Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF ASSAYING COMPOUNDS WHICH AFFECT THE A(~ l lVl'l ~ OF
G PROTEIN-COUPLED RECEPrORS BASED ON MEASUREMENT OF
RECEPTOR OLIGOl~'IF.~T7.~TION
5 FIELD OF THE INVENTION
The present invention relates to a method of assaying compounds for the ability to modulate
the function of G protein-coupled receptors based on mea~ulen~ of rec~Lol oligol~ Lion
state.
BACKGROUND OF THE INV~NTION
The class of receptors known as G protein-coupled receptors (GPCRs) are typically
characterized by a 7-helix org~ni~ on~ whereby the receptor protein is believed to traverse
15 the "Rl~l~le seven times. They also share a common sign~lling m~orl~ ll, whereby signal
tr~n.ccl~ction across the membrane involves intracellular tr~n~ cer elements known as G
plu~ s. When a chemir~1 m~ nger binds to a specific site on the extracellular surface of
the rec~Lor, the conru~lndLion of the rec~lor changes so that it can interact with and activate
a G protein. This causes a molecule, guanosine diphosphate (GDP), that is bound to the
2 o surface of the G protein, to be replaced by another molecule, guanosine triphosphate (GTP),
triggering another co,lro",~tional change in the G protein. When GTP is bound to its surface,
the G protein regulates the activity of an effector. These effectors include enzymes such as
adenylyl cyclase and phospholipase C, channels that are specific for calcium ions (Ca ),
+ +
p~L~ssium ions (K ), or sodium ions (Na ) and certain l~ po~l plvtei~s.
In general, activation of GPCRs by tr~nc.,.i~e,s will induce one or another of the following
effector lespo~ses: activation of adenylyl cyclase, inhibition of adenylyl cyclase or stim~ tion
of phospholipase C activity. When the effector adenylyl cyclase is either activated or inhibited
it produces changes in the concentration of the molecule cyclic adenosine monophosphate
3 o (cAMP). Another erreclol~, phospholipase C, causes one molecule of phosphatidylinositol-
bisphosphate (PIP2) to be cleaved into one molecule each of inositol triphosphate (IP3) and
diacylglycerol (DAG); IP3 then causes calcium ions (Ca ) to be released into the cytoplasm.
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Alterations in cellular levels of cAMP and Ca are two of the most important intracellular
messages that in turn act to alter the behaviour of other target proteins in the cell.
GPCRs may be classified according to the type of sign~lling ~alhwdy they activate in cells.
5 This occurs at the level of the G proteins, which detect and direct signals from diverse
r.,ce~ to the a~loplial~ effector-response pathway. The three main groups of G prO~t~lllS
are: Gs-like, which m~ tP adenylyl cyclase activation; Gi-like, which m~Ai~te inhibition of
adenylyl cyclase; and Gq-like, which mP~ te activation of phosphoplipase C. Since one
receptor can activate many G proteins, the signal can be greatly amplified through this signal
10 tr~n~d~ction palllwdy~
A wide variety of ~ rnir~l messengers involved in regulating key functions in the body act
through GPCRs. These include n~ulot~ rs such as dop~"ine, acetylcholine and
serotonin, hormones of the endocrine system such as so-~to~lhli-~, glucagon and~15 adrenocorticotropin, lipid mediators such as prost~gl~n-lin.~ and leukotrienes, and
odulatory ploleills such as interleukin-8 and monocyte-rh~ ttractant polypeptide.
The family of GPCRs also includes the receptors for light (rhodopsin), for odours (olfactory
receptol~) and for taste (gustatory receptors). Over one hundred different G protein-coupled
receptors have been i(1entified in humans, and many more are expected to be discovered. All
2 o or most of these receptors are believed to utilize one of the three principal G protein-effector
sign~lling paLhwdys (stimul~tion or inhibition of adenylyl cyclase or activation of
phospholipase C).
Examples of G Protein-Coupled N~ olldllsmitter Receptors
Inhibits AC Stim~ tes AC Stim~ tes PLC Neuiotl~ )ille
m2, m4, m" m3, mS Acetylcholine
A" A2 Adenosine
CRF-R Corticotropin-Releasing Factor
3 0 Rc Cannabinoids
D2 D" Ds Dopamine
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H2 Histamine
Y" Y2. Y3 Neuropeptide Y
a~-AR ~2-AR cc,-AR Norepinephrine, epinephrine
- ~, O, K K Opioids
S-HT,A, 5-HT,B S-HT4 S-HT2 Serotonin
S-HT",
The development of a method of testing compounds for their abilities to affect GPCRs has
great utility for many industries whose goal is to develop chemical compounds that interact
0 with GPCRs. Since GPCRs are ubiquitous and widely used in nature to transmit cellular
signals, this invention has utility for different industries including: the pharmaceutical
industry, the pest-control industry, the aquaculture industry, the food industry and the
fragrance industry.
5 In view of the diverse functions of G protein-coupled receptors in the human body, it is not
surprising that the pharmaceutical sector has great interest in the development of new drugs
which target G protein-coupled receptors for potential therapeutic applications in a wide range
of human pathologies, including psychiatric disorders (depression, psychoses, bipolar
disorder), metabolic disorders (diabetes, obesity, anorexia nervosa), cancer, autoimml-ne
20 disorders, cardiovascular disorders, neurodegenerative disorders (Alzheimer's disease) and
pain disorders.
