U.S. patent application number 09/117345 was filed with the patent office on 2001-12-20 for methods and compositions for identifying modulators of g-protein-coupled receptors.
Invention is credited to CONKLIN, BRUCE R., KOSTENIS, EVI, WESS, JURGEN.
Application Number | 20010053532 09/117345 |
Document ID | / |
Family ID | 21987565 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053532 |
Kind Code |
A1 |
CONKLIN, BRUCE R. ; et
al. |
December 20, 2001 |
METHODS AND COMPOSITIONS FOR IDENTIFYING MODULATORS OF
G-PROTEIN-COUPLED RECEPTORS
Abstract
The present invention provides methods and compositions that can
be used to identify modulators of G-protein-coupled receptors.
Inventors: |
CONKLIN, BRUCE R.; (SAN
FRANCISCO, CA) ; KOSTENIS, EVI; (BETHESDA, MD)
; WESS, JURGEN; (ROCKVILLE, MD) |
Correspondence
Address: |
TYLER M. DYLAN
MORRISON & FOERSTER
755 PAGE MILL ROAD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
21987565 |
Appl. No.: |
09/117345 |
Filed: |
July 27, 1998 |
PCT Filed: |
July 27, 1998 |
PCT NO: |
PCT/US98/15567 |
Current U.S.
Class: |
435/7.2 ;
435/325; 536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 14/4722 20130101; C07K 14/723 20130101; G01N 2333/726
20130101 |
Class at
Publication: |
435/7.2 ;
536/23.5; 435/325 |
International
Class: |
G01N 033/53; G01N
033/567; C07H 021/04; C12N 005/00; C12N 005/02 |
Claims
1. A method for identifying a modulator of a G-protein-coupled
receptor (GPCR), comprising the steps of: (i) contacting a
candidate GPCR modulator with a mutant q-type G protein comprising
an N-terminally altered .alpha..sub.q subunit, wherein said altered
.alpha..sub.q subunit renders the G protein responsive to at least
one non-q-type GPCR; and (ii) assaying for a q-type G protein
response.
2. A method according to claim 1, wherein said contacting step
occurs in a cell comprising a recombinant polynucleotide encoding
the N-terminally altered .alpha..sub.q subunit.
3. A method according to claim 2, wherein said cell firther
comprises a recombinant polynucleotide encoding a non-q-type
GPCR.
4. A method according to claim 3, wherein said cell has been
transformed with a vector encoding the N-terminally altered ocq
subunit and the non-q-type GPCR.
5. A method according to one of the preceding claims, farther
comprising, prior to step (i), the step of providing a host cell
over-expressing said non-q-type GPCR and said mutant q-type G
protein.
6. A method according to one of the preceding claims, wherein
assaying for a q-type G protein response comprises assaying for
activation of phospholipase C.
7. A method according to one of the preceding claims, wherein
assaying for a q-type G protein response comprises assaying for
hydrolysis of phosphatidylinositols.
8. A method according to one of the preceding claims, wherein
assaying for a q-type G protein response comprises assaying for
calcium mobilization.
9. A method according to one of the preceding claims, wherein said
modulator is a GPCR agonist that binds to and activates a
non-q-type GPCR.
10. A method according to claim 9, wherein said modulator is a GPCR
agonist that binds to and activates a Gi-type GPCR.
11. A method according to claim 9, wherein said modulator is a GPCR
agonist that binds to and activates a Gs-type GPCR.
12. A method according to one of claims 1-8, wherein said modulator
is a GPCR antagonist that binds to and competitively inhibits a
non-q-type GPCR.
13. A method according to claim 12, wherein said modulator is a
GPCR antagonist that binds to and non-competitively inhibits a
non-q-type GPCR.
14. A method according to claim 12, wherein said method further
comprises contacting said mutant q-type G protein with an agonist
of said GPCR.
15. A method according to one of the preceding claims, wherein said
N-terminally altered .alpha.q subunit has a mutation affecting at
least one of the first ten amino acid residues of .alpha..sub.q,
wherein said mutation impairs the ability of the N-terminal region
to restrict the G protein to interactions with q-type receptors and
effectors.
16. A method according to one of the preceding claims, wherein said
mutation is selected from the group consisting of a deletion,
substitution and insertion.
17. A method according to one of the preceding claims, wherein said
mutation maintains the alpha-helical nature of the N-terminal
region.
18. A method according to one of the preceding claims, wherein said
mutation comprises a substitution of at least one of the first six
residues of .alpha.q.
19. A method according to one of the preceding claims, wherein said
mutation comprises a substitution of at least two of the first six
residues of .alpha.q.
20. A method according to claim 16, wherein said mutation is a
deletion removing at least one of the first six residues of
.alpha.q.
21. A method according to claim 16, wherein said mutation is a
deletion removing at least two of the first six residues of
.alpha.q.
22. A method according to claim 16, wherein said mutation is a
deletion removing the first six residues of .alpha..sub.q.
23. A method according to claim 16, wherein said N-terminally
altered .alpha..sub.q subunit has a deletion or substitution
affecting at least one of the first six amino acid residues of
.alpha..sub.q, and an additional N-terminal mutation affecting at
least one of residues 7-10.
24. A method according to claim 23, wherein said additional
mutation comprises a substitution wherein at least one of residues
7, 8, 9 or 10 is substituted with an amino acid found at residues
1, 2, 3 or 4 of a non-q-type .alpha. subunit.
25. A method according to claim 16, wherein said N-terminally
altered .alpha.q subunit has a deletion or substitution affecting
at least one of the first six amino acid residues of .alpha..sub.q,
and an additional C-terminal mutation in which one or more residues
at the C-terminus of .alpha..sub.q is replaced with residues from
the C-terminus of a non-q-type .alpha. subunit.
26. A recombinant polynucleotide encoding an N-terminally altered
.alpha..sub.q subunit, wherein said altered .alpha..sub.q subunit
renders the G protein responsive to at least one non-q-type
GPCR.
27. A recombinant polynucleotide according to claim 26, wherein
said N-terminally altered .alpha..sub.q subunit has a mutation
affecting at least one of the first ten amino acid residues of
.alpha..sub.q, wherein said mutation impairs the ability of the
N-terminal region to restrict the G protein to interactions with
q-type receptors and effectors.
28. A recombinant polynucleotide according to claim 26, wherein
said mutation is selected from the group consisting of a deletion,
substitution and insertion.
29. A recombinant polynucleotide according to claim 26, wherein
said mutation maintains the alpha-helical nature of the N-terminal
region.
30. A recombinant polynucleotide according to claim 26, wherein
said mutation comprises a substitution of at least one of the first
six residues of .alpha..sub.q .
31. A recombinant polynucleotide according to claim 26, wherein
said mutation comprises a substitution of at least two of the first
six residues of .alpha..sub.q .
32. A recombinant polynucleotide according to claim 26, wherein
said mutation is a deletion removing at least one of the first six
residues of .alpha..sub.q.
33. A recombinant polynucleotide according to claim 26, wherein
said mutation is a deletion removing at least two of the first six
residues of .alpha..sub.q.
34. A recombinant polynucleotide according to claim 26, wherein
said mutation is a deletion removing the first six residues of
.alpha..sub.q.
35. A recombinant polynucleotide according to claim 26, wherein
said N-terminally altered .alpha..sub.q subunit has a deletion or
substitution affecting at least one of the first six amino acid
residues of .alpha..sub.q, and an additional N-terminal mutation
affecting at least one of residues 7-10.
36. A recombinant polynucleotide according to claim 26, wherein
said additional mutation comprises a substitution wherein at least
one of residues 7, 8, 9 or 10 is substituted with an amino acid
found at residues 1, 2, 3 or 4 of a non-q-type a subunit.
37. A recombinant polynucleotide according to claim 26, wherein
said N-terminally altered .alpha..sub.q subunit has a deletion or
substitution affecting at least one of the first six amino acid
residues of .alpha..sub.q, and an additional C-terminal mutation in
which one or more residues at the C-terminus of .alpha..sub.q is
replaced with residues from the C-terminus of a non-q-type .alpha.
subunit.
38. A cell transformed with a recombinant polynucleotide according
to one of claims 26-37.
39. A cell of claim 38, further transformed with a recombinant
polynucleotide encoding a non-q-type GPCR.
40. A cell of claim 38, wherein said non-q-type GPCR is a Gi-type
GPCR.
41. A cell of claim 38, wherein said non-q-type GPCR is a Gs-type
GPCR.
Description
TECHNICAL FIELD
[0001] This invention relates to signal transduction via
G-protein-coupled receptors (hereinafter GPCRs).
[0002] More specifically, this invention relates to compositions
and methods for screening modulators of GPCRs. The compositions and
methods involve mutated G proteins that can be used to shift signal
transduction of certain classes of GPCRs to an alternative effector
pathway that can be measured by high throughput screening
techniques. Generation of such mutants therefore facilitates
large-scale rapid screening for candidate GPCR modulators having
diagnostic and/or therapeutic benefit.
