activation
sequence of said cell surface receptor.
33. A method according to claim 31, wherein said sequence of an
.alpha.1-domain of an MHC Class I antigen is SEQ ID NO:1.
34. A method for screening for an bioactive agent capable of
modulating the internalization of a type-1 cell-surface receptor,
said method comprising the steps of: a) adding a ligand bound by
said cell surface receptor and a candidate bioactive agent to a
cell comprising said cell surface receptor; and b) determining the
effect of the candidate bioactive agent on the internalization of
said receptor.
35. A method for screening for an bioactive agent capable of
modulating the internalization of a type-2 cell-surface receptor,
said method comprising the steps of: a) adding a candidate
bioactive agent to a cell comprising said cell surface receptor;
and b) determining the effect of the candidate bioactive agent on
the internalization of said receptor.
36. A composition comprising a cell-surface receptor with an
exogeneous compound bound to the activation sequence.
Description
[0001] This application is a continuing application of U.S. Ser.
No. 08/788,820, filed Jan. 23, 1997, which is a continuing
application of U.S. Ser. No. 08/701,382, filed Aug. 22, 1996, which
is a continuing application of U.S. Ser. No. 08/612,999, filed Mar.
8, 1996.
INTRODUCTION
[0002] 1. Technical Field
[0003] The field of this invention is the modulation of activity of
cell surface receptors, including both modulation of
internalization of the receptor and modulation of activation of the
receptors in the presence or absence of ligand.
[0004] 2. Background
[0005] The complex regulatory balance between hormones, receptors
and responding cells is critical to the correct functioning of
multicellular organisms. Subtle environmental and genetic factors
can disrupt this balance, sometimes resulting in disease. The
advent of molecular biology has meant that medically important
hormones can be made available in therapeutically useful amounts.
Among them are human growth hormone, EPO, TPO, insulin-like growth
factor, insulin, epidermal growth factor, and numerous others.
[0006] A condition of great economic and medical significance is
insulin resistance, which is an essential feature of a great
variety of clinical disorders, such as diabetes mellitus, obesity
and certain types of hypertension. Individuals with non-insulin
dependent diabetes present with insulin resistance in peripheral
tissues. They have a subnormal glucose utilization in skeletal
muscle, where glucose transport across the cell membrane of
skeletal muscle is the rate limiting step in glucose metabolism. It
is possible that a defect exists in insulin-dependent glucose
transport in skeletal muscle in diabetic states, where decreased
levels of the glucose transporter 4 protein (GLUT4) have been
observed. In adipose and muscle cells, insulin stimulates a rapid
and dramatic increase in glucose uptake, primarily by promoting the
redistribution of the GLUT4 glucose transporter from its
intracellular storage site to the plasma membrane.
[0007] Insulin resistance may also be attributed to a defect in
insulin action at the cellular level. The insulin receptor is
activated by binding of insulin to the alpha-subunit of the
receptor, which causes autophosphorylation of the intracellular
beta-subunit region. The activated insulin receptor couples to
cytosolic receptor substrates that can affect signaling cascades,
resulting in the pleiotropic hormone response. Most proteins
involved in the signal transduction pathway are not known yet, but
each of them might play a role in the various forms of insulin
resistance. The heterogeneous nature of insulin resistance makes
treatments that can act "upstream" of the signal transduction
pathways very attractive, because a number of different pathologies
could be treated with a single drug.
[0008] Specific peptides have been previously shown to enhance the
cellular response to certain hormones. This effect has been
attributed to inhibition of the internalization of the
corresponding hormone receptors. Insulin-stimulated glucose uptake
is increased by adding the peptides to responding cells, offering
the possibility of improved therapy for insulin dependent and
insulin resistant diabetes. The enhanced response may also be
exploited in therapies involving other hormones. Improvements in
the specificity of agents that enhance the activity of insulin and
other hormones are of considerable interest for their therapeutic
benefits. The site of action for such peptides on receptors
molecules is of interest for drug evaluation and design.
[0009] In addition, there is great interest in finding ligand
replacements, or mimetics, that could be used in place of the
naturally-occuring ligand. This is of particular interest for
ligand hormones that may have a number of biological functions of
which only a subset are to be regulated.
RELEVANT LITERATURE
[0010] Several groups have examined the glucose transporter and
insulin receptor for residues that are involved in internalization.
Rajagopalan et al. (1995) Biochem. Biophys. Res. Commun. 211:714-8
found that residues GPYL950-953 served as the predominant
endocytosis signal and the sequence NPEY957-960 as a secondary
signal. Levy-Toledano et al. (1993) Biochem. Bioph s. Acta.
1220:1-14 suggest that the structural domain located 43-113 amino
acids from the C-terminus is required in intact cells for
insulin-stimulated autophosphorylation and signal transmission.
Verhey et al. (1995) J. Cell Biol. 130:1071-9 identified sequences
involved in the differential subcellular localization and
hormone-responsiveness of glucose transporter isoforms. The
COOH-terminal 30 amino acids of GLUT4 are sufficient for its
correct localization to an intracellular storage pool that
translocates to the cell surface in response to insulin. In
addition, there is a report of important leucine residues in
insulin receptor endocytosis. See Hamer et al., J. Biol. Chem.
272:21685 (1997).
[0011] U.S. Pat. No. 5,385,888, issued Jan. 31, 1995, describes
Class I MHC peptide modulation of surface receptor activity. Data
presented in International patent application PCT/US94/09189
suggest that these peptides must be in an ordered conformation to
be biologically active. The composition and uses of such peptides
are further described in International application PCT/US93/01758.
The peptides are further disclosed in International application
PCT/US89/00876.
[0012] Regulation of receptor internalization by the major
histocompatibility complex class I molecule is shown by Olsson et
al. (1994) Proc. Natl. Acad. Sci. 91:9086-90, and by a peptide
derived from the insulin receptor in Naranda et al. Proc. Natl.
Acad. Sci. 94:11692 (1997). Peptides derived from the alpha 1
domain of the major histocompatibility complex class I protein
(MHC-I) inhibit internalization of some receptors, thereby
increasing the steady-state number of active receptors on the cell
surface. It is suggested that MHC-I participates in the regulation
of cell surface receptor activity. Stagsted et al. (1993) J. Biol.
Chem. 268:22809-13 demonstrate that such peptides inhibit the
internalization of glucose transporters (GLUT4) and insulin-like
growth factor II (IGF-II) receptors in insulin-stimulated
cells.
SUMMARY OF THE INVENTION
[0013] It is an object of the invention to provide compositions and
methods useful in the modulation of receptor activity. Accordingly,
the present invention provides oligopeptides comprising at least
about 8 amino acids and less than about 40 amino acids which has an
amino acid sequence corresponding to the activation sequence of the
extracellular domain of a cell surface receptor. Also provided are
oligopeptides at least about 90% homologous to an activation
sequence of a cell-surface receptor.
[0014] In an additional aspect, the invention provides methods of
modulating the internalization of a cell-surface receptor
containing an activation sequence comprising binding an exogeneous
compound to said activation sequence.
[0015] In a further aspect, the invention provides mammalian cells
comprising a modified cell surface receptor, wherein the
modification comprises an amino acid sequence substitution,
insertion or deletion in an activation sequence of the region of
the extracellular domain.
[0016] In an additional aspect, the invention provides methods of
determining an activation sequence of a cell surface receptor
comprising searching for a region of sequence similarity between
the cell surface receptor and the sequence of an .alpha.1-domain of
an MHC Class I antigen.
[0017] In a further aspect, the invention provides methods for
screening for a bioactive agent capable of binding to the
activation sequence of a cell surface receptor, comprising
combining a cell surface receptor and a candidate bioactive agent,
and determining the binding of the candidate agent to the
activation sequence of the cell surface receptor.
[0018] In an additional aspect, the invention provides methods for
screening for an bioactive agent capable of modulating the
internalization of a type-1 cell-surface receptor, the method
comprising the steps of adding a ligand bound by the cell surface
receptor and a candidate bioactive agent to a cell comprising the
cell surface receptor. The effect of the candidate bioactive agent
on the internalization of the receptor is then determined.
[0019] In a further aspect, the invention provides methods for
screening for an bioactive agent capable of modulating the
internalization of a type-2 cell-surface receptor, the method
comprising the steps of adding a candidate bioactive agent to a
cell comprising the cell surface receptor. The effect of the
candidate bioactive agent on the internalization of the receptor is
then determined.
[0020] In an additional aspect, the invention provides compositions
comprising a cell-surface receptor with an exogeneous compound
bound to the activation sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A and 1B: Synergistic effect of EPO receptor derived
peptide (EPO-Rp, SEQ ID NO:11) on the effect of EPO as measured by
phosphorylation of the EPO receptor (EPO-R). IP,
immunoprecipitation; WB, Western blot. FIGS. 1A and 1B are digital
images of western blots. Cells were incubated for 20 minutes with
EPO and/or EPO-Rp prior to lysis and IP and WB. The panels show
that EPO-Rp in itself stimulates the EPO-R, but also that EPO-Rp
has a strong synergistic effect as 0.01 U/ml EPO alone has an
effect, 10 nM EPO-Rp a weak effect, but that 0.01 U/ml EPO plus 10
nM EPO-Rp has a strong effect (FIG. 1B to the right). This result
thus indicates that the EPO-Rp can strongly enhance the endogenous
levels of EPO, e.g., in patients with kidney disease where the
production of EPO is reduced significantly.
[0022] FIG. 2: Kinetics for the effect of EPO and the EPO receptor
derived peptide (EPO-Rp, SEQ ID NO:11). FIG. 2 is a digital image
of a western blot. Doses for the two compounds were chosen to give
a strong signal on their own. The EPO-induced signal is faster than
EPO-Rp (5 minutes with peak at 15 minutes versus 15 minutes and
with a duration of at least 90 minutes for EPO-Rp). Thus, the
duration of the effect of EPO-Rp is much longer than that of
EPO.
[0023] FIG. 3: Demonstration of the stimulation of EPO-R with
EPO-Rp. The cells were stimulated for 20 minutes. FIG. 3 is a
digital image of a western blot. Growth hormone receptor derived
peptide (GHRp) was used as control in a concentration of 10 .mu.M
that strongly enhances growth hormone receptor activity. 2.5 U/ml
EPO in activity corresponds to about 30-100 nM EPO-Rp.
[0024] FIG. 4: Dimerization of EPO-R is assumed to induce
intracellular association between JAK2 and EPO-R. Using an antibody
to JAK2 as precipitating antibody, both EPO and EPO-Rp results in
association of EPO-R with JAK2. FIG. 4 is a digital image of a
western blot.
[0025] FIG. 5 depicts that EPO-Rp selectively activates
EPO-Receptor. TF 1 cells were stimulated for 20 min with 0.1 U/ml
of EPO, 10 nM TPO, different concentrations of EPO-Rp, and
combinations of TPO or EPO (at the same concentrations) together
with EPO-Rp. Cells were lysed and immunoprecipitated with
anti-phosphotyrosine antibody (PY). Western blot analysis was
performed by using anti-EPOR antibody that specifically recognizes
EPO-R and anti-JAK2 antibody that will recognize a kinase that
specifically associates only with activated receptors. FIG. 5 is a
digital image of western blot. EPO and EPO-R peptide acts
synergistically in activating EPOR (bottom panel) and JAK2 kinase
(upper panel). TPO together with EPO-R peptide does not enhance the
signal; activation of JAK2 with TPO and EPORp is additive (top
panel), because TPO activates its own receptor that also associates
with JAK2. As well, bottom panel shows that TPO with EPORp does not
activate the EPOR, as the signal is due to presence of the peptide
only. FIG. 6 depicts the inhibition of EPO-R internalization with
EPO-Rp. Internalization of [.sup.125] EPO was measured in TF1 cells
in the presence or absence of EPORp. Cells were preincubated for 30
min at 37.degree. C., followed by addition of [.sup.125]-EPO and 15
.mu.M peptide concentration (EPORp). Cells were further incubated
at 37.degree. C. for different times and internalized ligand was
measured by the method of acid wash. Cells were spun through oil
mixture to separate bound from unbound ligand. Internalized ligand
is calculated as percent of [.sup.125]-EPO resistant to acid wash
(intracellular) versus the total mount of hormone bound to the
cells. Thus, EPORp inhibits internalization of EPO-Receptor and
therefore extends its cell surface time.
[0026] FIG. 7 demonstrates the activation (phosphorylation) of JAK2
kinase by TPO receptor derived peptide (TPO-Rp; SEQ ID NO:10).
Different concentrations of peptide (as indicated) were added to
cells for 20 min and IP with anti-PY antibody was performed,
followed by western blot using anti-JAK2 antibody. This kinase will
get activated only if it can bind to dimerized (biologically
active) TPO-Receptors. Thus, TPORp enhances signaling through
TPO-Receptor.
[0027] FIG. 8 shows that TPORp selectively activates TPO-Receptor.
Cells were stimulated for 20 min with 0.1 U/ml of EPO, 10 nM TPO,
different concentrations of TPOR-peptide, and combinations of TPO
or EPO (at the same concentrations) together with TPO-R peptide.
Cells were lysed and immunoprecipitated with anti-PY antibody.
Western blot analysis was performed by using anti-EPOR antibody
that specifically associates only with activated receptors (EPOR or
TPOR). Figure is a digital image of western blot. TPO and TPO-R
peptide act synergistically in activating JAK2 kinase through the
TPO-R (upper panel). EPO together with TPO-R peptide does not
enhance the signal in the same way as activation of JAK2 with EPO
and TPORp as additive. In addition, EPO with TPORp does not
activate the EPOR, as the signal seen on the bottom panel is due to
presence of the EPO only.