The process of discovering and developing new therapeutic drugs which act on G protein-
coupled receptors involves the systematic testing of drug candidate compounds in biological
2 5 assay systems which contain the targeted G protein-coupled receptor in a functional state. The
goal of this testing is to identify those compounds, among a very large number of c~n~ rrs,
which can modulate the function of the targeted G protein-coupled receptor in a predictable
and therapeutically-relevant manner. Most assay systems used for drug screening classify
compounds into three broad categories: 1) inactive, i.e. the compounds have no effect on
3 o receptor function at relevant doses; 2) agonists, i.e. the compound mimics the natural cllrmir~
mrssenger by activating the receptor; and 3) antagonists, i.e. the compound inhibits receptor
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In general, two types of assay system are used by the pharmaceutical industry for screening
compounds which target G protein-coupled receptors: ligand binding assays and functional
5 bioassays.
The ligand binding assay detects compounds that can interact with and bind to the receptor at
the same site as the natural chemical messenger. This usually involves the use of radioactive
derivatives of either the natural chemical messenger or of known drugs which bind to the same
0 receptor site, and measurement of the ability of test compounds to block the binding of the
radioactive drug to the targeted receptor present in a biological preparation (e.g. a tissue
extract). In addition to detecting compounds which bind to the receptor, the radioligand
binding assay also permits the ranking of compounds based on binding affinity, i.e. the
concentration of the compound which results in occupation of half of the receptors in the
15 preparation. In general, the lower the concentration of compound necessary to occupy half
of the sites (i.e. the higher the affinity), the better the candidate. Radioligand binding assays,
while widely employed in the first steps of drug screening, have a number of limitations, the
most severe being the inability of this assay to discriminate between agonists and antagonists.
20 The functional bioassay tests the effect of the compounds on receptor activity, i.e. the ability
of the receptor to transmit signals across the cell membrane to control cellular response
pathways. Since G protein-coupled receptors control a wide spectrum of cellular functions,
the functional bioassays used in drug screening for G protein-coupled receptors include a large
variety of different tests which monitor any one of a series of biochemical or cellular processes
2 5 which are under the control of receptor activity. These assays all permit the discrimination of
agonists from antagonists, i.e. agonists will activate receptor sign~lling pathways, while
antagonists will block activation of sign~llin~ pathways by receptor agonists (such as the
natural chemical transmitter). In addition, most functional hioassays can also rank agonist
compounds based on efficacy, i.e. the maximum level of activation of the sign~lling pathway
30 achieved by the agonist compound. Full agonists result in full activation of the receptor-
controlled process, whereas partial agonists can induce only fractional activation of the
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receptor-controlled process even at full receptor occupancy.
All of the functional bioassay systems ~;ullelllly used to screen drugs for GPCRs have a
- cornmon element in that they all rely on the measurement of post-receptor processes as an
5 indication of the direct effect of the compound on the receptor. Examples of functional
bioassays performed to test the effect of compounds on the activity of G protein-coupled
receptors cover the full spectrum of receptor-controlled processes including: (1) activation of
G proteins in cell membrane preparations by measuring the rate of binding of the guanyl
nucleotide analogue GTPrS35 or the hydrolysis of GTP; (2) modulation of effector activity
0 as a result of G protein activation, e.g. activation or inhibition of adenylyl cyclase activity or
activation of phospholipase activity; (3) modulation of post-effector sign~lling proteins, such
as kinase and phosphatase enzymes. ion channels, transcription factors, etc.; (4) modulation
of integrated cellular responses such as secretion (e.g. for glandular cells), contraction (e.g.
for smooth muscle), electrical activity (e.g. for neurons), growth and proliferation (e.g. for
5 endothelial cells) .
All of these functional bioassays require the use of cells and/or cellular preparations that have
a high degree of biological integrity. Until recently functional bioassays relied on the use of
animal tissues. With the enormous progress in the cloning, sequencing and expression of genes
2 o which encode G protein-coupled receptors (and drug target proteins in general) there has been
a major shift from the use of animal tissues to using recombinant receptors. Recombinant
receptors are produced by expression of the cloned gene in cultured cells. In those cases
where the receptor cDNA has been isolated and cloned such recombinant receptors are now
the principal source of receptors for drug screening in ligand binding assays and in functional
25 bioassays. The use of recombinant receptors has many advantages over tissue sources,
including the ability to use human receptors expressed from human genes, the facility with
which large amounts of the protein can be produced, and the fact that a single receptor subtyp e
can be tested and compared against closely related subtypes (receptor subtypes are receptors
that are closely related but distinct, yet which use the same natural tr~n~mittt~r).
Bioassay systems for recombinant G protein-coupled receptors that are known in the art are
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based on the ability of the expressed receptor to activate endogenous si_n~ling pathways in
the host cell. Early assays measured the activity of effectors (adenylyl cyclase and
phospholipase C) using known biochemical assays originally used for tissue-based assays.
These generally employ m~mm~ n cell lines which have been made to express the cloned
5 receptor DNA using techniques (eg. transfection, transformation) which are well known to and
routinely practised by technicians trained in the art. One example uses fluorescent dyes
sensitive to the concentrations of specific ions~ primarily calcium, to measure changes in the
intracellular ion concentrations associated with activation of receptors coupled to Gq-
phospholipase C sign~lling.