BACKGROUND OF THE INVENTION
[0003] The interaction between extracellular ligands and cell
surface receptors is of central importance in the sensitivity and
responsiveness of eukaryotic cells to exogenous signals and other
environmental stimuli. Over the past decades, numerous cell surface
receptors have been discovered, characterized and cloned. Among
them, G-protein-coupled receptors (GPCRs) plays a pivotal role in a
wide range of physiological responses.
[0004] G-protein-coupled receptors form a large family of related
proteins. Members of each subfamily are coupled to different
subsets of downstream signal transduction components that transduce
and amplify distinct arrays of intracellular signals. In
particular, GPCRs generally comprise a characteristic group of
seven transmembrane spanning regions, and are further characterized
by their ability to interact with heteromultimeric G-protein
complexes, which are generally trimeric complexes comprised of
.alpha., .beta. and .gamma. subunits. Typically, activation of a
GPCR by an agonist (i.e. a positive modulator such as a specific
ligand) causes the GPCR to interact with and activate a particular
class of G protein. In the activated G protein, GDP bound to the a
subunit is replaced with GTP followed by the dissociation of
.alpha..sub.GTP from the .beta..gamma. dimer. The activated G
protein subunits (.alpha..sub.GTP or free .beta..gamma.) are then
able to modulate downstream signal transduction events via one or
more "effector" molecules (typically via effector enzymes and/or
the modulation of ion channels. Many effectors can be regulated by
the .alpha. subunit and independently co-regulated (either
positively or negatively) by the .beta..gamma. units (see, e.g.,
E.J. Neer, Cell 80: 249-257, 1995).
[0005] At least four subfamilies of G protein .alpha. subunits have
been identified to date, namely
.alpha..sub.q,.alpha..sub.s,.alpha..sub.i and .alpha..sub.12 (as
illustrated in FIG. 6), and multiple .beta. and .gamma. subunits
have been found. More than half of GPCRs appear to couple to the
heterotrimeric G proteins Gs or Gi in which the ax subunit is
.alpha..sub.s, or .alpha..sub.i. Both Gs and Gi signal through the
cyclic AMP (cAMP) pathway. In particular, Gs stimulates adenylyl
cyclase, resulting in an increase in intracellular cAMP
concentration, whereas Gi inhibits adenylyl cyclase, causing a
decrease in intracellular cAMP level. A smaller number of GPCRs
activate a phospholipase C (PLC) pathway by coupling with a
distinct group of heterotrimeric G proteins, Gq in which the
.alpha. subunit is .alpha..sub.q. Phospholipase C hydrolyzes
phosphoinositides to generate two classes of well-characterized
second messengers, namely, diacylglycerol and inositol phosphates.
Diacylglycerol activates certain protein kinase Cs (PKCs) and
certain inositol phosphates stimulate the mobilization of calcium
from intracellular stores.
[0006] Among the physiological responses involving signal
transduction via GPCRs are the dilation/constriction of blood
vessels, bronchi and organs within the gastrointestinal tract, the
modulation of endocrine secretions, and the control of heart rate.
Indeed, in mammals it is believed that more than 1000 genes encode
GPCRs of one type or another. Of those, several hundred GPCRs are
likely to be involved in various disease processes, and thus are
potential diagnostic and/or therapeutic targets. While GPCR
modulators (including agonists and antagonists for example) have a
great potential as diagnostic/therapeutic agents, the search for
such agents is often both time consuming and labor intensive.
[0007] While some screening assays have been described, many assays
are either inconvenient to perform on a large scale, or their uses
are limited to only a subset of GPCRs. For example, while calcium
mobilization (a downstream event associated with Gq activation) is
amenable to high throughput screening using robotic assays, the
majority of GPCRs are not coupled to Gq. Conversely, while many
GPCRs are coupled to G proteins affecting changes in cyclic AMP
(i.e. Gs or Gi as noted above), changes in cAMP level are not
particularly amenable to high throughput or robotic screening
assays.
[0008] There thus remains a considerable need for compositions and
methods that can be used to facilitate the large-scale and rapid
screening of GPCR modulatory agents, particularly agents that
modulate GPCRs that are associated with alterations in
intracellular cAMP (which collectively represent the majority of
known GPCRs).
SUMMARY OF THE INVENTION
[0009] The present invention provides compositions and methods for
identifying agents that modulate G-protein-coupled receptors,
particularly agents that modulate non-Gq-coupled receptors (such as
Gi- or Gs-coupled receptors), using high throughput screening
methods based on Gq signaling.
[0010] In essence, the compositions and methods of the present
invention effectively couple non-q upstream signaling events (such
as those initiated by ligand binding to Gi- or Gs-coupled
receptors) to q-type downstream signaling, such as calcium
mobilization, that is readily amenable to high throughput screening
techniques. The effective shift in signal transduction pathways
greatly facilitates screening for GPCR modulatory compounds which
would be of major diagnostic and therapeutic potential.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 depicts the comparison of the N-terminal amino acid
sequences of selected G protein .alpha. subunits. Gaps were
introduced to allow for maximum sequence identity. The position of
the N-terminal portion of the .alpha. N helix, as revealed by X-ray
crystallography is indicated. "-6q" denotes a mutant G.alpha..sub.q
construct lacking the first six amino acids.
[0012] FIG. 2 depicts the functional interaction of different G
protein-coupled receptors with the G.alpha. subunits, WTq and -6q.
COS-7 cells co-expressing WTq or -6q and different Gi/o-coupled
receptors (A) or Gs-coupled receptors (B) were incubated for 1 h
(at 37.degree. C.) in the absence or presence of the indicated
agonist ligands. The resulting increases in intracellular IP.sub.1
levels were determined as described under "Example 3". Data are
given as means .+-. S.E. of three to seven independent experiments,
each carried out in triplicate. The following receptors: ligands
were used: (A) m2 muscarinic receptor: carbachol (100 .mu.M); D2
dopamine receptor: (-)-quinpirole (10 .mu.M); ic-opioid receptor
(K-OR): (-)-U50488 (10 .mu.M); somatostatin SSTR1 receptor:
somatostatin-14 (1 .mu.M); (B) Al adenosine receptor: (-)-PIA
(R(-)-N.sup.6-(2-Phenylisopropyl)-adenosine (10 .mu.M); D1 dopamine
receptor: dopamine (1 mM); V2 vasopressin receptor: [Arg.sup.8]
vasopressin (1 nM); .beta.2-adrenergic receptor: (-)-isoproterenol
(200 .mu.M).
[0013] FIG. 3 depicts the effect of pertussis toxin on inositol
phosphate accumulation in cells co-transfected with WTq or -6q and
Gi/o-coupled receptors. COS-7 cells were cotransfected with
expression plasmids coding for the wild type m2 or the D2 dopamine
receptor and vector DNA (pcDNAI), WTq, or -6q. Transfected cells
were incubated for 1 h (at 37.degree. C.) in the absence or
presence of the appropriate agonist ligands. The resulting
increases in intracellular IP.sub.1 levels either in the absence or
in the presence of PTX (500 ng/ml). Data are given as means .+-.
S.E. of four independent experiments, each carried out in
triplicate. The following ligands were used: m2 muscarinic
receptor, carbachol (100 .mu.M); D2 dopamine receptor,
(-)-quinpirole (10 .mu.M).
[0014] FIG. 4 depicts the functional interaction of the m2
muscarinic and the D2 dopamine receptor with -6q. COS-7 cells
co-transfected with vector DNA (pcDNAI), WTq, or -6q and m2
muscarinic (A) or D2 dopamine receptor (B) expression plasmids were
incubated for I h (at 37.degree. C.) with increasing concentrations
of carbachol (A) or (-)-quinpirole (B). The resulting increases in
intracellular IP.sub.1 levels were determined as described under
Example 3. Results (mean values) from a representative experiment,
carried out in triplicate, are shown; four additional experiments
gave similar results.
[0015] FIG. 5 depicts the subcellular localization and
palmitoylation of WTq and -6q. COS-7 cells were transfected with
vector DNA alone (V), WTq, or -6q. (A) Cellular proteins were
separated into particulate (P) and soluble (S) fractions, and
G.alpha. subunits were detected by Western blotting using the
HA-epitope specific 12CA5 antibody, as described under "Example 5.
" (B) Following labeling of cells with [.sup.3H]palmitic acid,
proteins were immunoprecipitated with the 12CA5 antibody. The
immunoprecipitates were resolved by SDS-PAGE and analyzed by
fluorography. WTq and -6q proteins run at approximately 44 kDa.
Protein molecular weight standards (in kDa) are indicated.
[0016] FIG. 6 illustrates the relationship of various G protein
families and also shows the relative percent amino acid identity
shared by various G protein families and individual types.
DETAILED DESCRIPTION OF THE INVENTION
[0017] As noted above, the present invention provides compositions
and methods for identifying agents that modulate G-protein-coupled
receptors, particularly agents that modulate non-Gq-coupled
receptors (such as Gi- or Gs-coupled receptors), using high
throughput screening methods based on downstream events normally
associated with Gq signaling. The compositions and methods of the
present invention effectively couple non...q upstream signaling
events (such as those initiated by ligand binding to Gi- or
Gs-coupled receptors) to q-type downstream signaling, such as
calcium mobilization, that is readily amenable to high throughput
screening techniques. The effective shift in signal transduction
pathways greatly facilitates screening for GPCR modulatory
compounds which would be of major diagnostic and therapeutic
potential.