[0028] FIG. 9 demonstrates that TPORp activates JAK2 kinase through
TPO-Receptor. Experiment was performed as on FIG. 8, except that
cells were IP with anti-JAK2 antibody. WB probed with anti-PY
antibody (top panel) shows that TPORp activates JAK2 kinase in two
ways: mimicking TPO activation and acting synergistically with TPO.
WB and anti-JAK2 antibody (bottom panel) shows that all
immunoprecipitates contained the same amount of JAK2 protein.
[0029] FIG. 10 depicts the activation of Leptin Receptor (OB-R)
signaling with Leptin Receptor derived peptide (LRp). COS-1 cells
were transiently transfected with long form of Leptin Receptor
(OB-R.sub.1) and stimulated for 20 min with 6 nM Leptin, 15 .mu.M
LRp or combination thereof. Expression of Leptin Receptor was
confirmed by western blot analysis using specific anti-OBR antibody
and by binding of [.sup.125]-Leptin to the transfected cells. Cells
were lysed and immunoprecipitation was performed with anti-JAK2
antibody. WB with the same antibody (left panel) shows that equal
amount of proteins are present in all the immunoprecipitates. WB
with anti-PY antibody (right panel) demonstrates that LRp activates
JAK2 through Lepin Receptor, because there is identical enhancement
of protein phosphorylation signal in both cases; cells stimulated
with Leptin or LRp. As indicated on the figure upper band
represents phosphorylated JAK2 molecule, middle band represents
phosphorylated STAT5 molecule (both specific proteins in a
signaling pathway of OB-R), and bottom band is an unidentified
protein whose phosphorylation does not change between
non-stimulated and stimulated cells. Two additional peptides were
used as a control of specificity: Insulin receptor derived peptide
(IRp; SEQ ID NO:3) a peptide that at 15 .mu.M concentration
strongly activates signaling through the Insulin receptor, and Cp,
a peptide derivative of Leptin Receptor as well, but without any
similarity to MHC-I peptide. None of these peptides activated JAK2
or STAT5 proteins.
[0030] FIG. 11 demonstrates that the LRp mimics Leptin and shows a
synergistic effect together with Leptin in activation of Leptin
Receptor signaling. COS-1 cells were transfected with long form of
Leptin receptor and stimulated for 20 min with different
concentrations of Leptin (OB), LRp and the lowest dose of OB (1
ng/ml) together with different peptide concentrations, as indicated
on the figure. IP was performed with anti-JAK2 antibody. Bottom
panel WB shows that all the immunoprecipitates contain the same
amount of JAK2 protein. WB with anti-PY (upper panel) shows
activation (phosphorylation) of JAK2 and STAT5 by OB and LRp. Both,
natural ligand and the peptide activate JAK2 to the same extent.
When combined, there is a synergistic effect between them; e.g.,
the activation of JAK2 is stronger when 1 ng/ml of OB and 30 .mu.M
LRp were added together (16 arbitrary units), than when the cells
were stimulated with the same compounds but separately (2 and 8
units). CP and IRp showed no effect on protein phosphorylation when
alone or combined with OB. Numbers in the box below the panel show
quantification of western blots. For that purpose, the membranes
were scanned and intensity of the protein bands was quantitated
using the NIH 1.5 Image program. Upper values are corresponding to
JAK2 protein, lower to STAT5.
[0031] FIG. 12 shows the effect of LRp on activation of STAT5
through Leptin Receptor. STAT5 is transcription factor that
specifically will bind to activated JAK2, which previously needs to
be phosphorylated by interacting with dimerized receptors.
Experiments were performed as described in FIG. 11, except that
anti-STAT5 antibody was used instead of JAK2. WB with anti-PY
antibody (upper panel) shows that LRp mimics the action of Leptin
(OB) and when combined with it acts synergistically. Lower panel
shows that all the immunoprecipitates contain the same amount of
STAT5 protein.
[0032] FIG. 13 demonstrates the effect of LRp on the signaling of
the Leptin Receptor short form (OB-R.sub.s). COS-1 cells were
transfected with short form of Leptin receptor and stimulated for
20 min with different concentrations of Leptin (OB), LRp and two
different doses of OB (1 ng/ml and 10 ng/ml) together with
different peptide concentrations, as indicated on the figure. IP
was performed with anti-OBR antibody and subsequent blot was probed
with JAK2 antibody. WB on the upper panel shows that JAK2 protein
associates with Leptin receptor only when the receptor has been
activated. LRp shows that same effect as natural ligand. On the
bottom panel, cells were IP with anti-JAK2 antibody and the
membrane was probed with anti-PY. Phosphorylation (activation) of
JAK2 is observed only when both LRp and Leptin are present. Thus,
LRp and Leptin together can activate short form of leptin receptor
(form of the receptor that is expressed in db/db animals), the
effect that is not observed when only one of the compound is
added.
[0033] FIG. 14 demonstrates the activation of Growth Hormone
Receptor (GHR) by GHR-derived peptide (GH-Rp; SEQ ID NO:9). IM9
cells were stimulated for 20 min with 10 nM GH, 30 .mu.M GHRp and
combination thereof. IP was performed with anti-JAK2 antibody. WB
and JAK2 antibody (right panel) shows the same amount of the
protein present in all the immunoprecipitates. WB with anti-PY
demonstrate equal activation (phosphorylation) of JAK2 by GH and
GHR-peptide.
[0034] FIG. 15 shows the kinetics for the effect of GH and GHRp.
Doses for the two compounds are as indicated on the top of the
panels. Upper panel shows that GH and GHRp phosphorylate JAK2 to
the same extent; most likely by inducing dimerized conformation
between GH-Receptors (receptor dimerization is assumed to induce
intracellular association between JAK2 and GHR). The GH-induced
signal of JAK2 phosphorylation is faster than GHRp (1 min with
duration of approximately 30 min, versus 5 min and with duration of
at least 40 min for GHRp). Thus, the duration of the effect of GHRp
is much longer than that of GH.
[0035] FIG. 16 demonstrates that GHRp (P) broadens the range of GH
action. IM9 cells were stimulated with 1 nM, 10 nM and 2 .mu.M GH
in the presence or absence of GHRp.
[0036] Those concentrations of GH were use, because it is known
that GH action has the bel-shaped dose response curve with an
IC.sub.50 at 2 .mu.M GH concentration. IP was performed with
anti-JAK2; WB with the same antibody (right panel) or with anti-PY
(left panel). Blot probed with anti-JAK2 shows that the same amount
of protein is present in all immunoprecipitates. Anti-PY WB
demonstrates that peptide enhances JAK2 activation approximately
three times in the presence of 1 nM GH. In the GH inhibitory
concentration of 2 .mu.M GHRp has increased natural hormone
response approximately two times. Therefore, GHRp broadens the
range of GH action by increasing its activity at low concentrations
and extending the activity to higher hormone concentrations.
[0037] FIG. 17 demonstrates synergistic effect of GHRp on the GH
action. Cells were stimulated for 20 minutes with different
concentrations of the GHR-peptide alone (top panel), or in
combination with 1 nM (middle panel) and 0.1 riM GH (bottom panel).
IP was performed with anti-JAK2 and WB with anti-PY antibody. GHRp
shows strong synergistic effect with both concentrations of GH; (i)
no effect on JAK2 phosphorylation is observed with low (up to 5
.mu.M) peptide concentrations, but when combined with 1 nM GH,
activation of JAK2 is significantly increased (1 versus 12
arbitrary units); (ii) 0.1 nM concentration of GH shows no effect
on JAK2 phosphorylation, but when combined with low 0.3 .mu.m GHRp
(that as well has no effect on its own), JAK2 activation is
increased approximately four times.
[0038] FIG. 18 demonstrates that GHRp enhances the phosphorylation
of STAT5. STAT5 is molecule downstream of JAK2 in a signaling
pathway of the GH. The putative mechanism is that the receptors
dimerize (or multimerize), JAK2 associates with the multimerized
receptors and becomes phosphorylated. JAK2 can then associate with
STAT5, which also becomes activated by phosphorylation. Cells were
stimulated with different concentrations of GHRp or GH as indicated
at the top of the panel, IP with anti-STAT5 and then WB with
anti-PY (upper panel) and anti-STAT5 (lower panel). Increasing
phosphorylation of STAT5 is observed as the dose of added GHRp was
increasing. The same band of phosphorylation was observed when
cells were stimulated with GH indicating specific signaling through
GHR. Lower panel demonstrates that all the immunoprecipitates
contained the same amount of STAT5 protein.
[0039] FIG. 19 demonstrates that GHRp together with GH has a
synergistic effect on activation (phosphorylation) of STAT5.
Stimulation of the cells with 0.2 nM GH in the presence or absence
of GHRp shows significant increase in STAT5 phosphorylation, e.g.,
no phosphorylation is observed with 0.2 nM GH or 1.25 .mu.M GHRp
alone, but the signal is increased when both compounds are present.
In addition, a weak signal of STAT5 protein phosphorylation with
2.5 .mu.M peptide is strongly increased by presence of 0.2 nM GH.
Activity was measured with anti-PY antibody (upper panel). The
lower panel demonstrates that the same amount of STAT5 is present
in all the immunoprecipitates. Thus, GHRp has very strong
synergistic effect on GH, measured by STAT5 phosphorvlation.
[0040] FIG. 20 shows the selectivity of GHRp for the GH-Receptor.
Internalization of [125]-GH was measured in IM9 cells in the
presence of GHRp, IGF-1RP (peptide selective and active on IGF-1R;
SEQ ID NO:5) and Pl-Rp (peptide specific for Prolactin receptor,
SEQ ID NO:35; the prolactin receptor has a high level of identity
(approximately 50%) to the GHR). Cells were preincubated for 30 min
at 37.degree. C., followed by addition of [.sup.12I]-GH and
different peptide concentrations as indicated. After the incubation
of 20 min at 37.degree. C., internalized ligand was measured by the
method of acid wash. Cells were spun through oil mixture to
separate bound from unbound ligand. Internalized ligand is
calculated as percent of [.sup.125I]-GH resistant to acid wash
(intracellular) versus the total amount of hormone bound to the
cells. Thus, GHRp, selectively inhibits internalization of
GH-receptor and therefore extends receptor's cell surface time.
DATABASE REFERENCES FOR NUCLEOTIDE AND AMINO ACID SEQUENCES
[0041] The complete mRNA sequence encoding the human insulin
responsive glucose transporter (GLUT4) has the Genbank accession
number M20747, published by Fukumoto et al. (1989) J. Biol. Chem.
264:7776-7779. The complete mRNA sequence encoding the human
insulin receptor has the Genbank accession number A18657, published
in International Patent Application No. WO/91/17253. The complete
mRNA sequence encoding the human leptin receptor has the Genbank
accession number U43168, and was published by Tartaglia et al.
(1995) Cell 83:1263-1271. The DNA sequence encoding the human
granulocyte colony stimulating factor (G-CSF) receptor has the EMBL
accession numbers M59820, M38027, X55720 and X55721, and was
published by Larsen et al. (1990) J. Exp. Med. 172:1559-1570. The
complete sequence of the human interleukin 2 (IL-2) receptor has
the Swissprot accession number P01589, and was published by Leonard
et al. (1984) Nature 311:626-631. The complete sequence of the
human epidermal growth factor (EGF) receptor has the Swissprot
accession number P00533, and was published by Ullrich et al. (1984)
Nature 309:418-425. Additional Swissprot accession numbers are as
follows, with the remainder of the numbers easily obtained:
[0042] interleukin-6 receptor, P08887; interleukin-8 receptor-B,
P25025; interleukin-8 receptor-A, P24024; interleukin-11 receptor,
U32324; interleukin-12, P42701; interleukin-17 receptor, U31993;
EPO receptor, P19235; and TPO receptor, P40238. The sequences for
other cell surface receptors are known, and easily ascertainable by
those in the art.
[0043] The sequences of known HLA and H-2 alleles may be found in
Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, N.I.H. publication no. 91-3242, vol. 1, pp. 738-740, 761,
770-771, 779-780, 788-789 and 802-804.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0044] Generally, cell surface receptors of interest are
internalized or are recycled into the cytoplasm in response to
ligand binding. The present invention is based on the initial
discovery that sequences on the extracellular portion of cell
surface receptors, termed "internalization sequences" or
"activation sequences" herein, are involved in modulation of
receptor responses. Fortuitously, the activation sequences are not
directly involved in ligand binding; that is, the ligand binding
site is separate from the activation sequence of the receptor.
[0045] Without being bound by theory, it appears that these
activation sequences are important in two distinct ways; in the
modulation of receptor internalization, and/or in the modulation of
activation of the receptor.
[0046] First, for some receptors, it appears that these activation
sequences are involved in internalization of the receptor. That is,
the addition of oligopeptides corresponding to the activation
sequence of a receptor can modulate the internalization of the
receptor; for example, to retard or inhibit the internalization of
the receptor (although in some cases, as outlined below,
antagonists could be created). This inhibition of internalization
of the receptors effectively can provide for a greater number of
receptors on the cell surface. This increase or stabilization of
the number of receptors at the cell surface can result in increased
signalling per unit of ligand. This has therapeutic relevance in a
number of disease conditions where decreased ligand binding or
signalling is a problem, or where the hormone is expensive or
difficult to produce. For example, there are a number of diseases
where hormone sensitivity is reduced or the production of the
hormone is decreased, such that increased efficiency of ligand
signalling is desirable. Non-insulin dependent diabetes mellitus
(NIDDM) is an example of such a condition.