An increasing number of new assay systems involve genetic engineering of the host cell to
facilitate measurement of the effector response to receptor activation. In gene reporter assays,
the gene for an enzyme that is readily assayed, such as beta-galactosidase, is inserted into the
host cell genome under the control of a gene promoter element which is normally under the
15 control of a receptor sign~lling pathway. Receptors which activate the specific sign~lling
pathway (will activate expression of the beta-galactosidase reporter gene. Measurement of
the enzyme activity in a simple assay thus provides a measure of receptor activity and provides
a functional bioassay to monitor the activity of compounds on the receptor. A variation of thi s
type of assay uses the yeast Saccharomvces cerevisiae as a microbial host cell to express
20 human G protein-coupled receptors which are coupled to an endogenous yeast sign~lling
pathway controlling the response to sex pheromones. In this case, the receptor activates a yeas t
promoter which in turn controls the expression of a reporter enzyme (e.g. beta-galactosidase).
Another approach is to express the receptors in specialized cells that have endogenous respons e
2 5 mtocll~ni.~m~ that allow convenient assay of ligand activation of the receptor. For example,
receptors that change cAMP levels have been cloned in melanophores ~cultured pigment cells)
wherein altered cAMP levels alter cellular colour, a response that is a conveniently measured
response (Potenza et al., 1992, Anal. Biochem. 206:315). The limitations of these types of
assays are that only certain functional types of receptors can be measured, in addition to
3 o limitations in the endogenous responses and cells that are used.
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Another assay strategy draws upon the observation that a wide diversity of receptors are able
to alter the pH of the me(lium that is used for cell culture when exposed to ligands. These pH
changes are small in magnitude and therefore require expensive instrumentation for
measurement (Cytosensor, Molecular Dynamics Co.). Moreover, samples must be inrllb~tecl
5 within the instrument for several minutes which severely limits sample throughput.
Yet another assay strategy is based upon the ability of certain receptors to alter cellular
growth. Cells of the NIH 3T3 fibroblast cell line have been extensively used to evaluate the
activity of a large diversity of gene products that control cell growth, and a number of
10 receptors are able to control the activity of these cells when stimul:~tt~d by individual ligands.
For example, carbachol (a muscarinic agonist) stim~ tes cells transfected with certain
muscarinic receptors (Gutkind et al., 1991, Proc. Natl. Acad. Sci. USA, 88:4703; Stephens
et al., 1993, Oncogene, 8:19), and Norepinephrine stim~ tes cells transfected with certain b-
adrenergic receptors (Allen el al., 1991, Proc. Natl. Acad. Sci. USA, 88:11354). In the
5 course of long-term stimulation with agonist ligands, several characteristics of the cells are
altered, including cellular growth, loss of contact inhibition, and formation of macroscopic
colonies termed foci. Proprietary methods have been developed in the art in order to facilitat e
detection of such foci.
20 All of the functional bioassay systems used in screening share the common point that they
measure post-receptor events~ i.e. processes which are under the control of the receptor. This
has several major drawbacks: (1) the measurement of compound efficacy is indirect and
therefore subject to artifacts of the particular assay system used because post-receptor events
may be modulated in unpredictable ways by unrelated cellular processes (e.g. other sign~lling
25 pathways); (2) since the family of GPCRs is divided into functionally distinct groups, the
particular functional bioassay will be limited in utility to a subset of the receptor family; (3)
the functional bioassay systems based on post receptor events rely on complex biological
systems which require a high degree of biological integrity to function effectively and they are
therefore expensive and unstable.
Thus, there is great need for functional bioassays to assay compounds as potential drugs
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affecting GPCRs which is simple, which is broadly applicable to functionally distinct receptors
and which permits the direct measurement of compound efficacy on the receptor itself.
Accordingly, it is an object of this invention to provide a functional bioassay method for
assaying c~n~ e drugs in virro for their activity on GPCRs which meets these criteria. The
5 assay system is based on the ability to monitor directly compound efficacy on GPCRs through
a novel indicator of receptor activity: receptor oligomerization.
The background information is provided for the purpose of making known information
believed by the applicant to be of possible relevance to the present invention. No admission
10 is necessarily intended, nor should be construed, that any of the preceding information
con~tit~-tes prior art against the present invention. Moreover, publications referred to in the
following discussion are hereby incorporated by reference in their entireties in this application.
SUMMARY OF THE INVENTION
These objects are accomplished by the use a novel method of assaying compounds in vitro for
their ability to interact with and modulate the functional properties of GPCRs. In particular,
a techniques are described constituting a new method of testing compounds for their abilities
to alter the relative amounts of monomeric and multimeric receptors. The invention is based
2 0 on the discovery that GPCRs undergo reversible association-dissociation between monomeric
and multimeric states (homo-multimers or hetero-multimers) as a normal part of their activity
cycle, and that drugs affect this process, both in nature and extent, in a predictable marmer.
By using techniques generally known in the art to measure the relative amounts of monomeric
and oligomeric receptors in a receptor l lepa,a~ion, the method of this invention permits direct
2 5 measurement of the pharmacological efficacy of drug c~n~ tes at GPCRs.