[0018] This invention encompasses nucleic acids encoding G protein
.alpha..sub.q mutants, cell lines expressing such mutants, and
methods of using such mutants for the screening of GPCR modulators,
particular modulators of Gi- and Gs-coupled GPCRs. The uses
described include methods of high throughput screen of compounds
that bind to or modulate the activities of non-Gq-coupled (such as
Gi- and Gs-coupled GPCRs which collectively constitute the majority
of known GPCRs). The invention also includes compounds for
modulating these receptors identified by such methods of
screening.
[0019] Definitions
[0020] The terms "polypeptide", "peptide" and "protein" are used
interchangeably to refer to polymers of amino acids and do not
refer to any particular lengths of the polymers. Also included
within the definition are, for example, proteins containing one or
more analogs of an amino acid (including, for example, unnatural
amino acids, etc.), proteins with substituted linkages, as well as
other modifications known in the art, both naturally occurring and
non-naturally occurring. It is also known in the art that many
proteins are post-translationally modified in eukyotic cells in a
number of ways, including but not limited to glycosylation,
acetylation, phosphorylation, myristoylation, and palmitoylation.
For instance, many G proteins described herein, for example,
G.alpha..sub.q, G.alpha..sub.s and G.alpha..sub.i are either
palmitoylated or myristoylated.
[0021] "Domain" refers to a portion of a protein or peptide that is
physically or functionally distinguished from other portions of the
protein or peptide. Physically-defined domains include those amino
acid sequences that are exceptionally hydrophobic or hydrophilic,
such as those sequences that are membrane-associated or
cytoplasm-associated. Domains may also be defined by internal
homologies that arise, for example, from gene duplication.
Functionally-defined domains have a distinct biological
function(s). The ligand-binding domain of a receptor, for example,
is that domain that binds ligand. Functionally-defined domains need
not be encoded by contiguous amino acid sequences.
Functionally-defined domains may contain one or more
physically-defined domain. Receptors, for example, are generally
divided into the extracellular ligand-binding domain, a
transmembrane domain, and an intracellular effector domain.
[0022] As used herein, a "polynucleotide" or "nucleic acid" is a
polymeric form of nucleotides of any length, which contain
deoxyribonucleotides, ribonucleotides, and/or their analogs. The
terms "polynucleotide" and "nucleotide" as used herein are used
interchangeably. Polynucleotides may have any three-dimensional
structure, and may perform any function, known or unknown. The term
"polynucleotide" includes doublestranded, single-stranded, and
triple-helical molecules. Unless otherwise specified or required,
any embodiment of the invention described herein that is a
polynucleotide encompasses both the double-stranded form and each
of two complementary singlestranded forms known or predicted to
make up the double stranded form. Not all linkages in a
polynucleotide need be identical. The following are non-limiting
examples of polynucleotides: a gene or gene fragment, exons,
introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors,
isolated DNA of any sequence, isolated RNA of any sequence, nucleic
acid probes, and primers. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide
analogs.
[0023] A polynucleotide or polynucleotide region has a certain
percentage (for example, 80%, 85%, 90%, or 95%) of "sequence
identity" to another sequence means that, when aligned, that
percentage of bases are the same in comparing the two
sequences.
[0024] A nucleotide is said to "encode" a polypeptide if, in its
native state or when manipulated by methods known to those skilled
in the art, it can be transcribed and/or translated to produce the
polypeptide or a fragment thereof. For purposes of this invention,
and to avoid cumbersome referrals to complementary strands, the
anti-sense (or complementary) strand of such a polynucleotide is
also said to encode the sequence; that is, a polynucleotide
sequence that "encodes" a polypeptide includes both the
conventional coding strand and the complementary sequence (or
strand).
[0025] An "open reading frame" (or "ORF") is a region of a
polynucleotide sequence that can encode a polypeptide or a portion
of a polypeptide (i.e., the region may represent a portion of a
protein coding sequence or an entire protein coding sequence).
[0026] "Fused" or "fusion" refers to the joining together of two or
more elements, components, etc., by whatever means (including, for
example, a "fusion protein" made by chemical conjugation (whether
covalent or non-covalent), as well as the use of an in-frame fusion
to generate a "fusion protein" by recombinant means, as discussed
infra). An "inframe fusion" refers to the joining of two or more
open reading frames (ORFs), by recombinant means, to form a single
larger ORF, in a manner that maintains the correct reading frame of
the original ORFs. Thus, the resulting recombinant fusion protein
is a single protein containing two or more segments that correspond
to polypeptides encoded by the original ORFs (which segments are
not normally so joined in nature). Although the reading frame is
thus made continuous throughout the fused segments, the segments
may be physically separated by, for example, in-frame flexible
polypeptide linker sequences.
[0027] A "transcriptional regulatory region" or "transcriptional
control region" refers to a polynucleotide encompassing all of the
cis-acting sequences necessary for transcription, and may include
sequences necessary for regulation. Thus, a transcriptional
regulatory region includes at least a promoter sequence, and may
also include other regulatory sequences such as enhancers,
transcription factor binding sites, polyadenylation signals and
splicing signals.
[0028] "Operably linked" or "operatively linked" refers to a
juxtaposition wherein the components so described are in a
relationship permitting them to function in their intended manner.
For instance, a promoter sequence is operably linked to a coding
sequence if the promoter sequence promotes transcription of the
coding sequence.
[0029] A "vector" is a self-replicating nucleic acid molecule that
transfers an inserted nucleic acid molecule into and/or between
host cells. The term includes vectors that function primarily for
insertion of a nucleic acid molecule into a cell, replication of
vectors that function primarily for the replication of nucleic
acid, and expression vectors that function for transcription and/or
translation of the DNA or RNA. Also included are vectors that
provide more than one of the above functions.
[0030] "Expression vectors" are defined as polynucleotides which,
when introduced into an appropriate host cell, can be transcribed
and translated into a polypeptide(s). An "expression system"
usually connotes a suitable host cell comprised of an expression
vector that can function to yield a desired expression product.
[0031] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient for vector(s) or for
incorporation of nucleic acid molecules and/or proteins. Host cells
include progeny of a single host cell, and the progeny may not
necessarily be completely identical (in morphology or in genomic of
total DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation. A host cell includes cells
transfected in vivo with a polynucleotide(s) of this invention.
[0032] "Transformation" or "transfection" refers to the insertion
of an exogenous polynucleotide into a host cell, irrespective of
the method used for the insertion, for example, lipofection,
transduction, infection or electroporation. The exogenous
polynucleotide may be maintained as a non-integrated vector, for
example, a plasmid, or alternatively, may be integrated into the
host cell genome.
[0033] "Mutant" forms of a protein or polypeptide are those which
have alterations in amino acid sequence and/or structure caused,
for example, by site-directed mutagenesis (on the nucleotide level)
or other chemical modification (on the peptide level); by errors in
transcription or translation; or which are prepared synthetically
by rational design. Minor alterations are those which result in
amino acid sequences wherein the biological activity of the
polypeptide is retained and/or wherein the mutant polypeptide has
at least 90% homology with the native form. As used herein, the
biological activity of a mutant G protein is findamentally defined
by its ability to interact with GPCRs and with downstream targets.
For example, certain mutant G proteins as described herein are
functionally "promiscuous" with respect to upstream signaling (i.e.
they have acquired the ability to be activated by GPCRs other than
those to which the "parental-type" protein is normally coupled)
while retaining their specificity with respect to downstream
signaling (i.e. the retain the ability to activate the same set or
sets of downstream effectors as the parental-type protein). As
described herein, an exemplary group of such mutant G proteins has
one or more alterations affecting the N-terminus of the
.alpha..sub.q subunit which allows the G protein to interact with
(and be activated by) Gi- and Gs-coupled GPCRs, but to convey the
downstream signal that is normally associated with Gq
signaling.
[0034] "Recombinant," as applied to a polynucleotide, means that
the polynucleotide is the product of various combinations of
cloning, restriction and/or ligation steps resulting in a construct
that is distinct from a polynucleotide found in nature. Recombinant
may also be used to refer to the protein product of a recombinant
polynucleotide. By way of illustration, a nucleic acid sequences
encoding a GPCR or a G protein subunit (such as a G.alpha.
subunit), can be assembled from cDNA fragments and short
oligonucleotide linkers, or from a series of oligonucleotides, to
provide a synthetic gene which is capable of being expressed when
operably linked to a transcriptional regulatory region. The coding
sequences are preferably provided in the form of an open reading
frame uninterrupted by internal non-translated sequences (i.e.
"introns"), such as those commonly found in eukaryotic genes. These
and other nucleic acid sequences referred to in the context of the
present invention can also be generally obtained by PCR
amplification using viral, prokaryotic or eukaryotic DNA or RNA
templates in conjunction with appropriate PCR amplimers.