[0047] In addition, it has surprisingly been found that for a
particular class of cell-surface receptors, termed "type 2 cell
surface receptors" herein, the activation sequence can be important
in the activation of the receptor, i.e. the activation of the
signalling pathway of the receptor. Thus, oligopeptides
corresponding to a receptor's activation sequence can actually
replace the requirement for the ligand, and will cause receptor
activation even in the absence of ligand. That is, even though the
binding site for the hormone ligand and the activation sequence are
different, the addition of the activation sequence oligopeptide
will cause receptor activation. Without being bound by theory, it
appears that type 2 cell surface receptors occur as monomeric
units, each with a distinct ligand binding site and an
internalization or activation site. Generally, two monomeric
receptors are brought together by the binding of a single ligand
molecule; this non-covalent "dimerization" is what activates the
receptor and allows the downstream biological function which is the
result of ligand binding. Surprisingly, the present invention
reveals that this dimerization and subsequent receptor activation
can occur upon the binding of the activation sequence oligopeptide,
in the absence of ligand. In addition, the effect of the ligand and
the effect of the activation oligopeptide is signficantly
synergistic, and can allow maximum signal using reduced
concentrations of each. These oligopeptides can thus be utilized
either as a ligand replacement, or to increase the response of a
given amount of ligand. In addition, as more fully described below,
the present invention also allows the creation of antagonists to
receptor signalling.
[0048] Preferably, an internalization or activation peptide will be
derived from the sequence of the receptor that is to be modulated.
The sequence of interest corresponds to the region of the receptor
on the extracellular surface, but usually is not directly involved
in ligand binding, i.e. contact is not made with the ligand (that
is, there is no effect on the K.sub.D of the ligand). It should be
noted that the activation sequences from different receptors are
highly specific; in general, an activation sequence from one
receptor will not activate a different receptor. Sequences of
receptors, and positioning of the receptors in the cell membrane
are known in the art. Such information may be accessed through
public databases, as previously cited.
[0049] In a preferred embodiment, the sequences and receptors
described herein are from humans; although as will be appreciated
by those in the art, the present invention may be useful in the
creation and elucidation of animal models of human disease.
Accordingly, sequences and receptors from rodents (rats, mice,
hamsters, guinea pigs, etc.), primates, farm animals (cows, sheep,
pigs, goats, etc.) may also be used. In addition, sequences from
one organism may be tested in other organisms; for example, rat
sequences may be tested in humans, etc.
[0050] Accordingly, the present invention provides regulatory
oligopeptides comprising activation sequences that have an amino
acid sequence at least substantially identical to the sequence of a
portion of a cell surface receptor extracellular domain. Generally,
these oligopeptides also have sequence similarity to bioactive
oligopeptides of the major histocompatibility locus class I
antigens (described in U.S. Pat. No. 5,385,888, herein incorporated
by reference). The oligopeptides modulate the effect of ligand
binding to the corresponding receptor, thereby enhancing the
physiological effect of the ligand, and may, as outlined above, act
to replace the ligand requirement entirely, depending on the
characterization of the receptor.
[0051] The activation sequences are initially identified by
homology to the sequence of an .alpha..sub.1-domain of an MHC Class
I antigen. MHC Class I antigens include human MHC Class I antigens
and mammalian equivalents thereof, such as Class I antigens of the
H-2 locus of mice, in particular H-2 D and K. Human MHC Class I
antigens include HLA-A, B and C. Of more particular interest are
the amino acid sequences in the polymorphic regions of the
.alpha.-1 domain, more particularly amino acids 55 to 90, usually
60 to 90, more particularly 62 to 90. The region 60-85 of the
.alpha.-1 domain, more particularly 62-85 or 72-82 are found to be
of particular interest. One MHC sequence of particular interest is
ERETQIAKGNEQSFRVDLRTLLR, (SEQ ID NO:1; U.S. Pat. No. 5,385,888).
Thus, oligopeptides with sequence similarity to these regions are
preferred.
[0052] Using these sequences, and in particular SEQ ID NO:1, the
sequences of any number of cell surface receptors are scanned for
homologous regions. Suitable cell surface receptors include, but
are not limited to, insulin receptor, insulin-like growth factor
receptor, growth hormone receptor, glucose transporters
(particularly GLUT 4 receptor), transferrin receptor, epidermal
growth factor receptor, low density lipoprotein receptor, high
density lipoprotein receptor, epidermal growth factor receptor,
leptin receptor, interleukin receptors including IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and
IL-17 receptors, human growth hormone receptor, VEGF receptor, PDGF
receptor, EPO receptor, TPO receptor, ciliary neurotrophic factor
receptor, prolactin receptor, and T-cell receptors. In addition,
there are a number of "orphan" receptors for which biological
function has not yet been fully assigned; these are referenced
herein by their SwissProt reference numbers and include, but are
not limited to, SwissProt ML1B (rat melatonin receptor type 1B);
SwissProt SCRC (human secretin receptor precursor); SwissProt NY1R
(Xenopus nueropeptide Y receptor); SwissProt PAFR (rat platelet
activating factor receptor); and SwissProt BLR1 (Burkitt's Lymphoma
receptor for human, mouse and rat). Algorithms for sequence
analysis are known in the art, and include, but are not limited to,
the Best Fit sequence program described by Devereux et al., Nucl.
Acid Res. 12:387-395 (1984), with default settings preferred;
BLAST, described in Altschul et al. (1990) J. Mol. Biol.
215:403-10; ADVANCE and ADAM, described in Torelli and Robotti
(1994) Comput. Appl. Biosci. 10:3-5; and FASTA, described in
Pearson and Lipman (1988) P.N.A.S. 85:2444-8. The sequence
similarity may be determined using the Wisconsin Package, version
8.0-OpenVMS, Genetics Computer Group.
[0053] Preferably, the amino acid sequence of the receptor region
of interest will have at least about 10% sequence identity, and
frequently at least about 15-20% sequence identity. The sequence
similarity will be at least about 30%, with at least about 35%
being preferred, and frequently at least about 45-50%. The examples
provide the results of exemplary similarity searches.
[0054] Exemplary activation amino acid sequences of interest
include, but are not limited to:
[0055] 1. TWLGRQGPEGPSSIPPGTLTTLW (SEQ ID NO:2) from human glucose
transporter, GLUT4, which is 13% identical and 39% similar to SEQ
ID NO:1;
[0056] 2. KTDSQILKELEESSFRKTFEDYLH (SEQ ID NO:3) from human insulin
receptor, which is 35% identical and 56% similar to SEQ ID
NO:1;
[0057] 3. EAEAAVATQETSTVRLKVSSTAVRT (SEQ ID NO:4) from human LDL
receptor, which is 16% identical and 88% similar to SEQ ID
NO:1;
[0058] 4. KTEAEKQAEKEEAEYRKVFENFLH (SEQ ID NO:5) from human insulin
like growth factor receptor, which is 32% identical and 54% similar
to SEQ ID NO:1;
[0059] 5. KKENKIVPSKEIVWWMNLAEKIP (SEQ ID NO:6) from human leptin
receptor, which is 17% identical and 43% similar to SEQ ID
NO:1;
[0060] 6. EKKPVPWESHNSSETCGLPTLVQTY (SEQ ID NO:7) from human GCSF
receptor;
[0061] 7. GPHCVKTCPAGVMGENNTLVWKY (SEQ ID NO:8) from human
epidermal growth factor receptor, which is 17% identical and 43%
similar to SEQ ID NO:1;
[0062] 8. EYELQYKEVNETKWKMMDPILTTSVPVY (SEQ ID NO:9) from human
growth factor receptor, which is 32% identical and 48% similar to
SEQ ID NO:1;
[0063] 9. ARGGTLELRPRSRYRLQLRARLN (SEQ ID NO:10) from human
thrombopoietin receptor, which is 22% identical and 43% similar to
SEQ ID NO:1;
[0064] 10. QRVEILEGRTECVLSNLRGRTRY (SEQ ID NO:11) from human
erythropoietin receptor, which is 26% identical and 43% similar to
SEQ ID NO:1;
[0065] 11. EMQSPMQPVDQASLPGHCREPPPW, (SEQ ID NO:12) from
interleukin-2 (IL-2) receptor alpha chain, which is 12% identical
and 37% similar to SEQ ID NO:1;
[0066] 12. DPDEGVAGAPTGSSPQPLQPL, (SEQ ID NO:13) from IL-2 receptor
beta chain, which is 19% identical and 38% similar to SEQ ID
NO:1;
[0067] 13. QEEGANTRAWRTSLLIALGTLL (SEQ ID NO:14) from interleukin-3
(IL-3), which is 27% identical and 50% similar to SEQ ID NO:1;
[0068] 14. EPSLRIAASTLKSQISYRARVRAWAQCY (SEQ ID NO:15) from
interleukin-4 (IL-4) receptor, which is 28% identical and 52%
similar to SEQ ID NO:1;
[0069] 15. DYETRITESKCVTILHKGFSASVRTILQ (SEQ ID NO:16) from
interleukin-5 (IL-5), which is 30% identical and 52% similar to SEQ
ID NO:1;
[0070] 16. PAQEVARGVLTSLPGDSVTL (SEQ ID NO:17) interleukin-6 (IL-6)
receptor, which is 30% identical and 50% similar to SEQ ID
NO:1;
[0071] 17. GKSNICVKVGEKSLTCKKIDLTTIVK (SEQ ID NO:18) from
interleukin-7 (IL-7), which is 26% identical and 61% similar to SEQ
ID NO:1;
[0072] 18. EDMGNNTANWRMLLRILPQSF (SEQ ID NO:19) from interleukin-8
(IL-8) receptor-B, which is 24% identical and 43% similar to SEQ ID
NO:1;
[0073] 19. EVLGNDTAKWRMVLRILPHTF (SEQ ID NO:20) from interleukin-8
(IL-8) receptor-A, which is 19% identical and 52% similar to SEQ ID
NO:1;
[0074] 20. ELDPGFIHEARLRVQMATL (SEQ ID NO:21) from interleukin-9
(IL-9) receptor, which is 32% identical and 63% similar to SEQ ID
NO:1;
[0075] 21. EVITDAVAGLPHAVRVSARDFL (SEQ ID NO:22) from
interleukin-11, which is 32% identical and 45% similar to SEQ ID
NO:1;
[0076] 22. EQPTQLELPEGCQGLAPGTEVTYRLQLHML (SEQ ID NO:23) from
interleukin-12, which is 43% identical and 67% similar to SEQ ID
NO:1;
[0077] 23. EWSDKQCWEGEDLSKKTLLRFW (SEQ ID NO:24) from
interleukin-13, which is 27% identical and 59% similar to SEQ ID
NO:1;
[0078] 24. KQDKKIAPETRRSIEVPLNERI (SEQ ID NO:25) from
interleukin-13 (a second sequence), which is 23% identical and 41%
similar to SEQ ID NO:1;
[0079] 25. DPNITVETLEAHQLRVSFTLWNESTHYQILLTSF (SEQ ID NO:26) from
interleukin-17;
[0080] 26. EITTDVEKIQEIRYRSKLKLI (SEQ ID NO:27) from hunan platelet
derived growth factor (PDGF) receptor;
[0081] 27. EARCDFCSNNEESFILDADSNM (SEQ ID NO:28) from human
vascular endothelial growth factor (VEGF) receptor, which is 27%
identical and 50% similar to SEQ ID NO:1;
[0082] 28. TWQTPSTWPDPESFPLKFFLRY (SEQ ID NO:29) from human ciliary
neurotrophic factor receptor-alpha, which is 27% identical and 59%
similar to SEQ ID NO:1;
[0083] 29. DSQTNVSQSKDSDVYITDKTVL (SEQ ID NO:30) from T-cell
receptor alpha chain, which is 14% identical and 50% similar to SEQ
ID NO:1;
[0084] 30. EWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:31) from T-cell
receptor beta chain, which is 17% identical and 37% similar to SEQ
ID NO:1;
[0085] 31. SQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRH (SEQ ID NO:32) from
T-cell receptor gamma chain, which is 29% identical and 50% similar
to SEQ ID NO:1;
[0086] 32. VHTEKVNMMSLTVLGLRMLF (SEQ ID NO:33) from T-cell receptor
delta chain, which is 37% identical and 58% similar to SEQ ID
NO:1;
[0087] 33. EKTDRFVMKKLNDRVMRVEYHFLSPY (SEQ ID NO:34) from human
transferrin receptor, which is 29% identical and 54% similar to SEQ
ID NO:1;
[0088] 34. EWEIHFAGQQTEFKILSLHPGQKYL (SEQ ID NO:35) from human
prolactin receptor, which is 20% identical and 48% similar to SEQ
ID NO:1.
[0089] In addition, as outlined below, there are a number of
"orphan" receptors, for which specific function has not yet been
associated, that may be included in the invention. The activation
sequences of these are as follows:
[0090] 35. ARRKAKAERKLRLRPSDLRSFLTMF (SEQ ID NO:38) from rat
melatonin receptor type 1B.
[0091] 36. KLRTQETRGNEVSHYKRLARSTLLLIP (SEQ ID NO:39) from human
secretin receptor.