TABLES AND FIGURES
While the specification concludes with claims particularly pointing out and distinctly claiming
3 0 the subject matter regarded as forming the present invention, it is believed that the invention
will be better understood from the following plef~lled embodiments of the invention taken in
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connection with the accompanying drawings in which:
Figure 1 shows immunoblotting of human ~2AR expressed in Sf9 cells. Crude membrane
preparations (lane 1), digitonin-solubilized membrane proteins (lane 2) and afflnity puri~led
5 receptors (lane 3) derived from Sf9 cells expressing either c-myc tagged (lane 3) or HA-tagged
(lanes 1 and 2) ~2AR were immunoblotted following SDS-PAGE using the appropliateantibody (9E10 and 12CAS, respectively). The blots reveal imrnunoreactive bands
corresponding to the expected monomeric form (43-50kDa) as well as a higher molecular
weight species (85-95 kDa). The right panel illustrates immunoblots of crude membrane
10 preparations derived from Sf9 cells expressing HA-tagged ~2AR treated (lane 5) or not (lane
4) with the membrane-permeant photoactivatible crosslinker BASED. Position of receptor
bands are denoted by arrows and molecular weight markets are as shown.
Figure 2 shows effects of various peptides and ~2AR ligands on receptor dimerization. Co-
15 imml~nnprecipitation of ~2ARs bearing two different immllno}ogical tags. Lanes 1 and 2: c-
myc (lane 1 ) or anti-HA (lane 2) mAbs . The two imm~lnQprecipitates were then
immunoblotted with the anti-HA mAb. The occurrence of dimerization between the HA- and
c-myc-tagged receptors is revealed by the fact that the HA-tagged ~2AR is co-
im~nunoprecipitated with the c-myc tagged receptor by the anti-c-myc rnAb (lane 1). Lanes
2 o 3 and 4: c-myc tagged ~2AR was expressed in Sf9 cells and immlmoprecipitated with anti-c-
myc mAb. The immunoprecipitates were then immunoblotted with either anti-HA (lane 3) or
anti-c-myc or anti-c-myc (lane 4) rnAbs. Lanes 5 and 6: HA-tagged ~2AR was expressed in
Sf9 cells, immllnoprecipitated with anti-HA rnAb and then immunoblotted with either anti-c-
myc (lane 5) or anti-HA (lane 6) mAbs. These controls demonstrate the specificity of each
25 antibody towards their respective targets. Lane 7 and 8: HA-tagged ~2AR and c-myc tagged
M2 muscarinic receptors were co-expressed in Sf9 cells, immunoprecipitated with either anti-
HA (lane 7) or anti-c-myC (lane 8) mAbs. Immunoblotting with the anti-c-myc mAb did not
reveal the presence of a ~2AR/M2 muscarinic receptor heterodimer (lane 8). Results shown
are representative of three separate experiments.
Figure 3 demonstrates immllnnblotting of V2-vasopressin receptors (V2-R) expressed in COS-
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7 cells. Crude membrane preparalions from COS-7 cells transientlv transfected with c-mvc
tagged V2-R (lane 1 ) or c-mvo tagged V2-R truncation mutant 0-11 (lane 2) were
immunoblotted with the anti-c-1nvc mAb. The molecular weight markets are as shown.
Square brackets highli~ht the dimeric species of both wildtype and O-ll V2 vasopressin
5 receptors while asterisks denote the monomeric species. Data are representative of three
independent experiments.
Figure 4 shows effects of various peptides on receptor dimerization. A, Time course of the
effect of the TM VI peptide on ~2AR dimerization. Membranes derived from S~9 cells
expressing ~2AR were treated at room te~ ,eld~ule with TM VI peptide [residues 276-296:
NH ,-GIIMGTFTLCWLPFFIVNIVH-COOH] at a concentration of 0.15 ug/,uL for 0 (lane 1) ,
lS (lane 2), 20 (lane 3) or 30 minutes (lane 4). Membranes were then subjecled to SDS-
PAGE, transferred to nilrocellulose and immunoblotted with the ami-c-mvc antibody. A
representative immnnnblot is shown. B, Densilometric analvses of three experiments similar
to that shown in the Figure 4A demonstrating the effects of treatment for 30 minutes with
either vehicle (CON, lane l ), TM VI peptide (TM VI, lane 2) TM VI-Ala [NH 2-
AIIMATFTACWLPFFIVNIVH-COOH] (TM VI-Ala~ lane 3), or D2 dopamine receptor TM
VII peptide [residues 407-426 NH2-YIIPNVASNVYGLWTFASYL-COOH) (D2 TM VII, lane
4). All peptides were used at a concentration of 0.15 ,ug/~L. The relative intensity of the
2 o dirner is expressed as percent of total receptor (monomer I dimer) irr~nunoreactivity. Data
shown are mean + /- SEM (n = 3) .