[0035] "Heterologous" means derived from (i.e., obtained from) a
genotypically distinct entity from the rest of the entity to which
it is being compared. For example, a polynucleotide may be placed
by genetic engineering techniques into a plasmid or vector derived
from a different source, thus becoming a heterologous
polynucleotide. A promoter which is linked to a coding sequence
with which it is not naturally linked is a heterologous
promoter.
[0036] "Signal transduction" refers to a series of intercellular
and subcellular events by which a chemical or physical signal is
transmitted to a cell and is conveyed to a subcellular component or
components that are involved in a response to the signal. Signal
transduction mediated by G proteins can be conveniently divided
into two stages in the context of the present invention. In
particular, the G protein can be regarded as playing a pivotal role
between: (i) "upstream" signal transduction events (including, for
example, binding of a "first messenger" such as a ligand or agonist
to a GPCR and consequent activation of a coupled G protein), and
(ii) "downstream" signal transduction events (including, for
example, the interaction of the activated G protein and/or G
protein subunits with "effectors" such as ion channels or enzymes
involved in the synthesis of "second messengers" like cyclic AMP or
inositol phosphate).
[0037] A "G-protein-coupled receptor" or "GPCR" refers to a member
of a class of cellular proteins involved in the receipt of an
extracellular signal and the transmission of that signal to a G
protein to which they are coupled. GPCRs can be organized into
families based on structural and functional relatedness. As
discussed herein, GPCRs can be grouped into families which
collectively transduce a variety of different upstream signals
(e.g. a variety of different chemical or physical signals) into a
downstream pathway that is mediated by a particular class of G
protein. For example, a large variety of GPCRs are coupled to G
proteins of the Gi or Gs type, which effectively convey
extracellular signals to effectors resulting in the inhibition or
stimulation, respectively, of intracellular cyclic AMP.
[0038] A "G protein" refers to a member of a class of cellular
proteins that bind to GTP/GDP and which regulate the activity of a
wide variety of cellular enzymes and/or ion channels. Many G
proteins interact with GPCRs and effectively transduce a signal
from the GPCR to downstream effectors. Such GPCR-interactive G
proteins are typically heterotrimeric proteins (comprising .alpha.,
.beta. and .gamma. subunits) that are activated by GPCRs having a
characteristic group of seven transmembrane spanning regions.
However, despite these commonalties, the G proteins differ in the
nature of the GPCRs and effectors that they interact with or "talk"
to, the specificity of which interaction appears to be essentially
dependent on the nature of the G protein cc subunit. For example, G
proteins of the Gs type (which have an .alpha..sub.s subunit) are
coupled to and activated by a class of GPCRs and, upon activation,
mediate signal transduction to effectors that stimulate the
production of cyclic AMP within the cell (typically as a result of
dissociation of the activated G protein into a-GTP and a Py dimer,
as described above). Whereas G proteins of the Gi type (which have
an ac subunit) associate with another class of GPCRs to mediate
inhibition of adenylyl cyclase, resulting in a decrease in cyclic
AMP level. G proteins of the third class, namely the Gq type (which
have an a subunit selected from a group of .alpha..sub.16,
.alpha..sub.14, .alpha..sub.11, .alpha..sub.q (see FIG. 6)) are
coupled to another distinct class of GPCRs and, upon stimulation,
mediate signaling via PLC-pathway.
[0039] A "promiscuous" G protein refers to a G protein that has
acquired the ability to interact with receptors and/or effectors
other than those with which it normally interacts; a phenomenon
sometimes referred to as "cross-talk." By way of illustration, a
promiscous Gq protein may exhibit the ability to interact with
non-q-type receptors (such as Gi-type or Gs-type GPCRs, as
described below).
[0040] An "effector," as used herein, refers to an intracellular
protein or complex of proteins that mediate cellular responses to a
signal such as that tranduced by GPCRs and their coupled G
proteins. Effectors of G protein and/or G protein subunits may
include adenylyl cyclases, phospholipase C enzymes, and
phospholipase A2 enzymes (and other enzymes involved in the
synthesis of second messengers), as well as ion channels that can
be opened or closed as a result of activation of GPCRs and their
associated G proteins.
[0041] As used herein, a receptor is considered to be "biologically
active" if it is capable of binding ligand, and transmitting an
intracellular signal or response. "Intracellular signal" and
"intracellular response" are used interchangeably herein. The
intracellular responses characteristic Gq-coupled receptor include
coupling with G.alpha..sub.q subunit, a subsequent activation of
phospholipase pathway.
[0042] A "modulator" refers to a compound which modulates a
receptor, including agonists, antagonists, allosteric modulators,
and the like. Typically, the modulator binds to the receptor (i.e.
it acts as a ligand for the GPCR). GPCR modulators thus refer to
agents that modulate (e.g. stimulate or inhibit) the activity of
G-protein-coupled receptors. As is known in the art, inhibitors can
bind reversibly (in which case they can usally be "competed off" by
increasing the levels of the normal agonist), or they can bind
irreversibly (in which case the inhibitory effect is observed to be
non-competitive).
[0043] A "ligand" refers to a molecule capable of being bound by
the ligand-binding domain of a receptor. The molecule may be
chemically synthesized or may occur in nature. "Agonist" refers to
a ligand capable of stimulating the biological activity of a
receptor. "Antagonist" refers to a ligand capable of inhibiting the
biological activity of a receptor.
[0044] Altered G Proteins that engage in promiscuous signaling
[0045] Experiments carried out on several distinct classes of
heterotrimeric G proteins have suggested the general principle that
G-protein coupling specificity is determined primarily by the
.alpha. subunit of the G protein.
[0046] The majority of known GPCRs influence the levels of cyclic
AMP as a "second messenger" within the responding cell, and fall
into one of two general classes depending on the nature of their
.alpha. subunit and the consequent effector activity (i.e. either
stimulation or inhibition of cyclic AMP). In particular, G proteins
of the Gs type (which have an .alpha..sub.s subunit) are coupled to
and activated by a class of GPCRs and, upon activation, mediate
signal transduction to effectors that stimulate the production of
cyclic AMP within the cell (typically as a result of dissociation
of the activated G protein into .alpha.-GTP and a .beta..gamma.
dimer, as described above and in the art). Conversely, G proteins
of the Gi type (which have an .alpha..sub.i subunit) are coupled to
and activated by a class of GPCRs and, upon activation, mediate
signal transduction to effectors that inhibit the production of
cyclic AMP within the cell.
[0047] Another class of G proteins, of the Gq type (which have an
.alpha..sub.q subunit), activate a phospholipase C (PLC) pathway,
resulting in the hydrolysis of phosphoinositides to generate two
classes of different second messengers, namely, diacylglycerol and
inositol phosphates. Diacylglycerol activates certain protein
kinase Cs (PKCs) and certain inositol phosphates stimulate the
mobilization of calcium from intracellular stores.
[0048] Previously-described mutants capable of switching signaling
from a non-q pathway to a q pathway comprised C-terminal
alterations that appeared to switch signaling specificity from Gi
to Gq (see B. R. Conklin et al., Nature 363: 274-276, 1993). More
recently, C-terminal alterations that appeared to switch signaling
specificity from Gs to Gq were described (see B. R. Conklin et al.,
Molecular Pharmacology 50: 885-890, 1996).
[0049] Published reports describing N-terminal alterations of
Gq.sub..alpha.indicated that while the first six amino acids could
be deleted without destroying the ability of the Gq to transduce
its normal signal, alterations that impacted the cysteine residues
(at positions 9 and 10) tended to disrupt both receptor and
effector interaction (see, e.g., P. B. Wedegaertner, J. Biol.
Chem., 268: 25001-25005, 1993; J. R. Hepler et al., J. Biol. Chem.,
271: 496-504, 1996; and M. D. Edgerton et al., FEBS Letters 354:
195-199, 1994).
[0050] We have made the surprising discovery that N-terminal
alterations of Gq.sub..alpha.subunits can be used to render Gq
relatively promiscuous with respect to upstream signaling events in
that such Gq proteins incorporating N-terminally altered
Gq.sub..alpha.subunits acquire the ability to be activated by one
or more non-Gq receptors, and yet still retain the downstream
signaling capacity that is characteristic of the Gq receptor. Such
N-terminal alterations in Gq.sub..alpha.can also be combined with
C-terminal alterations to further modulate the ability of the
mutant G proteins to engage in "cross-talk" with non-q GPCRs as a
means of shunting non-q signaling to q-type effector events (such
as calcium mobilization) that are amenable to high throughput
screening.
[0051] The present invention provides compositions and methods by
which non-q upstream signaling events (such as those arising from
the activation of Gs-type or Gi-type GPCRs) are "shunted" to q-type
downstream signaling events (such as the mobilization of
intracellular calcium). Among other things, such compositions and
methods allow for the screening of non-q modulators (such as a
ligands or agonists capable of activating Gs-type or Gi-type GPCRs)
using rapid and efficient high throughput screening methods that
can be applied to the detection of downstream events such as
calcium mobilization.