[0092] 37. GKYVCLEDFPEDKRFLSYTTLLFIL (SEQ ID NO:40) from Xenopus
neuropeptide Y receptor type 1.
[0093] 38. SFRVDSEFRYT (SEQ ID NO:41) from rat platelet activating
factor receptor.
[0094] 39. CLNPMLYTFAGVKFRSDLSRLLTKL (SEQ ID NO:42) from human
Burkitt's lymphoma receptor.
[0095] 40. CLNPMLYTFAGVKRFSDLSRLLTKL (SEQ ID NO:43) from mouse
Burkitt's lymphoma receptor.
[0096] 41. CLNPMLYTFAGVKRFSDLSRLLTKL (SEQ ID NO:44) from rat
Burkitt's lymphoma receptor.
[0097] The receptor sequence of interest, i.e. the activation
sequence, will comprise, as an active motif sequence, at least 8
amino acids, usually at least about 12 amino acids, more usually at
least about 18 amino acids, and fewer than about 40 amino acids,
more usually fewer than 30 amino acids.
[0098] In a preferred embodiment, oligopeptides are made, either
synthetically or through recombinant means, which correspond to the
activation sequence of the extracellular domain of the cell surface
receptor. By "corresponds" herein is meant either that the
oligopeptide is identical to all or part of the activation
sequence, or that the oligopeptide has substantial homology to the
activation sequence; that is, as described below, the oligopeptide
may have amino acid substitutions, insertions or deletions as
compared to the activation sequence.
[0099] As will be appreciated by those in the art, the activation
sequences may be modified, either as modified oligopeptides or as
modified receptors, where the receptors are made with modified
activation sequences.
[0100] In a preferred embodiment, the activation sequences of the
regulatory oligopeptides are altered. Preferably, any modifications
do not substantially alter the biological activity, i.e. they do
not inhibit internalization or aggregation, or prevent activation,
of the activation sequence for the corresponding receptor. This is
easily tested using the binding assays described herein. For
example, amino acid substitutions, insertions and deletions may be
made.
[0101] In one embodiment, amino acid substitutions are made. In
general, it is preferable that residues critical for biological
activity are either not altered or conservatively altered. Critical
residues may be elucidated using known mutagenesis techniques
followed by activity or binding assays; for example, using scanning
mutagenesis techniques, wherein single amino acid residues within
the activation sequence are modified by substitution with an
aliphatic amino acid, e.g. serine, alanine, glycine, valine,
etc.
[0102] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the oligopeptide are desired, substitutions are
generally made in accordance with the following chart:
1 Chart I Original Residue Exemplary Substitutions Ala Ser Arg Lys
Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln
Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met,
Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
[0103] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those shown in Chart I, although these generally are not preferred.
For example, substitutions may be made which more significantly
affect: the structure of the polypeptide backbone in the area of
the alteration, for example the alpha-helical or beta-sheet
structure; the charge or hydrophobicity of the molecule at the
target site; or the bulk of the side chain. The substitutions which
in general are expected to produce the greatest changes in the
polypeptide's properties are those in which (a) a hydrophilic
residue, e.g. seryl or threonyl, is substituted for (or by) a
hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or
alanyl; (b) a cysteine or proline is substituted for (or by) any
other residue; (c) a residue having an electropositive side chain,
e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an
electronegative residue, e.g. glutamyl or aspartyl;
[0104] or (d) a residue having a bulky side chain, e.g.
phenylalanine, is substituted for (or by) one not having a side
chain, e.g. glycine.
[0105] In a preferred embodiment, not more than about three
substitutions or deletions will be made, and that the change will
not be more than about 20 number %, usually not more than about 10
number %, of the number of amino acids in the active motif,
although in some instances higher numbers of alterations may be
made. In some cases, it may be desirable to make antagonist
peptides, that will bind but not activate the receptor, in which
case more alterations may be made. In a preferred embodiment, the
present invention provides oligopeptides that have at least about
60% identical homology to the activation sequence of each receptor
described herein, with at least about 75% being preferred and at
least about 80% being especially preferred; in some instances the
identity will be as high as 90 to 95 or 98%.
[0106] However, if only non-critical residues are altered, this may
be higher. Similarly, if the biological function of the activation
sequence is to be decreased, the amount of changes may also be
greater. Preferred are conservative substitutions, as known in the
art, including substitutions within the large hydrophobic group:
isoleucine, leucine, valine and phenylalanine; between serine and
threonine; glycine and alanine; asparagine and glutamine; aspartic
acid and glutamic acid; or lysine, arginine and histidine. In some
embodiments, non-conservative alterations are done.
[0107] In addition to modifications within the activation
sequences, the oligopeptides may contain additional sequences, as
will be appreciated by those in the art. For example, the
oligopeptides may be extended to: 1) provide convenient linking
sites, e.g. cysteine or lysine; 2) to enhance stability; 3) to bind
to particular receptors; 4) to provide for site-directed action; 5)
to provide for ease of purification (for example, epitope or
purification (His.sub.6) tags); 6) to alter the physical
characteristics (e.g. solubility, charge, etc.); or 7) to stabilize
the conformation; etc. The oligopeptides may be joined to
non-wild-type flanking regions as fused proteins, joined either by
linking groups or covalently linked through cysteine (disulfide) or
peptide linkages. The oligopeptide may be linked through a variety
of bifunctional agents, such as maleimidobenzoic acid,
methyidithioacetic acid, mercaptobenzoic acid, S-pyridyl
dithiopropionate, etc. The oligopeptides may be joined to a single
amino acid at the N- or C-terminus of a chain of amino acids, or
may be internally joined. For example, the subject peptides may be
covalently linked to an immunogenic protein, such as keyhole limpet
hemocyanin, ovalbwnin, etc. to facilitate antibody production to
the subject oligopeptides.
[0108] In a preferred embodiment, the oligopeptides may be shorter
than those depicted herein; that is, residues from either the N- or
C-terminus of the oligopeptide may be deleted with the retention of
biological activity, preferably full biological activity. In some
cases, internal residues may be removed from the oligopeptide.
Generally, this will be done by sequentially removing residues and
assaying for the ability to bind to the activation sequence of a
receptor; once binding has been established, activation may be
evaluated.
[0109] Alternatively, the subject oligopeptides may be expressed in
conjunction with other peptides or proteins, so as to be a portion
of the chain, either internal, or at the N- or C-terminus. Various
post-expression modifications may be achieved. For example, by
employing the appropriate coding sequences, one may provide
farnesylation or prenylation, such that the subject peptide will be
bound to a lipid group at one terminus, and will be able to be
inserted into a lipid membrane, such as a liposome.
[0110] The subject oligopeptides may be modified by the addition of
chemical moieties or groups. For example, the oligopeptides may be
PEGylated, where the polyethyleneoxy group provides for enhanced
lifetime in the blood stream. The subject oligopeptides may also be
combined with other proteins, such as the Fc of an IgG isotype to
enhance complement binding, or with a toxin, such as ricin, abrin,
diphtheria toxin, or the like, particularly the A chain. The
oligopeptides may be linked to antibodies for site directed action.
For conjugation techniques, see, for example, U.S. Pat. Nos.
3,817,837; 3,853,914; 3,850,752; 3,905,654; 4,156,081; 4,069,105;
and 4,043,989, which are incorporated herein by reference. As
outlined herein, the oligopeptides may be labelled as well.
[0111] Oligomers of the regulatory oligopeptides of the invention
may also be made. For example, oligopeptides of interest for drug
screening include, but are not limited to: 1) an oligopeptide
having at least substantially the sequence of the receptor region
of interest; 2) MHC/receptor oligopeptide heterodimers having the
sequence of the receptor region of interest and the amino acid
sequence of bioactive oligopeptides of the major histocompatibility
locus class I antigens; and 3) receptor derived oligopeptide
homodimers, generally as a head to tail dimer, where a spacer of
from 1 to 3 small neutral amino acids may be present between the
two active peptide sequences, as is generally described in WO
US96/15426, specifically incorporated herein by reference.
[0112] Once identified, the oligopeptides comprising the activation
sequences may be prepared in accordance with conventional
techniques, such as synthesis (for example, use of a Beckman Model
990 peptide synthesizer or other commercial synthesizer). Peptides
may be produced directly by recombinant methods (see Sambrook et
al. Molecular Cloning: A Laboratory Manual, CSHL Press, Cold Spring
Harbor, N.Y., 1989) or as a fuision protein, for example to a
protein that is one of a specific binding pair, allowing
purification of the fusion protein by. means of affinity reagents,
followed by proteolytic cleavage, usually at a site engineered to
yield the desired peptide (see for example Driscoll et al. (1993)
J. Mol. Bio. 232:342-350).
[0113] In a preferred embodiment, the activation sequence contained
within the receptor is altered, to form a modified receptor. In a
modified form of the receptor, the sequence corresponding to the
regulatory peptide (i.e. the activation sequence) contains an
insertion, substitution or deletion, such that the ability of the
receptor to internalize in response to ligand binding is altered.
The modification may include a deletion or substitution of the
complete oligopeptide sequence, or a portion thereof. Substitutions
of interest also include scanning mutations as outlined above.
[0114] Conveniently, the modification is performed using
recombinant DNA technology. The DNA sequence encoding the desired
receptor may be obtained from various sources, or may be obtained
from a cDNA library using probes derived from publically available
sequence information. Techniques for in vitro mutagenesis of cloned
genes are known; methods for site specific mutagenesis can be found
in Sambrook, et al. supra. pp 15.3-15.108; Weiner et al. (1993)
Gene 126:3541; Sayers et al. (1992) Biotechniques 13:592-6; Jones
and Winistorfer (1992) Biotechniques 12:528-30; Barton et al.
(1990) Nucleic Acids Res. 18:7349-55; Marotti and Tomich (1989)
Gene Anal. Tech. 6:67-70 and Zhu (1989) Anal. Biochem. 177:1204.
For example, to delete a sequence, primers are devised that span
the region. On hybridization, the region to be deleted forms a
single stranded loop. The loop may be excised by nuclease
digestion, or a suitable polymerase may be used to extend out from
the primer.
[0115] For expression, the DNA sequences are inserted into an
appropriate expression vector, where the native transcriptional
initiation region may be employed or an exogenous transcriptional
initiation region, i.e., a promoter other than the promoter which
is associated with the gene in the normally occurring chromosome.
The promoter may be introduced by recombinant methods in vitro, or
as the result of homologous integration of the sequence into a
chromosome. A wide variety of transcriptional initiation regions
are known for a wide variety of expression hosts. Generally a
selectable marker operative in the expression host will be present.
The promoter may be operably linked to the coding sequence of the
genes of interest so as to produce a translatable mRNA transcript.
Expression vectors have convenient restriction sites located near
the promoter sequence so as to provide for the insertion of nucleic
acid sequences encoding heterologous proteins. The promoters in
suitable expression vectors may be either constitutive or
inducible. Expression vectors for the production of fusion
proteins, where the exogenous fusion peptide provides additional
functionality, i.e. increased protein synthesis, stability,
reactivity with defined antisera, an enzyme marker, e.g.,
b-galactosidase, etc.
[0116] The expression vectors are transformed into a host cell. The
expression hosts may involve prokaryotes or eukaryotes,
particularly E. coli; B. sublilis; yeast cells; mammalian cells;
e.g., COS and CHO cells, HeLa cells, L(tk-), primary cultures;
insect cells; Xenopus laevis oocytes; and the like. Particularly
preferred host cells are mammalian cells.
[0117] Once made, the oligopeptides and modified receptors find use
in a number of applications.
[0118] In a preferred embodiment, the oligopeptides are used in
methods for inhibiting the internalization of a cell surface
receptor response of a mammalian cell. The methods comprises adding
oligopeptides as defined herein to mammalian cells expressing the
cell surface receptor. Upon addition (either simultaneous or
sequential) of the ligand which binds the receptor, the
oligopeptide inhibits the receptor internalization. Alternatively,
for ligand-independent type-2 receptors, as outlined below, the
ligand need not be added to alter receptor internalization.
[0119] In a preferred embodiment, the oligopeptides are used in
methods of activating receptors. As discussed above, for some
receptors, the addition of the oligopeptide can replace or augment
the requirement for ligand binding to effect receptor activation.
As outlined briefly above, cell-surface receptors appear to fall
into two general classes: type 1 and type 2 receptors. Type 1
receptors have generally two identical subunits associated
together, either covalently or otherwise, in the absence of bound
ligand; they are essentially preformed dimers, even in the absence
of ligand. The type 1 receptors include the insulin receptor and
the IGF receptor. The type-2 receptors, however, generally are in a
monomeric form, and rely on either binding of one ligand to each of
two monomers, or, as shown in the present invention, the binding of
one or more oligopeptides that result in multimer formation, to
achieve receptor activation. Type-2 receptors include the growth
hormone receptor, the leptin receptor, the LDL receptor, the GCSF
receptor, the interleukin receptors including IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15,
IL-17, etc., receptors, receptor, EPO receptor, TPO receptor, VEGF
receptor, PDGF receptor, T-cell receptor, transferrin receptor,
prolactin receptor, and the ciliary neurotrophic factor receptor.
Thus, the present invention provides receptors with exogeneous
compounds bound to their activation sequences, and specifically
with bound oligopeptides as defined herein.