Figure 5 demonslrates, in A, effects of increasing concentrations of TM VI peptide on the
amount of ~2AR dimer. Increasing concentrations (0-6.3 mM) of the peptide were added to
2 5 purified c-mvc tagged ~2AR and the amount of dimer assessed by immunoblotting using the
anti c-myc rnAb (lanes 1 - 8). In lanes 9 and 10 purified ~2AR was treated (lane 10) or not
(lane 9) with the D2 TM VII peptide. The data shown are representative of three distinct
experirnents. Other control peptides used to determine the selectivity of the effect observed
with the TM VI peptide inrl~ e~l one derived from the C-terminal tail of the ~2AR [residues
3 0 347-358 NH2-LKAYGNGYSSNG-COOH] or an additional control peptide unrelated to the
~2AR but of similar size as the TM VI peptide ~NH2-SIQHLSTGHDHDDVDVGEQQ-
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COOH] were also found to be without effect on the amount of dimer (data not shown). B,
Densitometric analyses of three experiments similar to that shown in B. The relative intensity
of the dimer is expressed as percent of total receptor (monomer + dimer) immnnoreactivity.
Inset shows superimposed densitometric scans of irnmunoblotted receptors which were
5 previously treated with increasing concentrations of the TM VI peptide. The monomer is
denoted by M while the dimeric species is marked by D. The concentration of peptide added
for the curves shown was: none (.. ), 0.07 mM (---.. ----), 0.05 rnM (--- ---), and 1.25
mM (------).
0 Figure 6 demonstrates effects of TM VI peptide on ~2AR stimnl~tec~ adenylyl cyclase activity
in Sf9 cells. A, Membrane preparations derived from ~2AR expressing Sf9 cells were either
not treated (open circles), or treated with TM VI peptide (closed squares), control peptide TM
VI Ala (closed circles), or second control peptide from TM VII of the D2 dopamine receptor
(open triangles). Isoproterenol stim~ t~cl adenylyl cyclase activity was then assessed for these
membranes. Data are expressed relative to the maximal stimulation obtained with the
untreated membranes and represent mean +/- SEM for 8 independent experiments. Peptides
were used at a concentration of 0.15 ,ug/~l. B, Effects of TM VI peptide (hatched bars) or
vehicle alone (open bars) on basal (n - 13), maximal isoproterenol-stimnl~t~d (ISO, n = 13),
forskolin-m~ ted (~SK, n = 13) and NaF-stimnl~ted (n = 6) adenylyl cyclase activity was
investigated. Data are expressed as pmol cAMP produced per mg membrane protein per
minute +/- SEM. Statistical significance of the difference are indicated by an asterisk and
represent a p < 0.05 as assessed by a non-paired student's t-test. None of the co ntrol peptides
discussed in figure 2 had effects on adenylyl cyclase stimulation in ~2AR e~less~lg cells nor
did any of the peptides have effects on adenylyl cyclase stim~ tion in Sf9 cells which were
infected with the wildtype baculovirus (data not shown). C, Effects of increasing
concentrations of peptide on isoproterenol and dopamine stimlll~terl adenylyl cyclase activity
were also investig~tP-I Membranes were prepared from Sf9 cells ~ylessing either the human
,B2AR (open circles) or the human D1 dopamine receptor (closed circles). Adenylyl cyclase
activity was measured using maximally ~tim~ ting concentrations of either isoproterenol (10
3 o M) or dopamine (10 4 M) in the presence of peptide concentrations ranging from 10-8 to 10-4
M. Data were analyzed by non-linear least squares regression using SigmaPlot (Jandel
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Scientific). The data are expressed as the mean +/- SEM (n = 3).
Figure 7 shows effects of ~2AR ligands on receptor dimerization. A, Ti-rne course of the
effect of 1 ,uM isoproterenol on ~2AR dimerization. Membranes derived from Sf9 cells
5 e~pressJllg the c-~yc ~2AR were treated at room temperature with 1 ~M isoproterenol for 0
(lane 1), 15 (lane 2), 20 (lane 3) or 30 minllt~s (lane 4). Membranes were then subjected to
SDS-PAGE, transferred to nitrocellulose and immunoblotted with the anti-c-myc antibody.
A representative immllnoblot is shown. B, Densitometric analyses of three experirnents where
membranes from Sf9 cells expressing the ~2AR were treated for 30 minutes at room0 tempel~Lllre with either vehicle (CON), 1 ,uM isoproterenol (ISO), 10 ~M timolol (TIM), TM
VI peptide at a concentration of 0.15 ,ug/~L (TM VI), or isoplo~elenol followed by 30 minutes
with TM VI peptide (ISO/PEP). The TM VI data (lane 4) is reproduced from Figure 4b for
comparison. The relative intensity of the dimer is expressed as percent of total receptor
(monomer ~ dimer) immunoreactivity. Data shown are mean +/- SEM (n=3).
Figure 8 depicts effects of TM VI peptide on ~2AR expressed in m~mm~ n cells. A, Effect
of 0.15 ug/ul TM VI peptide (hatched bars) or vehicle (open bars) on basal (n=2), maximal
isoproterenol-stiml]l~te~ (ISO, n=2) forskolin-me~i~te~ (FSK, n=2) and NaF-stimulate~
(NaF, n=2) adenylyl cyclase activity in CHW cells expressing 5 pmol ~2AR/mg protein.
2 o Data are expressed as pmol cAMP produced per mg membrane protein per minute + SEM.