[0052] One embodiment of this invention provides recombinant
nucleic acids encoding N-terminally altered .alpha..sub.q subunits,
which render the altered Gq protein relatively promiscuous in that
it acquires the ability to interact with one or more non-Gq-coupled
receptors (such as Gs-type or Gi-type GPCRs). Whereas these
promiscuous Gq mutants result in the ability to associate with one
or more non-Gq receptors, they still retain the downstream
signaling capacity that is characteristic of the Gq receptors (e.g.
the ability to transmit signals from an activated receptor to
downstream effectors such as PLCs, resulting in an augmentation of
inositol phosphate levels and subsequent calcium mobilization). As
a result, such altered G protein subunits (and the heterotrimeric G
proteins into which they are incorporated) can be used to shunt
non-Gq signaling (which is somewhat difficult to measure in a high
throughput screening context) into Gq-type downstream signaling
that can be readily measured using techniques that are amenable to
high throughput assays and robotic screens.
[0053] Preferred examples of such altered .alpha..sub.q subunits
comprise one or more alterations affecting the amino terminus,
preferably a deletion, substitution and/or insertion that affects
one or more of the first 10 amino acids, more preferably affecting
one or more the first six amino acids, most preferably a mutation
that deletes or disrupts the first six amino acids. As described
below, we have shown that such N-terminally altered .alpha..sub.q
subunits can be used to allow Gq to associate with, and transduce
the signal of, other GPCRs (such as Gi-type and Gs-type GPCRs).
[0054] Additional alterations can be introduced at the N-terminal
region and/or the C-terminal region to further influence the signal
transduction and/or to modulate the localization, activity or
stability of the G protein. By way of illustration, we describe
mutants below in which the first 6 amino acids have been removed,
and adjacent amino acids have been switched (from q-type to
non-q-type). We also describe mutants in which N-terminal
alterations have been combined with C-terminal switches.
[0055] It appears that the cysteine residues at position 9 and 10
of .alpha..sub.q can affect both receptor and effector interaction.
In particular, it has been reported that the loss of both cysteine
residues resulted in the impairment of both receptor interaction
and effector function (see, e.g., J. R. Hepler et al., J. Biol.
Chem., 271: 496-504, 1996; and M. D. Edgerton et al., FEBS Letters
354: 195-199, 1994). In our presently preferred examples, at least
one of these cysteine residues is retained in the .alpha..sub.q
mutants. However, Gi-type G proteins are generally myristoylated at
their amino termini (id.), which can be useful in connection with
the .alpha..sub.q mutarits of the present invention, particularly
where increasing cross-talk with the Gi-type GPCRs is desired.
[0056] In a first exemplary embodiment, a nucleic acid of an
.alpha..sub.q mutant encodes a truncated protein lacking the first
six N-terminal amino acids (hereafter referred to as the "6q"
mutant) (see Example 1 and FIG. 2A). We have found that such
truncation at the Nterminus confers receptor coupling promiscuity
to the G protein, which apparently results from an acquired ability
to respond to Gi-type and Gs-type GPCRs. Such altered G proteins
can be recognized and activated by, for example, GPCRs of the Gi/o
family (such as m2 muscarinic receptors and D2 dopamine receptors),
and GPCRs of the Gs family (such as D1 dopamine receptors, V2
vasopressin receptors and P2-adrenergic receptors), as described
below.
[0057] Another exemplary embodiment of this invention provides
nucleic acids encoding chimeric .alpha..sub.q subunits, in which
the N-terminal sequences are replaced with corresponding sequences
from non-q-type G proteins (such as Gi-type or Gs-type proteins).
The new N-terminal domain derived from other families of
G.sub..alpha.subunits allows the mutant to associate with certain
GPCRs in addition to G.sub.q-type GPCRs, while nevertheless
retaining the ability to engage in q-type downstream signaling
(e.g. through the PLC pathway). An exemplary nucleic acid encodes a
chimeric .alpha..sub.q mutant whose first N-terminal six amino
acids are removed, and the next four residues are replaced with the
corresponding .alpha. residues (hereafter referred to as "i4q").
Alteration of the N-terminal 10 amino acids allowed coupling to
G.sub.i-coupled m2 muscarinic receptor which normally does not
interact with G.sub.q. For instance, in the presence of i4q, the m2
muscarinic receptor exhibited the ability to productively couple to
.alpha..sub.q as indicated by an increase in inositol phosphate
level.
[0058] N-terminal mutations can also be made in combination with
C-terminal alterations such as those previously described by
Conklin et al. (see B. R. Conklin et al., Nature 363: 274-276,
1993; and B. R. Conklin et al., Molecular Pharmacology 50: 885-890,
1996). In the first report (Nature 1993), C-terminal chimeras were
generated by replacing C-terminal amino acids of .alpha..sub.q with
the corresponding residues of .alpha..sub.12 to create
.alpha..sub.q/.alpha..sub.i chimeras that can mediate stimulation
of phospholipase C by receptors otherwise coupled exclusively to
G.sub.i. A minimum of four .alpha..sub.12 amino acids from the
C-terminus sufficed to switch the receptor of the
.alpha..sub.q/.alpha..sub.12 chimeras. However, extensive
substitutions beyond 10 residues from the C terminus reduced the
ability of the chimeras to mediate a productive coupling with
G.sub.1-linked receptors, possibly due to disruption of the
tertiary structure of the proteins. Thus, N-terminally altered
proteins as described herein can be further modified at the
C-terminus according to the scheme outlined by Conklin et al. to
generate a host of double mutants (also referred to as "double
chimeras"). As described below, such double chimeras exhibit
significant improvement relative to the C-terminal chimeras.
Namely, they are better at activating phospholipase C by receptors
which have been previously shown to activate the C-terminal
chimeras, and by additional receptors that the C-terminal chimeras
apparently did not recognize.
[0059] An exemplary double chimera of the .alpha..sub.q subunit
(hereafter referred to as "i4qi4") has the first six residues
removed, the next four N-terminal residues changed to
G.sub..alpha.i (as in i4q), as well as four residues at the
C-terminus changed to G.sub..alpha.i residues. In the presence of
i4qi4, m2 muscarinic receptor can activate PLCs to a greater
extend. In addition, the somatostatin 1 receptor, which did not
interact with C-terminal chimeras, did recognize i4qi4 and mediated
a strong PLC response.
[0060] The subsequent report by Conklin et al. (in Molecular
Pharmacology 50: 885-890, 1996) demonstrated switching based on
C-terminal alterations from .alpha..sub.q to .alpha..sub.s, which
can also be employed in the context of the present invention.
[0061] Nucleic acids encoding G proteins and GPCRs as described
herein can be derived from native sources, including both
vertebrates and invertebrates, plants or fungi. Preferably, the
nucleic acid is from vertebrates, most preferably from a
mammal.
[0062] The invention also encompasses nucleic acids encoding for
functionally equivalent variants and derivatives of .alpha..sub.q
mutants. For instance, changes in a DNA sequence that do not change
the encoded amino acid sequence, as well as those that result in
conservative substitutions of amino acid residues, and substitution
of amino acid residues by amino acid analogs are those which will
not significantly affect properties of the encoded polypeptide.
Nucleotide substitutions that do not alter the amino acid residues
encoded can be useful for optimizing gene expression in different
systems.
[0063] Uses of nucleic acids encoding .alpha..sub.q mutants include
one or more of the following: producing mutated .alpha. subunits
which can he used, for example, for structure determination, to
assay a molecule's activity on a GPCRs, and to screen for molecules
useful as diagnostics and/or therapeutics. The .alpha..sub.q mutant
can also be expressed as a fusion protein in order to facilitate
its purification and/or identification.
[0064] As discussed herein, presently preferred G protein mutants
comprise promiscuous G.alpha..sub.q mutants having N-terminal
and/or C-terminal alterations that mediate shunting of non-q-type
upstream signaling events to q-type downstream effectors that can
be readily assayed using high throughput screening techniques.
However, analogous techniques can be used to generate promiscuous
mutants of other G protein a subunits. For example, a screening
technique applicable for assaying G.sub.12-type effector activity
could be applied to the screening for modulators of
non-G.sub.12-type GPCRs (such as Gs-, Gi- or Gq-type GPCRs) by
switching the corresponding N-terminal and/or C-terminal residues
of the G.alpha..sub.12 subunit to one of these other receptor types
using means analogous to those described herein.
[0065] Preparation of nucleic acids encoding .alpha..sub.q
mutants
[0066] The nucleic acids of this invention can be readily obtained
using chemical synthesis, recombinant methods, or PCR.
[0067] Methods of chemical polynucleotide synthesis are well known
in the art. For example, one of skill in the art can use the
sequence information provided herein and in gene banks such as
GenBank and a commercial DNA synthesizer to produce a desired DNA
sequence.