[0120] As is known in the art, the monomeric nature of these type 2
receptors can result in unusual saturation kinetics. For example,
it is known for growth hormone that saturation, leading to
reduction in activation, can occur at high levels of administered
growth hormone. This is presumably due to the fact that since there
is only one ligand binding site per monomeric receptor, at high
levels of ligand, each individual receptor monomer can bind a
ligand molecule, and thus will not be brought together into a dimer
form; that is, each monomer has a bound ligand, rather than two
monomers sharing a ligand.
[0121] A receptor may be classified as either type 1 (ligand
dependent) or type 2 (ligand independent) on the basis of several
tests. As noted above, type 2 receptors will exhibit a decrease in
receptor activation at high levels of ligand. Type 2 receptors may
also be classified as such using the present invention, since the
addition of the internalization oligopeptides of the invention will
result in receptor activation even in the absence of ligand.
[0122] As discussed herein, oligopeptides based on the activation
sequences of type 2 cell-surface receptors result in activation of
the receptor even in the absence of ligand. It is important to note
in this case that the ligand binding site is distinct from the
activation or activation sequence. Thus, the present invention
provides methods for activating a type 2 cell surface receptor by
binding an exogeneous compound to the activation sequence of the
receptor. As a result of this binding, two monomeric receptors
dimerize and activate the receptor. However, in an alternate
embodiment, activation may be prevented by binding an antagonistic
exogeneous agent to the activation sequence to prevent activation
of the receptor; that is, certain agents will prevent activation of
the receptor, even in the presence of ligand.
[0123] By "exogeneous compound" herein is meant a compound not
produced endogeneously by the cell or organism; that is, it is
artificially introduced to the cell or organism. Exogeneous
compounds include, but are not limited to, bioactive agents as
defined below such as chemical and small organic moieties; and the
oligopeptides described herein. As will be appreciated by those in
the art and described below, having determined the location of the
binding, and the mechanism of receptor activation by binding, it
will be routine to screen for other molecules that will accomplish
the same thing as the oligopeptides of the invention. This may be
done by finding exogeneous agents that will simultaneously bind at
least two monomeric receptors, causing activation, or by finding an
agent that will bind the internalization (activation) sequence of
the receptor and then making multimers (i.e. dimers) of this
binding agent. A preferred embodiment utilizes the oligopeptides of
the invention as the exogeneous compounds.
[0124] By "receptor activation" or grammatical equivalents herein
is meant the biological function associated with ligand binding to
a cell-surface receptor. As will be appreciated by those in the
art, this will vary widely depending on the identity of the
receptor. For example, cell-surface receptors frequently cause
phosphorylation as a result of ligand binding, that generally,
although not always, is due to a conformational change in the
receptor as a result of ligand binding. Thus "activation" may
comprise a conformational change either within the receptor, or as
a result of monomeric receptors becoming multimeric, which allows
the receptor do facilitate signalling. For example, this
conformational change may allow the receptor to become
phosphorylated, or allow the receptor to associate with a third
molecule which then results in phosphorylation of either the
receptor, the third molecule, or yet another molecule. In this way
signalling is accomplished.
[0125] In a preferred embodiment, the exogeneous compound is added
to the cell in the absence of exogeneous ligand. By "exogeneous
ligand" herein is meant ligand which is not produced endogeneously
by the cell or organism; i.e. exogeneous ligand is introduced to
the cell or organism. It will be appreciated that the composition
of the ligand is generally the same, whether produced endogeneously
or introduced exogeneously; the designation goes to the source of
the ligand, not its composition. As will be appreciated by those in
the art, there may or may not be endogeneous ligand present. The
exogeneous compound acts as a ligand replacement, although it does
not bind to the ligand binding site as a competitor; rather, as
outlined herein, it binds to the activation sequence of the
receptor. The presence or absence of binding of the exogeneous
compound may be tested in a variety of ways, as will be appreciated
by those in the art, including labeling the exogeneous compound and
detecting labelled receptors, competitive assays with known binding
agents, such as the oligopeptides of the invention, or by utilizing
modified receptors which do not contain activation sequences.
[0126] In a preferred embodiment, the exogeneous compound is added
to the receptor in the presence of the ligand which normally
activates the receptor. Again, as above, there may be endogeneous
ligand present, or exogeneous ligand added in addition to the
exogeneous compound. In this embodiment, the level of receptor
activation is greater with a combination of the ligand and the
exogeneous compound as compared with the same amount of ligand
alone. In fact, surprisingly, for some receptors there appears to
be a synergistic effect; that is, the effect of adding ligand and
exogeneous compound (in this case, an oligopeptide of the
invention), is greater than either ligand alone or exogeneous
compound alone, as is generally shown in the examples and Figures.
Without being bound by theory, it appears that this may be due to
the fact that the oligopeptides bind to receptors that do not have
bound ligand, and to those that do, and putatively retard
internalization as well. Accordingly, it may be desirable to add
some exogeneous ligand with the exogeneous compounds of the
invention.
[0127] Accordingly, in a preferred embodiment, the oligopeptides
are administered therapeutically. The subject oligopeptides act to
enhance the cellular response to hormones that bind to the surface
membrane receptor corresponding to the oligopeptide, e.g. insulin
response is enhanced by the oligopeptide SEQ ID NO:3, glucose
transport is enhanced by the oligopeptide SEQ ID NO:2, etc.
Hormones including insulin, insulin-like growth factor, human
growth hormone, glucose transporters, transferrin, epidermal growth
factor, EPO, TPO, low density lipoprotein, human growth hormone and
interleukins are herein referred to as "therapeutic hormones" or
"ligands". Enhancement of the cellular response to therapeutic
hormones by the subject oligopeptides provides a means of improving
the response of patients that are either unresponsive, e.g.
resistant, to the action of such hormones, or, in the case of the
type 2 receptors, the oligopeptides can actually serve as ligand
replacements. This is particularly desirable in some situations
where the administration of the hormone ligand has undesirable
side-effects, for example in the case of growth hormone; the use of
the oligopeptides or other exogeneous compounds may allow receptor
activation without significant side-effects.
[0128] In one embodiment, the subject oligopeptides may be
administered to patients requiring enhancement of the response to
naturally occurring levels of the therapeutic hormone.
[0129] Alternatively, the oligopeptides may be administered to
patients in conjunction with a therapeutic hormone. Of particular
interest is the treatment of insulin resistance, which may be
associated with defects in glucose transport, or in the cellular
response to insulin. Administration of the subject oligopeptides
improves the response to insulin therapy. Similarly, enhancement of
the effect of human growth hormone is also of particular interest.
Human growth hormone is current given in a number of clinical
situations as an injectible drug; alternative therapies may include
augmenting the response of endogeneous hormone.
[0130] Similarly, for type 2 cell surface receptors, exogeneous
compounds, including the oligopeptides of the invention, may be
administered to patients either as ligand replacements, or to
augment the response of a given amount of ligand hormone, as is
outlined above. Thus, the present invention provides for methods of
treating patients with disorders associated with a ligand that
normally activates a type 2 cell surface receptor. For example,
patients with either insufficient amounts of growth hormone, no
growth hormone, or insufficient response to a normal level of
growth hormone, may all be treated by the administration of an
exogeneous compound which binds to the activation sequence of the
growth hormone receptor and causes both receptor activation and
retarded receptor internalization. Similarly, other disorders such
as obesity or weight problems associated with leptin may be
treated. It should be noted that diseases associated with both not
enough ligand present in the patient, and too much ligand present
in the patient, may be treated using the exogeneous compounds of
the invention. For example, when there is too much ligand present
in the patient, for example into the ranges where ligand saturation
is seen, the addition of the exogeneous compounds, for example the
oligopeptides of the invention, can allow dimerization of the
receptor and activation can actually cause the body to make less
ligand.
[0131] When the exogeneous compound or the oligopeptide is to be
administered with exogeneous ligand, the administration can be
simultaneous or sequential, as will be appreciated by those in the
art.
[0132] For therapy, when the oligopeptides are to be used, the
oligopeptides may be administered topically or parenterally, e.g.
by injection at a particular site, for example, subcutaneously,
intraperitoneally, intravascularly, intranasally, transdermally or
the like. Formulations for injection will comprise a
physiologically-acceptab- le medium, such as water, saline, PBS,
aqueous ethanol, aqueous ethylene glycols, or the like. Water
soluble preservatives which may be employed include sodium
bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride,
chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl
alcohol and phenylethanol. These agents may be present in
individual amounts of from about 0.001 to about 5% by weight and
preferably about 0.01 to about 2%. Suitable water soluble buffering
agents that may be employed are alkali or alkaline earth
carbonates, phosphates, bicarbonates, citrates, borates, acetates,
succinates and the like, such as sodium phosphate, citrate, borate,
acetate, bicarbonate and carbonate. Additives such as
carboxymethylcellulose may be used as a carrier in amounts of from
about 0.01 to about 5% by weight. The formulation will vary
depending upon the purpose of the formulation, the particular mode
employed for modulating the receptor activity, the intended
treatment, and the like. The formulation may involve patches,
capsules, liposomes, time delayed coatings, pills, or may be
formulated in pumps for continuous administration. The specific
dosage can be determined empirically in accordance with known ways.
See, for example Harrison's, Principles of Internal Medicine, 11th
ed. Braunwald et al. ed, McGraw Hill Book Co., New York, 1987.
[0133] Generally, a therapeutically effective dose of the subject
oligopeptides will be in the range of about 0.005-10, more usually
from about 0.01-1 mg/kg of host weight, and preferably from about
0.1 to about 1 mg/kg; for example, from about 0.3 to about 0.9 is
preferred for the leptin receptor oligopeptide. Such a dose will be
sufficient to enhance the action of the therapeutic hormone,
usually by at least as much as 50%. Administration may be as often
as daily; usually not more than one or more times daily, or as
infrequent as weekly, depending upon the level of drug which is
administered. The oligopeptides may be administered alone, or in
combination with the therapeutic hormone. The hormone may be
administered at a normally therapeutically effective dose, or the
dose may be decreased by as much as 50%, usually by as much as 25%,
to compensate for the oligopeptide enhancement. The host may be any
mammal including domestic animals, pets, laboratory animals and
primates, particularly humans. The amount will generally be
adjusted depending upon the half life of the peptide, where dosages
in the lower portion of the range may be employed where the peptide
has an enhanced half life or is provided as a depot, such as a slow
release composition comprising particles, introduced in a matrix
which maintains the peptide over an extended period of time, e.g.,
a collagen matrix, use of a pump which continuously infuses the
peptide over an extended period of time over a substantially
continuous rate, or the like. Heller, Biodegradable Polymers in
Controlled Drug Delivery, in: CRC Critical Reviews in Therapeutic
Drua Carrier Systems, Vol. 1, CRC Press, Boca Raton, Fla., 1987, pp
39-90, describes encapsulation for controlled drug delivery, and Di
Colo (1992) Biomaterials 13:850-856 describes controlled drug
release from hydrophobic polymers.
[0134] In a preferred embodiment, the oligopeptides, modified
receptors and cells containing the modified receptors are used in
screening assays. Identification of the amino acid sequence in this
region of receptors permits the design of drug screening assays for
compounds that modulate receptor internalization or serve as ligand
replacements for type 2 receptors.
[0135] Drug screening assays utilize the subject sequence
information and peptide compositions, e.g., proteins, oligopeptides
and synthetic derivatives thereof, to identify agents that modulate
the internalization of cell surface receptors, or, in the case of
type 2 receptors, allow the identification of agents that serve as
ligand replacements.
[0136] Drug candidates capable of modulating surface receptor
internalization are identified by first screening the drug
candidates for the ability to compete with a bioactive oligopeptide
for association with the intact receptor or that interfere with the
binding of an oligopeptide to the subject receptor derived
oligopeptides.
[0137] Thus, in a preferred embodiment, the methods comprise
combining a cell surface receptor which contains an activation
sequence and a candidate bioactive agent, and determining the
binding of the candidate agent to the activation sequence. By "cell
surface receptor" herein is meant any of number of cell surface
receptors which are usually internalized upon ligand binding.
Suitable cell surface receptors are as outlined above. Preferred
embodiments utilize the human cell surface receptors, although
other mammalian receptors may also be used, including rodents
(mice, rats, hamsters, guinea pigs, etc.), farm animals (cows,
sheep, pigs, horses, etc.) and primates. This latter embodiments
may be preferred in the development of animal models of human
disease. Included within the definition of cell surface receptors
are amino acid substitions, insertions, or deletions of the
naturally occuring sequence. Preferably, these do not alter the
biological activity of the receptors, although as outlined herein,
in some instances it may be desirable to modify the biological
activity of the receptors.
[0138] Furthermore, included within the definition of cell surface
receptors are portions of cell surface receptors; that is, either
the full-length receptor may be used, or functional portions
thereof. In a preferred embodiment, at least for a type 1 receptor,
the functional domain comprises at least a ligand binding domain
and an activation sequence, such that the conformational change
that occurs upon ligand binding to the receptor will still occur.
However, as outlined herein, the function domain for a type 2
receptor may comprise only the activation sequence.