Statistical significance of the difference are indicated by an asterisk and represent a p < 0.05
as assessed by a non-paired student's t-test. Membranes were treated with either vehicle (lane
1) or the TM VI peptide at a concentration of 0.15 ug/ul (lane 2) for 30 minutes at room
temperature. Membranes from untransfected CHW cells had no ~letect~hle receptors (data not
2 5 shown). B, Effects of TM VI peptide on ~2AR stimulated adenylyl cyclase activity in mouse
Ltk- cells. Membranes were prepared from Ltk- cells stably expressing 200 fmol of human
~2AR/mg membrane protein. Isoproterenol-stimlllate~l adenylyl cyclase activity was then
assessed in membranes treated with vehicle (open circles), TM VI peptide (closed squares),
control peptide TM VI Ala (closed circles), or the D2 TM VII control peptide (open triangles) .
3 o Data are expressed relative to the maximal stim~ tion obtained with vehicle treated
membranes and r~pres~ mean + SEM for 3 independent experiments. Peptides were used
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at a concentration of 0.15 ug/ul.
~ DETAILED DESCRIPTION OF THE INVENTION
5 The following common abbreviations are used throughout the specification and in the claims:
The abbreviation, IP is inositol phosphate.
The abbreviation BASED is bis [~-(4 azidosalicylamindo) ethyl] disulphide
The abbreviation, 5-HT is 5-hydroxytryptarnine.
The abbreviation, DOI is 2,5-dimethoxy-4-iodoamphetamine hydrobromide.
10 The abbreviation, PBS is phosphate buffered saline.
The abbreviation"~2AR is ~2-adrenergic receptor.
The abbreviation, GPCR is G protein-coupled receptor.
The abbreviation, GpA is glycophorin A.
The abbreviation, HA is influenza hemagglutinin.
15 The abbreviation TM VI is transmembrane domain 6.
The abbreviation, NDI is nephrogenic diabetes insipidus.
Orphan receptors are receptors for which the natural ligands and/or biological function are
uncertain or unknown.
2 o The present invention resides in the discovery that certain GPCRs form oligomeric structures
(eg. homo-dimers) as part of their sign~lling activity, and that the effect of compounds o n this
process is predictive of the activity (agonist versus antagonist) and efficacy (partial and full
agonist and inverse agonist) of the compound on the specific GPCR. A working example is
provided, based on the human ~2 adrenergic receptor in which agonist promotes formation of
25 oligomers, inverse agonist promotes dissociation of oligomers and a peptide derived from
residues 276 - 296 of the ~32-adrenergic receptor inhibits agonist-promoted formation of
oligomer and also inhibits stimnl~tion of adenylyl cyclase activity. These results are
completely unexpected for receptor aggregation has not previously been considered to
influence or relate to activity of GPCRs.
This invention provides a method of testing compounds for activity and efficacy based on the
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ability of the compound to alter the monomer-multimer equilibrium. This invention permits
direct measurement of compound efficacy on the receptor independent of post-receptor
sign~lling pathways, and is applicable to G protein-coupled receptors of different functional
classes. Methods of screening compounds for activity and efficacy based on their ability to
5 alter association-dissociation of GPCRs are unknown in the art because GPCRs are not thought
to undergo association/dissociation as part of their activity.
It is surprising that measurements of multimer/monomer transitions in GPCRs would be
reflective of activity, because it is not thought that GPCRs form multimers for activity; thus,
10 testing for such a relationship is completely novel. The discoveries intim~t~ly relating
receptor-multimer formation to receptor-activity gave rise to this invention. Thus, the idea
of measuring multimer/monomer ratios instead of activities is an entirely new concept for a
GPCR drug screening test.
This invention can also apply to orphan GPCRs, for which ligand specificity or receptor
activities are not yet determined. The novel observations described in this invention indicate
that receptors in the GPCR family will undergo this fundamental oligomerization process as
an integral part of their activity, thereby allowing a novel method of assaying such receptors
2 o independent of knowledge regarding ligand specificity.
The method of this invention can also be used to test for compounds affecting the
oligomerization of homo-multimers and hetero-multimers (comprised of polypeptides from
dirr.,le.ll GPCR-types). For example it could be used to test for compounds that would affect
2 5 the activity of hetero-mllltimers formed between SHT-type receptor polypeptides and ,B-AR
receptor polypeptides.
The techniques of this invention, measuring the multimer/monomer transitions in GPCRs, can
either be correlated to receptor activity or used without such correlation. If they are so
3 o correlated for a particular type of GPCR, then assays can be conducted by determining the
mllltim~orlmonomer ratio of the receptor in order to obtain an indication of its activity without
SU~ ITE SHEET (RULE 26)
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having to measure that activity directly, thereby obviating time consuming and costly
procedures.
The principal goal of all the manifestations of the assay method embodied within this invention
5 is to measure the category of action and the efficacy of drug candidates by determining these
compounds' effects upon the ratio of monomeric receptor to oligomeric receptor (dimers,
trimers, homo-mulfimeric, hetero-multimeric, etc.) The change in ratio of the relative
amounts of monomer to multimer will reflect conversion of monomers to multimers or vice
versa, thus providing information on the activity and efficacy of drug candidates. Those
0 compounds which promote oligomerization would be predicted to have one activity (eg.
agonist or positive efficacy) while those which promote dissociation of oligomers would be
predicted to demonstrate the opposite activity (eg. inverse agonists or negative efficacy). The
m~gnitll~e of change in ratio and/or rate of change effected by the compound would provide
a measure of the compound's efficacy and/or potency in modulating receptor activity.