[0068] For preparing .alpha..sub.q mutant polynucleotides using
recombinant methods, a polynucleotide comprising a desired sequence
can be inserted into a suitable vector, and the vector in turn can
be introduced into a suitable host cell for replication and
amplification (see Example 1). Polynucleotides can be inserted into
host cells by any means known in the art. By way of illustration,
cells are transformed by introducing an exogenous polynucleotide by
direct uptake, endocytosis, transfection, or electroporation. Once
introduced, the exogenous polynucleotide can be maintained within
the cell as a nonintegrated vector (such as a plasmid) or
integrated into the host cell genome. The polynucleotide so
amplified can be isolated from the host cell by methods well known
within the art. See, e.g., Sambrook et al. (1989).
[0069] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art; see, e.g., PCR: The Polymerase
Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston
(1994).
[0070] RNA can be obtained by using the isolated DNA in an
appropriate vector and inserting it into a suitable host cell. When
the cell replicates and the DNA is transcribed into RNA, the RNA
can then be isolated using methods well known to those of skill in
the art, as set forth in Sambrook et al., (1989).
[0071] Vectors containing nucleic acids encoding .alpha..sub.q
mutants
[0072] The present invention further includes a variety of vectors
(i.e., cloning and expression vectors) having cloned therein
.alpha..sub.q mutant polynucleotide(s). These vectors can be used
for expression of recombinant polypeptides as well as a source of
.alpha..sub.q mutant polynucleotides. Cloning vectors can be used
to obtain replicate copies of the .alpha..sub.q mutant
polynucleotides they contain, or as a means of storing the
polynucleotides for future use. Expression vectors (and host cells
containing these expression vectors) can be used to obtain
polypeptides produced from the polynucleotides they contain.
[0073] Cloning and expression vectors generally contain a
selectable marker (for example, a gene encoding a protein necessary
for the survival or growth of a host cell transformed with the
vector), although such a marker gene can be carried on another
polynucleotide sequence co-introduced into the host cell. Only
those host cells into which a selectable gene has been introduced
will survive and/or grow under selective conditions. Typical
selectable marker genes encode protein(s) that (a) confer
resistance to antibiotics or other toxins substances, e.g.,
ampicillin, neomycyin, methotrexate, etc.; (b) complement
auxotrophic deficiencies; or (c) supply critical nutrients not
available from complex media. The choice of a suitable marker gene
will depend on the host cell, and appropriate genes for different
hosts are known in the art. Cloning and expression vectors also
typically contain a replication system recognized by the host.
[0074] Suitable cloning vectors can be constructed according to
standard techniques, or may be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected may vary according to the host cell intended to be used,
useful cloning vectors will generally have the ability to
self-replicate, may possess a single target for a particular
restriction endonuclease, and/or may carry genes for a marker that
can be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,
pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors
such as pSA3 and pAT28. These and many other cloning vectors are
available from commercial vendors such as BioRad, Stratagene, and
Invitrogen. The Examples provided herein also provide examples of
cloning vectors.
[0075] Expression vectors generally are replicable polynucleotide
constructs that contain a polynucleotide encoding an .alpha..sub.q
mutant polypeptide of interest. The polynucleotide encoding the
.alpha..sub.q mutant polypeptide is operatively linked to suitable
transcriptional controlling elements, such as promoters, enhancers
and terminators. For expression (i.e., translation), one or more
translational controlling elements are also usually required, such
as ribosome binding sites, translation initiation sites, and stop
codons. These controlling elements (transcriptional and
translational) may be derived from oq polynucleotides (i.e., the
.alpha..sub.q gene), or they may be heterologous (i.e., derived
from other genes and/or other organisms). A polynucleotide sequence
encoding a signal peptide can also be included to allow an
.alpha..sub.q mutant polypeptide to lodge in cell membranes (or to
be secreted from the cell as a means of facilitating protein
purification). A number of expression vectors suitable for
expression in eukaryotic cells including yeast, avian, and
mammalian cells are known in the art. The Examples provided herein
contain examples of expression vectors for mammalian systems.
[0076] The vectors containing the polynucleotides of interest can
be introduced into the host cell by any of a number of appropriate
means, including electroporation, transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (where the vector is an infectious agent, such as
vaccinia virus). The choice of means of introducing vectors or
.alpha..sub.q mutant polynucleotides will often depend on the host
cell.
[0077] Host cells transformed with .alpha..sub.q mutant
polynucleotides
[0078] Another embodiment of this invention provides host cells
transformed with vectors having .alpha..sub.q mutant
polynucleotide(s) sequences, as described above. For protein
expression, eukaryotic host cells should be used. Among eukaryotic
hosts are yeast, insect, avian, plant and mammalian cells, as are
known in the art.
[0079] The host cells of this invention can be used, inter alia, as
repositories of .alpha..sub.q mutant polynucleotides and/or
vehicles for production of .alpha..sub.q mutant polynucleotides
and/or polypeptides. In certain embodiments, host cells expressing
.alpha..sub.q mutants are used in screening methods, described in
detail below.
[0080] Screening procedures for identifying agents which modulate
GPCR activities using .alpha..sub.q mutants
[0081] Compounds targeted to GPCRs have several uses including
diagnostic uses and therapeutic uses. In one embodiment,
G.alpha..sub.q mutants of the present invention can be used for
identifying or characterizing agents that modulate the activities
of non-G.sub.q-interactive receptors. These agents include agonists
and antagonists of GPCRs (sometimes collectively referred to as
ligands) as well as agents capable of modulating interactions
between the receptors and heterotrimeric G proteins.
[0082] While crude receptor signaling studies can be performed
outside of cells using, for example, reconstituted vesicles (see,
e.g., J. R. Hepler, J. Biol. Chem. 271: 496-504, 1996), it is
preferable to employ cell-based functional assays.
[0083] A preferred method for identifying GPCR modulators in a
tested sample generally includes the following steps: (a) providing
a host cell expressing, or preferably over-expressing, a GPCR of
interest and an altered Gq protein according to the present
invention, (b) exposing the cell to a test sample containing a
candidate GPCR modulator, and (c) detecting a q-type G protein
response within the host cell. Suitable conditions to allow binding
of agent to a receptor are physiological conditions wherein the pH
is maintained between 6 and 8, and the temperature is between about
20-40 degrees C. As used herein, the binding of ligand to a
receptor is understood to denote an interaction of a molecule with
the ligand-binding domain of a receptor, which may result in a
conformational change in topology of the receptor. The binding of
ligand to a receptor may either trigger (in the case of agonist) or
block (in the case of antagonist) a detectable intracellular
response that is normally associated with signal transduction via
q-type G proteins. Such q-type intracellular responses for use in
the present invention include (1) activation of phospholipase C
proteins; (2) increase in phosphatidylinositol (PI) hydrolysis; (3)
and increase in intracellular calcium. Antagonists can be detected
by their ability to prevent or limit these responses in cells that
have been treated with a known agonist. A signaling modulator may
also be identified by its ability to alter the interaction between
the receptor and heterotrimeric G protein.
[0084] Methods of measuring intracellular inositol phosphates are
well known in the art and are exemplified in Example 3. Briefly,
cell membrane phospholipids can be labeled by incubating host cells
with [.sup.3H] myo-inositol for 20-24 hours. Cells are then
stimulated with appropriate agonist. Cell extracts can be collected
and inositol phosphates separated by ion-exchange chromatography
(e.g., by using AG1-X8 in either the chloride- or formate-form;
when only IP.sub.3 levels are to be determined, the chloride-form
is preferably used, whereas the formate form can be used to resolve
the major inositol phosphates (IP.sub.3, IP.sub.2 and
IP.sub.1).
[0085] Measuring intracellular calcium fluctuation can be rapidly
accomplished with the use of calcium-sensitive fluorescent probes,
including but not limited to Fura-2, Fluo-3 and Calcium Green-1.
Changes of calcium level are reflected by a change in fluorescence
of these probes, which can be measured by a high throughput assay
that is adaptable to robotic processing. For example, host cells
loaded with fluorescent probes can be monitored by FLIPR (Molecular
Devices Corp.), an instrument capable of performing stimulation in
all 96 wells of samples contained in a microplate simultaneously,
and providing real-time measurement and functional data once every
second. Typically, the assay is completed in less than fifteen
minutes. Since more than a hundred 96-microplates can be read in a
day, nearly 10,000 different compounds can be tested for GPCR
agonist or antagonist activities by using the mutants of the
present invention to shift non-q-type signaling to q-type signaling
which can be measured in high throughput assays. A variety of cell
types, both adherent and non-adherent, can be used in FLIPR.
[0086] Another exemplary high throughput calcium assay involves
induction of a reporter gene operatively linked to a
calcium-responsive promoter. In this method, a calcium flux
resulting from the activation of GPCR turns on the promoter which
subsequently drives the expression of a reporter gene encoding a
protein with an enzymatic activity that can be easily detected,
preferably by a colorimetric or fluorescent assay. Commonly used
reporter proteins include: .beta.-galactosidase, .beta.-lactamase,
chloramphenicol acetyltransferase (CAT), luciferase, green
fluorescent protein (GFP) and its derivatives, among others.