[0139] Generally, in a preferred embodiment of the methods herein,
the cell surface receptor is non-diffusably bound to an insoluble
support having isolated sample receiving areas (e.g. a microtiter
plate, an array, etc.). The insoluble supports may be made of any
composition to which peptide or receptor can be bound, is readily
separated from soluble material, and is otherwise compatible with
the overall method of screening. The surface of such supports may
be solid or porous and of any convenient shape. Examples of
suitable insoluble supports include microtiter plates, arrays,
membranes and beads. These are typically made of glass, plastic
(e.g., polystyrene), polysaccharides, nylon or nitrocellulose,
teflon.TM., etc. Microtiter plates and arrays are especially
convenient because a large number of assays can be carried out
simultaneously, using small amounts of reagents and samples. The
particular manner of binding of the peptide or other protein is not
crucial so long as it is compatible with the reagents and overall
methods of the invention, maintains the activity of the peptide and
is nondiffusable. Preferred methods of binding include the use of
antibodies (which do not sterically block either the ligand binding
site or activation sequence when the receptor is bound to the
support), direct binding to "sticky" or ionic supports, chemical
crosslinking, the synthesis of the receptor on the surface, etc.
Following binding of the peptide or receptor, excess unbound
material is removed by washing. The sample receiving areas may then
be blocked through incubation with bovine serum albumin (BSA),
casein or other innocuous protein.
[0140] In a preferred embodiment, particularly for type-1 receptor
assays, a ligand or analog bound by the cell surface receptor will
also be added to the assay. That is, when insulin receptors are
used, the ligand is insulin; when human growth hormone receptors
are used, the ligand is human growth hormone; etc. As will be
appreciated by those in the art, ligand analogs may also be used.
In a preferred embodiment, this is not required, for example when
assaying type-2 receptors.
[0141] A candidate bioactive agent is added to the assay. Novel
binding agents include specific antibodies, noh-natural binding
agents identified in screens of chemical libraries, peptide
analogs, etc. Of particular interest are screening assays for
agents that have a low toxicity for human cells. A wide variety of
assays may be used for this purpose, including labeled in vitro
protein-protein binding assays, electrophoretic mobility shift
assays, immunoassays for protein binding, functional assays
(phosphorylation assays, etc.) and the like.
[0142] The term "agent" or "exogeneous compound" as used herein
describes any molecule, e.g., protein, oligopeptide, small organic
molecule, polysaccharide, polynucleotide, etc., with the capability
of directly or indirectly altering cell surface receptor
internalization and/or activation, which can be in response to
ligand binding or in the absence of ligand binding. Generally a
plurality of assay mixtures are run in parallel with different
agent concentrations to obtain a differential response to the
various concentrations. Typically, one of these concentrations
serves as a negative control, i.e., at zero concentration or below
the level of detection.
[0143] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Particularly preferred are peptides.
[0144] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0145] The determination of the binding of the candidate bioactive
agent to the receptor may be done in a number of ways. In a
preferred embodiment, the candidate bioactive agent is labelled,
and binding determined directly. For example, this may be done by
attaching all or a portion of the cell-surface receptor to a solid
support, adding a labelled candidate agent (for example a
fluorescent label), washing off excess reagent, and determining
whether the label is present on the solid support. Various blocking
and washing steps may be utilized as is known in the art.
[0146] By "labeled" herein is meant that the compound is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g. radioisotope, fluorescers, enzyme,
antibodies, particles such as magnetic particles, chemiluminescers,
or specific binding molecules, etc. Specific binding molecules
include pairs, such as biotin and streptavidin, digoxin and
antidigoxin etc. For the specific binding members, the
complementary member would normally be labeled with a molecule
which provides for detection, in accordance with known procedures,
as outlined above. The label can directly or indirectly provide a
detectable signal.
[0147] In some embodiments, only one of the components is labeled.
For example, the oligopeptides may be labeled at tyrosine positions
using .sup.125I (for example, the activation sequences of the human
GLUT4, insulin, IGF-1, G-CSF, IL-2 and hGH receptors all contain
tyrosine residues), or with fluorophores. Alternatively, more than
one component may be labeled with different labels; using .sup.125I
for the oligopeptides, for example, and a fluorophor for the
candidate agents.
[0148] In a preferred embodiment, the binding of the candidate
bioactive agent is determined through the use of competitive
binding assays. In this embodiment, the competitor is an
oligopeptide as described herein.
[0149] In one embodiment, the candidate bioactive agent is labeled.
Either the candidate bioactive agent, or the oligopeptide, is added
first to the receptor for a time sufficient to allow binding, if
present. Incubations may be performed at any temperature which
facilitates optimal activity, typically between 4 and 40.degree. C.
Incubation periods are selected for optimum activity, but may also
be optimized to facilitate rapid high through put screening.
Typically between 0.1 and 1 hour will be sufficient. Excess reagent
is generally removed or washed away. The second component is then
added, and the presence or absence of the labeled component is
followed, to indicate binding.
[0150] In a preferred embodiment, the oligopeptide is added first,
followed by the candidate bioactive agent. Displacement of the
oligopeptide is an indication that the candidate bioactive agent is
binding to the activation sequence and thus is capable of
modulating the internalization of the receptor. In this embodiment,
either component can be labeled. Thus, for example, if the
oligopeptide is labeled, the presence of label in the wash solution
indicates displacement by the agent. Alternatively, if the
candidate bioactive agent is labeled, the presence of the label on
the support indicates displacement.
[0151] In an alternative embodiment, the candidate bioactive agent
is added first, with incubation and washing, followed by the
oligopeptide. The absence of binding by the oligopeptide may
indicate that the bioactive agent is bound to the receptor with a
higher affinity. Thus, if the candidate bioactive agent is labeled,
the presence of the label on the support, coupled with a lack of
oligopeptide binding, may indicate that the candidate agent is
capable of binding to the activation sequence and modulating the
internalization of the receptor.
[0152] In a preferred embodiment, the methods comprise combining a
cell surface receptor, a ligand bound by the receptor (if
required), and an oligopeptide as described herein, to form a test
mixture. The candidate bioactive agent is added to the test
mixture, and the binding of the candidate bioactive agent to the
activation sequence of the receptor is determined. In this
embodiment, either or both of the oligopeptide or the candidate
bioactive agent is labeled, with preferred embodiments utilizing
labeled oligopeptides, such that displacement of the label
indicates binding by the candidate bioactive agent.
[0153] In a preferred embodiment, the methods comprise differential
screening to identity bioactive agents that are capable of
modulating the internalization or activating receptors. In this
embodiment, the methods comprise combining a cell surface receptor,
a ligand (if required), and an oligopeptide in a first sample. A
second sample comprises a candidate bioactive agent, a cell surface
receptor, a ligand (if required), and an oligopeptide. The binding
of the oligopeptide is determined for both samples, and a change,
or difference in binding between the two samples indicates the
presence of an agent capable of binding the activation sequence and
potentially modulating the internalization of the receptor. That
is, if the binding of the oligopeptide is different in the second
sample relative to the first sample, the agent is capable of
binding the activation sequence.
[0154] Alternatively, a preferred embodiment utilizes differential
screening to identify drug candidates that bind to the native
receptor, but cannot bind to modified receptors, for example those
that have the activation sequences deleted. The structure of the
receptor sequence of interest may be modeled, and used in rational
drug design to synthesize agents that interact with that site. Drug
candidates that affect receptor internalization or receptor
activation are also identified by screening drugs for the ability
to either enhance or reduce the effect of the subject receptor
derived oligopeptides on the internalization or activation of a
selected surface receptor.
[0155] Screening may be performed to find agents that interfere
with the association of a bioactive MHC-derived oligopeptide with
the subject oligopeptides, where the agents will be capable of
modulating the internalization or activating the receptors from
which the subject oligopeptides are derived. This is done using the
methods described above, but replacing the oligopeptides with a
sequence from a .alpha.1-domain of an MHC Class I antigen, such as
SEQ ID NO:1.
[0156] The drug candidate and varying concentrations of the subject
receptor-derived oligopeptides are added to each of the sample
receiving areas containing support-bound peptide. The oligopeptide
added is of substantially the same amino acid sequence as the
oligopeptide bound to the support and is labeled.
[0157] Positive controls for binding of active peptide and
competitive binding of active peptide may include samples
containing labeled active peptide alone and a mixture of labeled
active peptide and unlabeled active peptide, respectively. Samples
containing labeled active peptide and unlabeled inactive peptide
that does not aggregate with the bound peptide may serve as a
negative control for competitive binding with peptide. Preferably
all control and test samples are performed in at least triplicate
to obtain statistically significant results. Incubation of all
samples is for a time sufficient for the binding of the labeled
active peptide to the support-bound peptide. Following incubation,
all samples are washed free of non-specifically bound material and
the amount of bound, labeled peptide determined. For example, where
a radiolabel is employed in labeling the peptide, the samples may
be counted in a scintillation counter to determine the amount of
bound, labeled peptide.
[0158] In test samples containing the drug candidate, if the amount
of labeled active peptide bound to the support-bound peptide or
receptor is in the range of values of the positive control samples
for competitive binding and is significantly less than the negative
control samples for competitive binding, then the drug candidate in
the test sample is able to successfully competitively bind the
support-bound peptide. Drug candidates capable of such competitive
binding may mediate modulation of cell surface expression of a
receptor.
[0159] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used.
[0160] The mixture of components may be added in any order that
provides for the requisite binding.
[0161] Screening for agents that activate type 2 receptors is done
in a manner similar to those outlined above. In addition, in some
embodiments it may be desirable to prevent activation of the type 2
receptors, using the same basic assays described herein, but
looking for agents that interfere with activation and/or
internalization, i.e. an antagonist. For example, agents which bind
to the activation sequences but do not allow multimerization of the
receptors could be antagonists. However, as will be appreciated by
those in the art, antagonists may be linked together, for example
via covalent bonds, to provide for a "dimer" of the antagonist that
now may act as an activator, since the dimer will now bind at least
two receptors. This can be done using linkers as will be
appreciated by those in the art.
[0162] In a preferred embodiment, methods of screening are
provided. In this embodiment, candidate bioactive agent as defined
above are added to a type 2 cell surface receptor with an
activation sequence. The receptor may be on the surface of a cell,
or may be done in vitro. The system is then assayed to determine
whether the candidate bioactive agent binds to the activation
sequence and activates the receptor.
[0163] Similarly, a preferred embodiment provides methods of
screening comprising adding a candidate bioactive agent to a
mammalian cell which has a type 2 cell surface receptor with an
activation sequence. The system is then assayed to determine
whether the candidate bioactive agent binds to the activation
sequence and activates the receptor.
[0164] In this way, bioactive agents are identified. Compounds with
pharmacological activity are able to enhance or interfere with the
internalization of cell surface receptors in response to ligand
binding, or can serve as ligand replacements as described herein.
Binding to the site on the receptor corresponding to the subject
oligopeptides is indicative of such activity, as is the ability to
interfere with the binding of the subject oligopeptides to the
cognate receptor. The compounds having the desired pharmacological
activity may be administered in a physiologically acceptable
carrier to a host, as previously described. The agents may be
administered in a variety of ways, orally, parenterally e.g.,
subcutaneously, intraperitoneally, intravascularly, etc. Depending
upon the manner of introduction, the compounds may be formulated in
a variety of ways. The concentration of therapeutically active
compound in the formulation may vary from about 0.1-100 wt. %. The
pharmaceutical compositions can be prepared in various forms, such
as granules, tablets, pills, suppositories, capsules, suspensions,
salves, lotions and the like. Pharmaceutical grade organic or
inorganic carriers and/or diluents suitable for oral and topical
use can be used to make up compositions containing the
therapeutically-active compounds. Diluents known to the art include
aqueous media, vegetable and animal oils and fats. Stabilizing
agents, wetting and emulsifying agents, salts for varying the
osmotic pressure or buffers for securing an adequate pH value, and
skin penetration enhancers can be used as auxiliary agents.
[0165] Accordingly, methods are provided for enhancing the
physiological effect of ligand binding to cell surface receptors by
administration of such bioactive, receptor derived oligopeptides,
oligopeptide homodimers, and MHC/receptor oligopeptide
heterodimers. The methods are used in diagnosis and therapy of
diseases that involve inadequate or inappropriate receptor
response. The data indicate that internalization of the receptor is
inhibited by the presence of the subject oligopeptides, thereby
providing for a greater number of receptors on the cell surface,
and increased effectiveness of ligand binding. In addition, as
outlined herein, the oligopeptides may also serve as ligand
replacements.
[0166] In a preferred embodiment, the oligopeptides of the
invention may be used to elucidate the function of unknown
receptors, so called "orphan receptors". That is, for receptors for
which no known function is associated, it is possible to add
oligopeptides to the receptors, either in vivo or in vitro, and
look for effects, as will be appreciated by those in the art.
[0167] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference in their entirety.
EXPERIMENTAL
EXAMPLE 1
[0168] In order to determine the region on the external domain of a
cell surface receptor that is involved in receptor internalization,
a sequence similarity comparison was performed. The comparisons
were performed with the commercially available Wisconsin Package,
version 8.0-openVMS, Genetics Computer Group. The complete receptor
sequences were obtained from public databases, as previously
described in the "Database References for Nucleotide and Amino Acid
Sequences".
[0169] The similarity is based on the evolutionary distance between
amino acids, as measured by Dayhoff and normalized by Gribskov and
Burgess (1986) Nucl. Acids Res. 14:6745-6763. The "local homology"
algorithm of Smith and Waterman (1981) Advances in Applied
Mathematics 2:482-289 finds the best segments of similarity between
the two sequences.
[0170] A similarity search between SEQ ID NO:1 and amino acid
sequences of the cell surface receptors outlined herein was done,
and the regions outlined above were identified, as shown in the
table below.