Measurin~ the ratio of monomeric receptor to multimeric receptor
There are many different techniques available for determining the relative amount of mbnomer
to multimer (eg. dimer) formed in the presence and absence of the test compound. For
20 example, different assay systems can be designed to measure the ability of compounds to
modify the ratio of monomers/multimers. In general, any procedure that permits measurement
of the relative amounts of monomer and oligomer in receptor preparations (eg. membranes,
solubilized receptor preparations, purified receptors, etc) can be used. Typically, a sample
cons~ining the compound to be tested or a control sample lacking the compound would be
2 5 added to a suspension or solution of receptor preparation. After an incubation period, the
receptor pre~a~ion would be analyzed to determine the relative amounts of monomeric and
oligomeric species such that changes in the ratio produced by the test compound could be used
~ to predict the activity and efficacy of the compound in regulating receptor function.
3 o Immunological methods can be used to measure compound efficacy. As demonstrated by the
working example provided herein (see Figures and Examples) dirr~ ial epitope tagging can
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.
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be used in combination with dirr~ lial co-imml~noprecipitation to demonstrate the formation
or absence of multimeric subunit aggregation. As each type of subunit bears a unique tag,
immunological techniques can be used to purify and identify the presence. of each subunit in
a m~ imer. If the complex is made up of two or more identical subunits (eg. homodimer or
homotrimer), each subunit is treated as if it is uni~ue, such that the subunits bear tags in
proportion to the number of units in the multimer. For example, if the complex is a
homodimer, one-half of the cDNA will be tagged with tag A and the other-half will be t agged
with tag B. The resulting dimers will form between A-A, AB, and BB subunits, but will be
observable by their migration in the SDS-PAGE gel, relative to the individual units. These
will be visualized by immunoblotting with either or both types of anti-A MAbs or anti-B
MAbs.
In a preferred embodiment of this invention, the following steps can be followed:
l) Synthesize sets of recombinant baculoviruses, wherein each set comprises cDNAencoding one subunit of a receptor and one unique immunologic tag, one set for each
subunit;
2) co-express the sets of receptor cDNA, each set bearing a unique tag, in Sf9 cells;
3) solubilize membranes and purify receptors;
4) add test compound to the receptor p-~aralion;
2 o 5) immunoprecipitate the receptors using anti-tag MAbs, one per unique tag;
6) separate the receptors using SDS PAGE;
7) immunoblot the SDS PAGE gel to observe resultant subunit aggregations;
An immunological method for measuring monomer/oligomer ratio entails separating monomers
and oligomers based on size and measurement of relative amounts of each using reporter
systems. In this embodiment the following steps would be followed:
1) receptor cDNA would be modified such that when expressed the expressed
receptor would be tagged with the epitope for a monoclonal antibody: this
expression would be performed in a heterologous system (eg. baculovirus-insect
3 o cell system);
2) the membrane prepa-dlion (or purified receptor) would be incubated with various
16
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concentrations of compound for defined period;
3) membranes (or pure receptor) would be solubilized in SDS sample buffer and
components separated by size on SDS-polacrylamide gels;
4) separated proteins would be transferred to nitrocellulose filter and relative positions
of the tagged receptors vi~ li7ed with anti-epitope antibody in an immunoblot reaction;
5) monomeric and oligomeric receptor species would be identified by size and relative
amounts of each species determined by densitometric SC~nning;
6) the ratio of monomer/oligomer species would be compared for different
concentrations of the test compound.
Using the techniques of this embodiment, alternate means of separating monomeric and
oligomeric receptor species by size can be used: eg. gel filtration, ul~l~ce~lLIifugation or others
followed by antibody detection of different size forms and determination of ratio of monomeric
to oligomeric species. Alternate means of labelling the receptor could entail labelling the
receptor with some reporter permitting specific detection of the receptor ( eg. fluorescent label
specifically incorporated into the receptor protein which can be quantified following size
separation of monomeric and oligomeric species.
In yet a further embodiment, the association of monomers into oligomeric receptor comp lexes
2 o can be measured directly using Fluorescence Resonance Energy Transfer, involving use of two
different fluorophores with distinct excitation and emission spectra, where the emission
spectrum of the first fluor overlaps with excitation spectrum of the second fluor. Two separate
al~tions of receptor would be labelled with one or the other fluor and these labelled
receptor ~ para~ions would be reconstituted together in solution or in phospholipid vesicles.
The mixture would then be irradiated at the excitation wavelength of the first fluor.
Monomers would show major emission and emission wavelength for the first fluor. Oligomers
would show increased emission at the emission wavelength of the second fluor due to close
proximity of the two fluors and energy transfer from the first to the second fluor. The ratio
of emission intensities at the emission wavelengths for the first and second fluors would
provide a measure of the relative amounts of monomeric ~no energy transfer) and the
oligomeric receptor species. Compounds which modify the ratio of monomeric and oligomeri c
SUBSllTUTE S~EET (RULE 26~
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species of the receptor will also modify the ratio of emission intensities at the two emission
wavelengths and permit prediction of activity and efficacy of the compound in regulating
receptor activity.