Reporter proteins can also be linked to other proteins whose
expression is dependent upon the stimulation of GPCRs. An
illustrative example would be a fusion protein comprising
luciferase sequence in frame with the open-reading frame of nuclear
factor of activated T cells (NFAT). Since the transcription of NFAT
requires the co-activation of calcium and protein kinases C
signaling pathways acting downstream of GPCRs, an effective
coupling of heterotrimeric G protein to the receptors can then be
measured by assaying NFAT mediated luciferase activity. This system
has been employed to demonstrate a carbachol dependent luciferase
response in cells co-expressing muscarinic m3 receptor and chimeric
.alpha..sub.q mutant i5q (Boss et al., J. Biol. Chem., 271:
10429-10482, 1996). In practice of this method, a preferred host
cell is one of lymphoid or neuronal origin, such as Jurkat cells
and pheochromocytoma PC12 cells. However, the choice of host cells
is not limited to these two types, as NFAT and NFAT isoforms are
present in a variety of cells including endothelial and myeloid
cells (id.).
[0087] In a preferred embodiment, the method of screening agents
acting on GPCR signal pathways involves host cells co-expressing
the GPCR of interest and suitable G.alpha..sub.q mutant so as to
enhance the signal. Even more preferably, expression of GPCRs or
G.alpha..sub.q mutant is controlled by an inducible promoter,
numerous examples of which have been described in the art and are
generally available. If an agent elicits or alters an intracellular
response characteristic of G.sub.q-coupled receptor in a cell in
which the inducible promoter is activated, an observation that the
agent fails to elicit the same result in a cell in which the
inducible promoter is not activated provides confirmation that the
agent is affecting at least one step or aspect of GPCR function.
Conversely, if the response is also observed in a cell in which the
inducible promoter is not activated, then it can be assumed that
the agent is not necessarily acting solely via the GPCR signal
transduction pathway.
[0088] The invention also encompasses therapeutic agents identified
by the method provided by the invention. Active agents may be
selected from large collections or libraries of molecules (e.g.
combinatorial libraries, proprietary compound libraries held by
large drug companies, etc.). A vast array of compounds can be
synthesized via biological or biochemical means. The selected agent
can be a biological or chemical compound such as a simple or
complex organic or inorganic molecule, a peptide, a protein or an
oligonucleotide or oligopeptides. In addition, various natural
sources can provide compounds for screening, such as plant or
animal extracts, and the like.
[0089] In another embodiment of the invention, .alpha..sub.q
mutants can be used for detailed structure-function studies of
mutant receptors using the same procedures mentioned above for
identifying agents active at GPCRs.
[0090] The following Examples are provided to further assist those
of ordinary skill in the art. Such examples are intended to be
illustrative and therefore should not be regarded as limiting the
invention. A number of exemplary modifications and variations are
described in this application and others will become apparent to
those of skill in this art. Such variations are considered to fall
within the scope of the invention as described and claimed
herein.
EXAMPLE 1
[0091] Generation of a first N-terminally altered G.alpha.
mutant
[0092] As a first example of a mutant G.alpha..sub.q subunit having
an alteration affecting the N terminus, we constructed an
.alpha..sub.q subunit (called "-6q") missing the first six amino
acids.
[0093] To create a construct coding for a mutant G.alpha..sub.q
subunit lacking the first six amino acids (-6q), a pcDNAI-based
expression plasmid coding for murine WTq, (Strathmann, M. et al.
(1990) Proc. Natl. Acad. Sci. 87:9113-9117, Wedegaertner, P.B. et
al. (1993) J. Biol. Chem. 268:25001-25008) was used. To generate
the -6expression plasmid, a 78 base pair synthetic BamHI-FspI
fragment containing the desired deletion was used to replace the
corresponding sequence in the wild type plasmid. In both plasmids
(WTq and -6), the BamHI site of the pcDNAI polylinker was
immediately followed by the initiating ATG codon. Both plasmids
contained a short sequence coding for an internal hemagglutinin
(HA) epitope tag (DVPDYA) which replaced WTq residues 125-130
(Wedegaertner, P. B. et al. (1993) J. Biol. Chem. 268:25001-25008).
The presence of the epitope tag did not affect the receptor and
effector coupling properties of WTq (Conklin, B. R. et al. (1993)
Nature 363:274-276, Liu, J. et al. (1995) Proc. Natl. Acad. Sci.
U.S.A. 92:11642-11646, Wedegaertner, P. B. et al. (1993) J. BioL
Chem. 268:25001-25008). The identity of the two G protein
constructs was verified by dideoxy sequencing (Sanger, F. et al.
(1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467).
[0094] The -6mutant was employed in various signal transduction
studies as described below.
EXAMPLE 2
[0095] Expression of Receptor and G.alpha..sub.q Mutant Constructs
in Mammalian Host Cells
[0096] This example illustrates the introduction and expression of
G-protein-coupled receptor constructs and mutant G proteins in
mammalian host cells suitable for the analysis of G
protein-mediated signal transduction.
[0097] COS-7cells were grown in Dulbecco's modified Eagle's medium,
supplemented with 10% fetal calf serum, at 37.degree. C. in a
humidified 5% CO.sub.2 incubator. For transfections, 1 .times.
10.sup.6 cells were seeded into 100-mm dishes. About 24 h later,
COS-7cells were cotransfected with expression plasmids coding for
WTq or -6(1 mg DNA/dish) and the indicated receptor cDNAs (4 mg
DNA/dish), by using a DEAE/dextran procedure (Cullen, B. R. Methods
Enzymol. (1987) 152:684-704).
[0098] The following receptor expression plasmids were used: m2
muscarinic receptor in pcD (Bonner, T. I. et al. (1987) Science
237:527-532), D2 dopamine receptor (Grandy, D.K. et al. (1989)
Proc. Natl. Acad. Sci. U.S.A. 86:9762-9766) in pcDNAI, k-opioid
receptor (Zhu, J. et al. (1995) Life Sci. 56:201-207) in pcDNA3,
somatostatin SSTR1 receptor (Yamada, Y. et al. (1992) Proc. Natl.
Acad. Sci. U.S.A. 89:251-255) in pCMV, Al adenosine receptor
(Mahan, L. C. et al. (1991) MoL PharmacoL 40:1-7) in CDM7, D1
dopamine receptor (Zhou, Q. Y. et al. (1990) Nature 347:76-80) in
pcDNAI, V2 vasopressin receptor in pcD-PS (Schoneberg, T. et al.
(1996) EMBO J. 15:1283-1291), and .beta.2-adrenergic receptor
(Gocayne, J. et al. (1987) Proc. Natl. Acad. Sci. USA 84:8296-8300)
in pSVL.
[0099] Signal transduction (in response to GPCR agonists) in the
transformed cells was analyzed as described below.
EXAMPLE 3
[0100] PI Hydrolysis Assay in Transformed Cells
[0101] Approximately 24 h after transfections, cells were split
into six-well dishes (ca. 0.4.times.10.sub.6 cells/well) in culture
medium supplemented with 3 .mu.Ci/m1 [.sup.3H]myo-inositol (20
Ci/mmol; American Radiolabeled Chemicals Inc.). After a 24-h
labelling period, cells were preincubated for 20 min at room
temperature with 2 ml Hank's balanced salt solution containing 20
mM HEPES and 10 mM LiCl. Cells were then stimulated, in the same
buffer, with the appropriate agonist ligands (1 h at 37.degree.
C.), and increases in intracellular inositol monophosphate
(IP.sub.1 ) levels were determined by anion exchange chromatography
as described (Kostenis, E. et al. (1997) Biochemistry
36:1487-1495).
[0102] In a subset of experiments, transfected cells were incubated
with pertussis toxin (pertussis toxin 500 ng/ml) for the last 18-24
h of culture.
EXAMPLE 4
[0103] Receptor Coupling Properties of wild-type q (WTq) and -6
[0104] A series of receptors that are preferentially coupled to G
proteins of the Gi/o family (m2 muscarinic, D2 dopamine,
.kappa.-opioid, SSTR1 somatostatin, and Al adenosine) were
coexpressed in COS-7cells with either WTq or -6. Transfected cells
were then incubated with the appropriate agonist ligands, and the
ability of the different receptors to couple to the two G proteins
was determined by measuring increases in inositol phosphate
production (due to WTq-mediated activation of PLC.beta.; Smrcka, A.
V. et al. (1991) Science 251:804-807; Berstein, G. et al. (1992) J.
Biol. Chem. 267:8081-8088). Co-expression of the different
Gi/o-coupled receptors with either vector DNA (pcDNAI) or WTq,
followed by ligand stimulation, resulted only in a rather small
increase in PLC.beta. activity (FIG. 2A). As shown in FIG. 3 for
the m2 muscarinic and D2 dopamine receptors, this small increase in
inositol phosphate production could be almost completely blocked by
pretreatment of cells with pertussis toxin (500ng/ml). Consistent
with previous findings (Ashkenazi, A. et al. (1987) Science
238:672-675, Offermanns, S. et al. (1994) Mol. PharmacoL
45:890-898), this observation suggests that the m2 muscarinic and
D2 dopamine receptors do not couple to WTq to a significant extent
and that the small increase in PI hydrolysis seen after stimulation
of these receptors is most likely due to activation of PLC.beta. by
G protein .beta..gamma. subunits released upon receptor-mediated
activation of endogenous Gi/o proteins (Katz, A. et al. (1992)
Nature 360:686-689, Camps, M. et al.(1992) Nature 360:684-686).