2TABLE 1 Peptides--modulators of cognate receptor activity Receptor
Sequence IR KTDSQILKELEESSFRKTFEDYLH IGF-IR ERETQIAKGNEQSFRVDLRTLLR
TPO-R ARGGTLELRPRSRYRLQLRARLN EPO-R QRVEILEGRTECVLSNLRGRTRY PDGF-R
EITTDVEKIQEIRYRSKLKLI VEGF-R EARCDFCSNNEESFILDADSNM GH-R
EYELQYKEVNETKWKMMDPILTTSVPVY PRL-R EWEIHFAGQQTEFKILSLHPGQKYL OB-R
KKENKIVPSKEIVWWMNLAEKIP EGF-R GPHCVKTCPAGVMGENNTLVWKY LDL-R
EAEAAVATQETSTVRLKVSSTAVRT Tf-R EKTDRFVMKKLNDRVMRVEYHFLSPY CNT-R
TWQTPSTWPDPESFPLKFFLRY GLUT-4 ERETQIAKGNEQSFRVDLRTLLR TC-R.alpha.
DSQTNVSQSKDSDVYITDKTVL TC-R.beta. EWTQDRAKPVTQIVSAEAWGRADC
TC-R.gamma. SQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCI TC-R.delta.
VRHVHTEKVNMMSLTVLGLRMLF IL-2R.alpha. EMQSPMQPVDQASLPGHCREPPPW
IL-2R.alpha. DPDEGVAGAPTGSSPQPLQPL IL-3R QEEGANTRAWRTSLLIALGTLL
IL-4R EPSLRIAASTLKSGISYRARVRAWAQCY IL-5R
DYETRITESKCVTILHKGFSASVRTILQ IL-6R PAQEVARGVLTSLPGDSVTL IL-7R
GKSNICKVKVGEKSLTCKKIDLTTIVK IL-8R.alpha. EVLGNDTAKWRMVLRILPHTF
IL-8R.beta. EDMGNNTANWRMILLRILPQSF IL-9R ELDPGFIHEARLRVQMATL IL-11R
EVITDAVAGLPHAVRVSARDFL IL-12R EQPTQLELPEGCQGLAPGTEVTYRLQLHML
IL-13R.alpha. KQDKKIAPETRRSIEVPLNERI IL-13R.beta.
EWSDKQCWEGEDLSKKTLLRFW IL-17R
DPNITVETLEAHQLRVSFTLWNESTHYQILLTSF
[0171]
3TABLE 2 Similarity between MHC-I peptide and certain sequences on
cell-surface receptors Receptor Identity (%) Similarity (%) IR 35
57 IGF-IR 32 55 TPO-R 22 44 EPO-R 26 44 PDGF-R 29 52 VEGF-R 27 50
GH-R 32 48 PRL-R 20 48 OB-R 18 48 EGF-R 17 44 LDL-R 16 88 Tf-R 29
54 CNT-R 27 59 GLUT-4 13 39 TC-R.alpha. 14 50 TC-R.beta. 17 38
TC-R.gamma. 29 50 TC-R.delta. 37 58 IL-2R.alpha. 13 38 IL-2R.beta.
19 38 IL-3R 27 50 IL-4R 28 52 IL-5R 40 52 IL-6R 30 50 IL-7R 26 61
IL-8R.alpha. 19 52 IL-8R.beta. 24 43 IL-9R 32 63 IL-11R 32 46
IL-12R 43 67 IL-13R.alpha. 23 41 IL-13R.beta. 27 59 IL-17R 24
52
EXAMPLE 2
[0172] Methods
[0173] Insulin Receptor modification and expression. The human
insulin receptor gene, as described in the database references and
in Ebina et al. (1985) Cell 40:747-758) with a pCR3 expression
vector (Invitrogen, catalog no. K3000-01) was transfected by
electroporation into HeLa cells. Methods of electroporation are
described in Boggs et al. (1986) Ex. Hematol. 149:988-994. In the
transfected cells the receptors show insulin dependent
internalization.
[0174] A mutated form of the insulin receptor was created by
deleting residues 713 to 740 (SEQ ID NO:36;
PKTDSQILKELEESSFRKTFEDYLHNV) using amplification primers that
spanned the region to be deleted. The deletion mutant, mIR, was
transfected into HeLa cells and internalization of the mIR was then
tested.
[0175] Measurement of IR internalization. Receptor internalization
was performed essentially as described in Stagsted et al. (1990)
Cell 62:297-307. Briefly, 50 .mu.l of the transfected cells at 106
cells/ml were incubated in a shaking water bath at 37.degree. C.
with 625 pM 1251-labeled insulin in the absence or presence of 10
.mu.M of peptide as shown in Table 1, and the final volume brought
to 100 .mu.l. The cells were then diluted with 50 .mu.l of KRHB
(pH7.2) (no acid wash) or 50 .mu.l of KRHB (pH 2.0) (acid wash) and
incubated on ice for 5 min. The cells were finally harvested by
centrifugation on top of silicone oil, and both free and
cell-associated radioactivity was measured.
[0176] Glucose Transport in Adipose Cells. The biological activity
of the peptides were measured by their effect on glucose uptake in
rat adipose cells as described (Stagsted et al. (1991) J. Biol.
Chem. 266:12844-12847). Briefly, rat adipose cells were obtained
from epididymal fat pads and suspended in Krebs-Ringer HEPES buffer
(KRH) with 5% bovine serum albumin at a lipocrit of 10% (final).
The peptide effect was measured in cells maximally stimulated with
insulin (10 nM). After equilibration at 37.degree. C. for 30 min
the cells were incubated for 30 min at 37.degree. C. with buffer
(basal), 10 nM insulin plus peptide. .sup.14C-D-glucose was added,
and the cells were incubated for an additional 30 min and harvested
on oil. Biological activity was measured by a dose-response curve
to interpolate the EC.sub.50 value, taking the maximum enhancement
of insulin effect (about 40% over the insulin-only maximum) as
100%. Most of the peptides were not tested at higher concentrations
than 30 .mu.M. Peptides that enhanced the maximum insulin effect by
less than 20% at 30 .mu.M were considered inactive.
[0177] Peptides. The peptides were assembled stepwise either on a
phenylacetamidomethyl (PAM) resin using the t-Boc NMP/HOBt protocol
of an Applied Biosystems Model 430A peptide synthesizer, or on a
p-alkoxy benzyl alcohol (Wang) resin using a modified Fmoc/BOP
protocol of a Milligen/Biosearch Model 9600 synthesizer. The
desired peptides were confirmed by sequence analysis, amino acid
composition, and fast atom bombardment mass spectrometry. The
peptides were activated by incubation of 1 mM stock solution at
37.degree. C. in 0.1 M NaCI overnight (Stagsted et al. (1991) J.
Biol. Chem. 266:12844-12847).
[0178] Results
[0179] Effect of peptides on receptor internalization. The kinetics
of internalization for insulin receptor and mutated insulin
receptor were determined in the absence or presence of the
peptides: SEQ ID NO:3, KTDSQILKELEESSFRKTFEDYLH (pepIR) and SEQ ID
NO:37, GNEQSFRVDLRTLLRYAGGGNEQSFRVDLRTLLRYA (DS-A85). The data are
shown in Table 2, where the numbers indicate percent internalized
receptor.
4TABLE 2 Time IR + mIR + IR + MIR + (min) IR mIR DS-A85 DS-A85
PEPIr pepIR 5 6 .+-. 4 4 .+-. 5 5 .+-. 4 -1 .+-. 5 6 .+-. 4 5 .+-.
4 15 39 .+-. 7 2 .+-. 2 9 .+-. 6 0 .+-. 3 2 .+-. 2 -2 .+-. 1 30 68
.+-. 6 4 .+-. 5 14 .+-. 6 2 .+-. 3 6 .+-. 4 0 .+-. 2 60 74 .+-. 8 5
.+-. 4 17 .+-. 3 1 .+-. 4 2 .+-. 4 2 .+-. 3
[0180] The data show that the mutated insulin receptor mIR does not
internalize upon insulin binding, whereas more than 50% of the wild
type IR is internalized within 30 minutes. The pepIR peptide
inhibits receptor internalization to the same extent as DS-A85.
[0181] Effect of peptides on glucose uptake. At maximal insulin
stimulation, the addition of peplR did not significantly affect
glucose uptake, indicating that pepIR does not affect GLUT4
internalization. Glucose uptake is enhanced 14.+-.3 fold by the
addition of 10 .mu.M insulin. Insulin+10 .mu.M of the DS-A85
peptide enhances glucose uptake 22.+-.4 fold, whereas addition of
insulin+10 .mu.M pepIR enhances glucose uptake 12.+-.4 fold, a
result not significantly different from insulin alone.
[0182] The GLUT4pep (SEQ ID NO:2), at a concentration of 10 .mu.M,
does not affect insulin receptor internalization by the transfected
cells. In the presence of peptide the per cent internalized
receptor is 6.+-.9, in the absence of peptide it is 64.+-.7. The
peptide does inhibit the internalization of GLUT4, as shown by the
effect on glucose uptake at maximal insulin stimulation. In the
presence of 10 nM insulin, the enhancement of glucose uptake was
12.+-.4 fold. The enhancement was increased to 24.+-.2 fold with
the addition of the GLUT4pep. The peptide therefore seems to
inhibit internalization of GLUT4, but not insulin receptor.
[0183] It is evident from the above results that oligopeptides
having the sequence of the extracellular domain of a cell surface
receptor, and having sequence identity with a region of an MHC
class I antigen, are effective in inhibiting the internalization of
the corresponding receptor. The peptides are therapeutically useful
in enhancing the cellular response to hormones such as insulin.
EXAMPLE 2
[0184] Ligand Replacement and Synergistic Addition
[0185] Protocol for Protein Phosphorylation and Activation of
Cognate Receptor Signaling Cascade
[0186] Materials
[0187] Appropriate cell lines have the following attributes: they
are responsive to the appropriate hormone, they expresses the
cognate receptor, they possess molecules required for down stream
signaling of hormone induced response. The growth medium is as
recommended for cell line.
[0188] Antibodies: Immunoprecipitation: 2-5 .mu.g of Anti PY-99
(Santa Cruz Biotechnology), Anti-Receptor or signaling molecule
specific antibody (e.g., Jak, STAT); Western blot: Anti Receptor
specific antibody, Signaling protein specific antibody (Santa Cruz
Biotechnology); or anti-PTyr (4G10, Upstate Biotech); with
secondary antibody being appropriate according to the primary.
Immunoprecipitation: Protein G beads (Pharmacia); 20-40 .mu.l of
slurry.
[0189] Protease inhibitor mix (100.times.) in water: 1 mg/ml
Aprotinin, 0.1 mg/ml Pepsatin A, 0.1 mg/ml Leupeptin, 0.1 mg/ml
Chymostatin, 0.1 mg/ml AEBSF.
[0190] Cell wash buffer: PBS with 1 mM ortho-vanadate, 50 mM NaF,
20 mM .beta.-glycerophosphate and 2 mM sodium phyrophosphate.
[0191] Lysis buffer: 50 mM HEPES, 150 mM NaCl, 1% Triton X-100,
1.times. protease inhibitor mix, 50 mM NaF, 5 mM EDTA, and 2 mM
sodium phyrophosphate.
[0192] Blocking buffer: Blotto: TST (0.01 M Tris pH 7.4, 0.15 M
NaCl, 0.075% Tween 20, 0.02% NaN.sub.3)+0.5% Dry Skim Milk
Powder
[0193] Procedure
[0194] Treatment of the Cells
[0195] The cells were grown cells to an approximate density of
1.times.10.sup.6 cells, centrifuged down and resuspended in
recommended media for growth. The cells were starved for 14-18
hours at 37.degree. C. (5% CO.sub.2) in a media without serum, spun
down and resuspended cells at a density of 1.times.10.sup.6
cells/ml in a media without serum. 10 ml of cell suspension per
P100 tissue culture dish was used. The cell suspension was treated
with 0.01-30 .mu.M peptide, 15-30 min at 37.degree. C. (5%
CO.sub.2); (thaw peptide very shortly before the assay). 10 ml of
ice cold cell wash buffer was added per dish, transfered to a
Falcon tube and centrifuged down quickly at 4.degree. C. and 3000
rpm. The media was carefully aspirated and the wash was repeated at
4.degree. C.
[0196] The following steps were all performed on ice: The media was
aspirated and 0.6 ml of 2.times.lysis buffer/tube was added. This
was pipetted up and down and transfered to Eppendorf tubes, let sit
on ice for ca. 30 min, and centrifuged at 14,000.times.g for 10
min, at 420 C. The supernatant (Lysate) was used in
Immunoprecipitations.
[0197] Immunoprecipitation: The protein G beads were washed with
0.5.times.lysis buffer in Eppendorf tubes, 1-4 .mu.g of Anti-PY-99
antibody was added, with receptor specific or signaling molecule
(JAK2, STAT5) specific antibody per IP. The tubes were incubated
with end-over-end rotation for 2 h at RT. The lysate was added, the
tubes incubated with end-over-end rotation over night at 4.degree.
C. The beads were washed 3 times with 1.times.lysis buffer, 40
.mu.l of SDS-sample buffer was added, and the samples were boiled
for 3 min.
[0198] Western blot analysis: ca 12 .mu.l/sample was run on 8%
Polyacrylamide mini gel, transferred to PVDF-membrane (Millipore),
blocked for 1 h in blocking buffer, incubated with the appropriate
primary antibody 1:1000, o/n at 4.degree. C. The membrane was
washed, incubated with appropriate secondary-alk. phosphatase
conjugated secondary antibody 1:2000, at RT for 2 hours. The
PVDF-membrane was washed with Blotto and developed with
NBT/BCIP.
[0199] Protocol for EPO-R Activation (Phosphorylation)
[0200] TF-1 cells from ATCC were used, with the growth medium being
RPM1 1640 with 1.times.Penicillin/Streptomycin, 2 mM glutamin, 10%
fetal bovine serum (Hyclone), and 1 ng/ml GM-CSF.
[0201] Treatment of the cells: Cells were grown to an approximate
density of 1.times.10.sup.6 cells, centrifuged down and resuspended
in media with 5% serum and no GM-CSF. The cells were starved for
14-18 hours at 37.degree. C. (5% CO.sub.2), spun down and
resuspended cells at a density of 1.times.10.sup.6 cells/m in a
media without serum and GM-CSF. 10 ml of cell suspension per P100
tissue culture dish was used. The cell suspension was treated with
1-30 .mu.M peptide, 15-30 min at 37.degree. C. (5% CO.sub.2); (thaw
peptide very shortly before the assay). 10 ml of ice cold cell wash
buffer was added per dish, transfered to Falcon tube and
centrifuged down quickly at 4.degree. C. and 3000 rpm. The media
was carefully aspirated and the wash repeated at 4.degree. C., with
the following steps performed on ice: The media was aspirated and
0.6 ml of 2.times.lysis buffer/tube was added; this was pipetted up
and down and transferred to Eppendorf tubes that sat on ice for ca.
30 min. The tubes were then centrifuged at 14,000.times.g for 10
min, at 4.degree. C., and the supernatant (Lysate) removed and used
in Immunoprecipitations and Western blots, as above, using
anti-EPO-receptor antibody (Santa Cruz Biotechnology).
[0202] The results are shown in the Figures.
[0203] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Sequence CWU 1
1
44 1 23 PRT Artificial Sequence MHC sequence 1 Glu Arg Glu Thr Gln
Ile Ala Lys Gly Asn Glu Gln Ser Phe Arg Val 1 5 10 15 Asp Leu Arg
Thr Leu Leu Arg 20 2 23 PRT Artificial Sequence human glucose
transporter 2 Thr Trp Leu Gly Arg Gln Gly Pro Glu Gly Pro Ser Ser
Ile Pro Pro 1 5 10 15 Gly Thr Leu Thr Thr Leu Trp 20 3 24 PRT
Artificial Sequence human insulin receptor 3 Lys Thr Asp Ser Gln
Ile Leu Lys Glu Leu Glu Glu Ser Ser Phe Arg 1 5 10 15 Lys Thr Phe
Glu Asp Tyr Leu His 20 4 25 PRT Artificial Sequence human LDL
receptor 4 Glu Ala Glu Ala Ala Val Ala Thr Gln Glu Thr Ser Thr Val
Arg Leu 1 5 10 15 Lys Val Ser Ser Thr Ala Val Arg Thr 20 25 5 24
PRT Artificial Sequence human insulin like growth factor receptor 5
Lys Thr Glu Ala Glu Lys Gln Ala Glu Lys Glu Glu Ala Glu Tyr Arg 1 5
10 15 Lys Val Phe Glu Asn Phe Leu His 20 6 23 PRT Artificial
Sequence human leptin receptor 6 Lys Lys Glu Asn Lys Ile Val Pro
Ser Lys Glu Ile Val Trp Trp Met 1 5 10 15 Asn Leu Ala Glu Lys Ile
Pro 20 7 25 PRT Artificial Sequence human GCSF receptor 7 Glu Lys
Lys Pro Val Pro Trp Glu Ser His Asn Ser Ser Glu Thr Cys 1 5 10 15
Gly Leu Pro Thr Leu Val Gln Thr Tyr 20 25 8 23 PRT Artificial
Sequence human epidermal growth factor receptor 8 Gly Pro His Cys
Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn 1 5 10 15 Asn Thr
Leu Val Trp Lys Tyr 20 9 28 PRT Artificial Sequence human growth
factor receptor 9 Glu Tyr Glu Leu Gln Tyr Lys Glu Val Asn Glu Thr
Lys Trp Lys Met 1 5 10 15 Met Asp Pro Ile Leu Thr Thr Ser Val Pro
Val Tyr 20 25 10 23 PRT Artificial Sequence human thrombopoietin
receptor 10 Ala Arg Gly Gly Thr Leu Glu Leu Arg Pro Arg Ser Arg Tyr
Arg Leu 1 5 10 15 Gln Leu Arg Ala Arg Leu Asn 20 11 23 PRT
Artificial Sequence human erythropoietin receptor 11 Gln Arg Val
Glu Ile Leu Glu Gly Arg Thr Glu Cys Val Leu Ser Asn 1 5 10 15 Leu
Arg Gly Arg Thr Arg Tyr 20 12 24 PRT Artificial Sequence
interleukin-2 receptor alpha chain 12 Glu Met Gln Ser Pro Met Gln
Pro Val Asp Gln Ala Ser Leu Pro Gly 1 5 10 15 His Cys Arg Glu Pro
Pro Pro Trp 20 13 21 PRT Artificial Sequence IL-2 receptor beta
chain 13 Asp Pro Asp Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser
Pro Gln 1 5 10 15 Pro Leu Gln Pro Leu 20 14 22 PRT Artificial
Sequence interleukin-3 receptor 14 Gln Glu Glu Gly Ala Asn Thr Arg
Ala Trp Arg Thr Ser Leu Leu Ile 1 5 10 15 Ala Leu Gly Thr Leu Leu
20 15 28 PRT Artificial Sequence interleukin-4 receptor 15 Glu Pro
Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys Ser Gln Ile Ser 1 5 10 15
Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr 20 25 16 28 PRT
Artificial Sequence interleukin-5 receptor 16 Asp Tyr Glu Thr Arg
Ile Thr Glu Ser Lys Cys Val Thr Ile Leu His 1 5 10 15 Lys Gly Phe
Ser Ala Ser Val Arg Thr Ile Leu Gln 20 25 17 20 PRT Artificial
Sequence interleukin-6 receptor 17 Pro Ala Gln Glu Val Ala Arg Gly
Val Leu Thr Ser Leu Pro Gly Asp 1 5 10 15 Ser Val Thr Leu 20 18 26
PRT Artificial Sequence interleukin-7 18 Gly Lys Ser Asn Ile Cys
Val Lys Val Gly Glu Lys Ser Leu Thr Cys 1 5 10 15 Lys Lys Ile Asp
Leu Thr Thr Ile Val Lys 20 25 19 21 PRT Artificial Sequence
interleukin-8 receptor-B 19 Glu Asp Met Gly Asn Asn Thr Ala Asn Trp
Arg Met Leu Leu Arg Ile 1 5 10 15 Leu Pro Gln Ser Phe 20 20 21 PRT
Artificial Sequence interleukin-8 receptor-A 20 Glu Val Leu Gly Asn
Asp Thr Ala Lys Trp Arg Met Val Leu Arg Ile 1 5 10 15 Leu Pro His
Thr Phe 20 21 19 PRT Artificial Sequence interleukin-9 receptor 21
Glu Leu Asp Pro Gly Phe Ile His Glu Ala Arg Leu Arg Val Gln Met 1 5
10 15 Ala Thr Leu 22 22 PRT Artificial Sequence interleukin-11 22
Glu Val Ile Thr Asp Ala Val Ala Gly Leu Pro His Ala Val Arg Val 1 5
10 15 Ser Ala Arg Asp Phe Leu 20 23 30 PRT Artificial Sequence
interleukin-12 23 Glu Gln Pro Thr Gln Leu Glu Leu Pro Glu Gly Cys
Gln Gly Leu Ala 1 5 10 15 Pro Gly Thr Glu Val Thr Tyr Arg Leu Gln
Leu His Met Leu 20 25 30 24 22 PRT Artificial Sequence
interleukin-13 24 Glu Trp Ser Asp Lys Gln Cys Trp Glu Gly Glu Asp
Leu Ser Lys Lys 1 5 10 15 Thr Leu Leu Arg Phe Trp 20 25 22 PRT
Artificial Sequence interleukin-13 25 Lys Gln Asp Lys Lys Ile Ala
Pro Glu Thr Arg Arg Ser Ile Glu Val 1 5 10 15 Pro Leu Asn Glu Arg
Ile 20 26 34 PRT Artificial Sequence interleukin-17 26 Asp Pro Asn
Ile Thr Val Glu Thr Leu Glu Ala His Gln Leu Arg Val 1 5 10 15 Ser
Phe Thr Leu Trp Asn Glu Ser Thr His Tyr Gln Ile Leu Leu Thr 20 25
30 Ser Phe 27 21 PRT Artificial Sequence human platelet derived
growth factor receptor 27 Glu Ile Thr Thr Asp Val Glu Lys Ile Gln
Glu Ile Arg Tyr Arg Ser 1 5 10 15 Lys Leu Lys Leu Ile 20 28 22 PRT
Artificial Sequence human vascular endothelial growth factor
receptor 28 Glu Ala Arg Cys Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe
Ile Leu 1 5 10 15 Asp Ala Asp Ser Asn Met 20 29 22 PRT Artificial
Sequence human ciliary neurotrophic factor receptor- alpha 29 Thr
Trp Gln Thr Pro Ser Thr Trp Pro Asp Pro Glu Ser Phe Pro Leu 1 5 10
15 Lys Phe Phe Leu Arg Tyr 20 30 22 PRT Artificial Sequence T-cell
receptor alpha chain 30 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp
Ser Asp Val Tyr Ile 1 5 10 15 Thr Asp Lys Thr Val Leu 20 31 24 PRT
Artificial Sequence T-cell receptor beta chain 31 Glu Trp Thr Gln
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala 1 5 10 15 Glu Ala
Trp Gly Arg Ala Asp Cys 20 32 36 PRT Artificial Sequence T-cell
receptor gamma chain 32 Ser Gln Glu Gly Asn Thr Met Lys Thr Asn Asp
Thr Tyr Met Lys Phe 1 5 10 15 Ser Trp Leu Thr Val Pro Glu Glu Ser
Leu Asp Lys Glu His Arg Cys 20 25 30 Ile Val Arg His 35 33 20 PRT
Artificial Sequence T-cell receptor delta chain 33 Val His Thr Glu
Lys Val Asn Met Met Ser Leu Thr Val Leu Gly Leu 1 5 10 15 Arg Met
Leu Phe 20 34 26 PRT Artificial Sequence human transferrin receptor
34 Glu Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met
1 5 10 15 Arg Val Glu Tyr His Phe Leu Ser Pro Tyr 20 25 35 25 PRT
Artificial Sequence human prolactin receptor 35 Glu Trp Glu Ile His
Phe Ala Gly Gln Gln Thr Glu Phe Lys Ile Leu 1 5 10 15 Ser Leu His
Pro Gly Gln Lys Tyr Leu 20 25 36 27 PRT Artificial Sequence mutated
form of insulin receptor 36 Pro Lys Thr Asp Ser Gln Ile Leu Lys Glu
Leu Glu Glu Ser Ser Phe 1 5 10 15 Arg Lys Thr Phe Glu Asp Tyr Leu
His Asn Val 20 25 37 36 PRT Artificial Sequence DC-A85 37 Gly Asn
Glu Gln Ser Phe Arg Val Asp Leu Arg Thr Leu Leu Arg Tyr 1 5 10 15
Ala Gly Gly Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg Thr Leu 20
25 30 Leu Arg Tyr Ala 35 38 25 PRT Artificial Sequence rat
melatonin receptor type 1B 38 Ala Arg Arg Lys Ala Lys Ala Glu Arg
Lys Leu Arg Leu Arg Pro Ser 1 5 10 15 Asp Leu Arg Ser Phe Leu Thr
Met Phe 20 25 39 27 PRT Artificial Sequence human secretin receptor
39 Lys Leu Arg Thr Gln Glu Thr Arg Gly Asn Glu Val Ser His Tyr Lys
1 5 10 15 Arg Leu Ala Arg Ser Thr Leu Leu Leu Ile Pro 20 25 40 25
PRT Artificial Sequence Xenopus neuropeptide Y receptor type 1 40
Gly Lys Tyr Val Cys Leu Glu Asp Phe Pro Glu Asp Lys Arg Phe Leu 1 5
10 15 Ser Tyr Thr Thr Leu Leu Phe Ile Leu 20 25 41 11 PRT
Artificial Sequence rat platelet activating factor receptor 41 Ser
Phe Arg Val Asp Ser Glu Phe Arg Tyr Thr 1 5 10 42 25 PRT Artificial
Sequence human Burkitt's lymphoma receptor 42 Cys Leu Asn Pro Met
Leu Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser 1 5 10 15 Asp Leu Ser
Arg Leu Leu Thr Lys Leu 20 25 43 25 PRT Artificial Sequence mouse
Burkitt's lymphoma receptor 43 Cys Leu Asn Pro Met Leu Tyr Thr Phe
Ala Gly Val Lys Arg Phe Ser 1 5 10 15 Asp Leu Ser Arg Leu Leu Thr
Lys Leu 20 25 44 25 PRT Artificial Sequence rat Burkitt's lymphoma
receptor 44 Cys Leu Asn Pro Met Leu Tyr Thr Phe Ala Gly Val Lys Arg
Phe Ser 1 5 10 15 Asp Leu Ser Arg Leu Leu Thr Lys Leu 20 25
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