[0105] However, co-expression of the different Gi/o-coupled
receptors with -6resulted in a significantly increased PI response
(as compared with WTq) which was most pronounced in the case of the
two biogenic amine receptors (m2 muscarinic and D2 dopamine; FIG.
2A). These responses could only be partly blocked by pertussis
toxin pretreatment (shown for the m2 muscarinic and D2 dopamine
receptors in FIG. 3), indicating that they were primarily due to
receptor-mediated generation of activated -6. Complete
concentrationresponse curves for m2 muscarinic and D2 dopamine
receptor-mediated activation of -6are given in FIG. 4. The EC50
values (means, S.E. of five independent experiments, each carried
out in triplicate) for these responses amounted to 7.4 .+-.0.3 mM
in the case of the muscarinic agonist, carbachol (FIG. 4A), and to
1.4 .+-.0.2 mM in the case of the dopaminergic ligand,
(-)-quinpirole (FIG. 4B), indicating that the interaction of the
two biogenic amine receptors with -6was highly efficient.
[0106] We also examined the ability of three Gs-coupled receptors
(DI dopamine, V2 vasopressin, and .beta.2-adrenergic) to
functionally interact with WTq or -6. Consistent with their known
coupling profiles (Watson, S. et al. (1994) in The G-Protein Linked
Receptor Facts Book (Watson, S., and Arkinstall, S., eds) pp.
1-291, Academic Press, London), these receptors were unable to
activate WTq to an appreciable extent (FIG. 2B). However, two of
the investigated G.-coupled receptors (D1 dopamine and
.beta.2-adrenergic) gained the ability to induce a significant
increase in inositol production when co-expressed with -6(as
compared with WTq; FIG.2B).
[0107] Our data therefore suggest that -6, in contrast to WTq, can
be activated by receptors that are members of all three major
functional classes of GPCRs. It should also be noted that
co-expressed -6did not improve coupling of the V2 vasopressin
receptor to PLC stimulation and that the absolute magnitude of
responses mediated by -6upon coexpression with the .kappa.-opioid
or the SSTR1 somatostatin receptor, respectively, was quite small
(FIG. 2), indicating that -6 is not universally promiscuous. One
possible reason for the observed differences in the ability of the
studied Gi/o- and Gs-coupled receptors to interact with -6 may be
that the relative functional importance of individual receptor/G
protein contact sites may vary among different GPCRs (e.g. peptide
receptors versus receptors for biogenic amines).
EXAMPLE 5
[0108] Expression Levels and Subcellular Distribution of
.alpha..sub.q Constructs
[0109] To exclude the possibility that the promiscuous nature of -6
was simply due to exceptionally high expression levels (as compared
to WTq), the subcellular distribution of the two G protein subunits
was studied by cell fractionation and immunoblotting.
[0110] Cells were fractionated into particulate and soluble
fractions as described (Degtyarev, M. Y. et al. (1994) J. Biol.
Chem. 269:30898-30903). The 12CA5mouse monoclonal antibody (BAbCo)
specific for the HA-epitope was used for immunoprecipitation and
immunoblotting. Immunoprecipitation studies were performed using
equivalent amounts of protein (2.5 mg) from total cell suspension
in solubilization buffer consisting of 50 mM Tris-HCl (pH 7.4), 150
mM NaCl, 1% (w/v) of Nonidet P-40, 0.5% (w/v) sodium deoxycholate,
and 0.1% (w/v) SDS in a total volume of 1 ml. Cell suspensions were
added to the solubilization buffer and incubated at 4.degree. C. on
a rotator for 30 min, followed by centrifugation for 10 min at
14,000 r.p.m. in an Eppendorf 5415 microcentrifuge to pellet
insoluble material. Supernatants were transferred to fresh
Eppendorf tubes, and 5 mg of 12CA5 antibody and 20 ml of a 50%
suspension of Protein A-agarose (Sigma) were added, followed by an
overnight incubation at 4.degree. C. on a rotator.
Immunoprecipitates were recovered by centrifugation at 1,000xg in a
microcentrifuge, washed twice in 1 ml solubilization buffer
containing {fraction (1/10)} of the original detergent
concentration, solubilized in 45 ml of gel sample buffer (Novex) in
the absence of reducing agents, boiled for 5 min, separated by
SDS-PAGE on 10% Tris-Gly gels (Novex), and prepared for
fluorography using Amplify (Amersham) according to the
manufacturer's instructions.
[0111] Fluorograms were exposed for four to six weeks at
--70.degree. C. Immunoblotting was performed by separating equal
amounts of protein (100 mg) from subcellular fractions solubilized
in gel sample buffer (Novex) with 2.5% (v/v) P-mercaptoethanol on
10% TrisGly gels (Novex), transfer to nitrocellulose membranes,
probing with the 12CA5 antibody conjugated to horse radish
peroxidase (Boehringer Mannheim), and development with enhanced
chemiluminescence reagents (Amersham). Protein concentrations were
determined using the Bio-Rad protein assay kit with IgG as the
standard.
[0112] Both G.alpha. subunits were detected with the monoclonal
antibody, 12CA5, which recognizes the internal HA-epitope tag
present in both proteins (see Example 1). As shown FIG. 5A, both G
protein constructs were found exclusively in the particulate
fraction; however, -6was expressed at lower levels (approximately
10-20% of WTq, as determined by scanning densitometry; data not
shown). The precise reason for this latter observation remains
unclear; however, possible factors may include reduced protein
stability or translation efficiency. In any case, these data
exclude the possibility that the ability of -6to be activated by
multiple classes of GPCRs is due to overexpression of this subunit
(as compared to WTq).
[0113] Moreover, they indicate that even relatively reduced levels
of a promiscuous G.alpha. mutant can be used to effectively shunt
GPCR signal transduction from non-q-type upstream pathways (such as
those mediated by Gs-type and Gi-type GPCRs) to q-type downstream
pathways (which are readily measurable using high throughput
screening techniques).
EXAMPLE 6
[0114] Palmitoylation Pattern of WTq and -6
[0115] Previous studies have shown that G proteins of the
G.alpha..sub.q family are palmitoylated at cysteine residues
located near the N-terminus of the proteins (corresponding to Cys9
and Cys10 in FIG. 1) Edgerton et al. (1994) FEBSLett. 354:195-199;
McCallum et al. (1995) Biochem. J. 310:1021-1027. To compare the
palmitoylation patterns of WTq and -6, transfected COS-7cells were
metabolically labeled with [.sup.3H]palmitic acid, followed by
immunoprecipitation of WTq and -6by the 12CA5 monoclonal antibody,
SDS-PAGE, and fluorography.
[0116] As shown in FIG. 5B, WTq and -6were the only
immunoprecipitated proteins, since no labeled proteins could be
precipitated when cells were transfected with "empty" vector DNA.
Consistent with published results (Edgerton et al. (1994) FEBS
Lett. 354:195-199; McCallum et al. (1995) Biochem. J.
310:1021-1027), both WTq and -6(16) incorporated considerable
amounts of [.sup.3H]palmitate (FIG. 5B). The reduction in signal
strength seen with -6correlated well with the reduction in -6levels
revealed by immunoblotting (FIG. 5A), as discussed above. This
observation suggests that -6is palmitoylated to an extent similar
to that of WTq. It is therefore unlikely that differences in
palmitoylation patterns are responsible for the different
functional properties of -6and WTq.
EXAMPLE 7
[0117] Generation of Additional N-Terminal G.alpha..sub.q
Mutants
[0118] As an illustrative example of another G.alpha..sub.q mutant,
we used techniques analogous to those described above to construct
an additional altered G.alpha..sub.q subunit (referred to as "i4q")
in which the first six N-terminal residues are deleted, and the
next four residues (i.e. residues 7-10) were replaced with residues
corresponding to residues 1-4 of a G.alpha..sub.q subunit.
[0119] Signal transduction studies conducted with this mutant
confirmed that it also acquired the ability to shunt non-q-type
upstream signaling to q-type downstream effector function.
EXAMPLE 8
[0120] Generation of Double G.alpha..sub.q Mutants Having Both
N-terminal and C-terminal Alterations
[0121] As an illustrative example of a double G.alpha..sub.q mutant
having both N-terminal and C-terminal alterations, we used
techniques analogous to those described above to construct an
additional altered G.alpha..sub.q subunit (referred to as "i4qi4")
in which the first six N-terminal residues are deleted, and the
next four residues (i.e. residues 7-10) were replaced with residues
corresponding to residues 1-4 of a G.alpha..sub.i subunit; and the
C-terminal region was altered by replacement of the last four amino
acids with corresponding residues from the C-terminal region of
G.alpha..sub.i.
[0122] Signal transduction studies conducted with this double
mutant confirmed that it also acquired the ability to shunt
non-q-type upstream signaling to q-type downstream effector
function, and that it appeared to be even more effective at signal
transduction than the single N-terminal mutants described
above.
* * * * *