5 Modifications to this Fluorescence Resonance Energy Transfer method can be made by using
receptors tagged with dirrerent epitopes and two corresponding monoclonal antibodies labelled
with first and second fluors. In this alternative method, two receptor populations (tag l and
tag 2) in the same plcl)al~Lion (by co-expression of two receptors in insect cells or m~mm~ n
lines; or by separate expression and reconstitution into single preparation) are incubated with
0 anti-tag l labelled with fluor l, and anti-tag 2 labelled with fluor 2. Monomers will n ot show
energy transfer between fluors l and 2 on different receptor monomers, whereas oligomers
will bring two receptor-bound antibodies into proximity and permit energy transfer, measured
as an increase in emission intensity at the emission wavelength of fluor-2. Compounds would
be added to the mixture and tested for their abilities to promote receptor oligomerization or
15 dissociation of oligomers into monomers, and this information would permit prediction of
compound activity and efficacy in regulating receptor function.
The specifics of assessment assays for test compounds would thus involve the following steps:
adding aqueous solution Cont~ining the test compound to be evaluated to solution Cont~ining
20 a GPCR preparation (tissue, cell or extract); adding agonist to the same solution; measuring
the response to agonist by means of an assay as described above; comparing th e magnitude of
the response to agonist in the presence of the peptide or peptidomimetic compound to that of
the response in the absence of test molecule under otherwise identical conditions. Decrease
in agonist-in~ red response in the presence of peptide or peptidomimetic compound intlir~tes
2 5 antagonist activity.
Activity of the test compound can be further characterized by testing: varying the
concentrations of test compound against a fixed concentration of agonist to determine the
potency of the antagonist-like test compound and then varying the concentration of the agonist
30 with fixed test compound concentration to determine competitive versus non-competitive
action. Finally, measuring the effect of test compound on progressively more distantly-related
18
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receptors can be performed in order to determine selectivity.
Activity of the test compounds can also be assessed by measuring the compound's effect on
spontaneous receptor activity (i.e., basal activity in absence of added agonist). In this case,
the same assay systems can be used but without agonist, and the decrease in receptor activity
in presence of the test compound is measured.
EXAMPLES
o Synthesis of peptides
Peptides were synthesized on solid-phase supports using f-moc chemistry (Merrifield,
R.B., Rec. Prog. Hormone Res. 23:451-482, 1967; Stewart. J. and Young, J., Solid Pllase
Peptide Synthesis, Pierce Chemical Company, Rockford, Illinois, 1984) on a BioLynx
4175 manual peptide synthesizer (LKB). Peptides were solubilized in the following buffer:
100 mM NaCl, 10 mM Tris-HCl pH 7.4, 2 mM EDTA (plus a protease inhibitor cocktail
consisting of 5 mg/ml leupeptin, 10 mg/ml benzamidine and Smg/ml soybean trypsininhibitor~, 0.05% digitonin and 10% DMSO. Peptide sequences were confirmed by either
mass spectrometry or amino acid analysis. Peptides used were as follows~ 2AR TM
2 o VI peptide consisting of residues 276-296; NH2-GIIMGTFTLCWLPFFIVNIVH-COOH,
(2) a second peptide with Ala residues substituted at positions 276, 280, and 284; NH 2-
AIIMATFTACWLPFFIVNIVH-COOH, (3) a peptide derived from residues 407-426 of
the D2 dopamine receptor TM VII; NH2-YIIPNVASNVYGLWTFASYL-COOH, (4) a
peptide derived from the C-terminal tail of the ~2AR consisting of residues 347-358; NH2-
2 5 LKAYGNGYSSNG-COOH, and (5) an additional peptide unrelated to the ~2AR but of
similar size as the TM VI peptide; NH2-SIQHLSTGHDHDDVDVGEQQ-COOH.
Analysis of monomer/multimer ratios
3 o To assess the effect of the different peptides on the ,B 2AR expressed in Sf9 and m~mm~ n
cells, the following experiments were performed. Generally, membrane preparations from
19
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m~mm~ n or Sfg cells infected with recombinant baculovirus expressing human ~ 2AR
were treated with increasing concentrations of the different peptides at room temperatures
and for various times as indicated below. Specifically, membrane preparations from
m~mm~ n or Sf9 cells or affinity purified receptors derived from Sf9 cells expressing c-
myc tagged ~2AR were treated at increasing concentrations of the different peptides at
room temperature for various times as infiir~tecl (see results). Samples were then run on
SDS-PAGE and then transferred to nitrocellulose. In some cases membrane preparations
were also treated with either 10 ,uM timolol or 1 uM isoproterenol instead of, or in
addition to the different peptides. Peptide antagonist activity was assessed by assaying
0 adenylyl cyclase activity. In these assays, membranes were also used to determine the
effect of various peptides on the ability of the ~2AR to stimnl~t~ adenylyl cyclase activity
described below.
Recombinant baculoviruses
The recombinant baculoviruses encoding the c-myc or hemaglutinin (HA) tagged wildtype
human ~ 2AR, the c-myc tagged human M, muscarinic receptor and c-mvc tagged D,
dopamine receptor (c-myc ,~2AR and HA-~2AR, c-myc M,-R, and c-myc Dl-R
respectively) were constructed as described (Mouillac, B., et al., J. Biol. Chem.,
2 o 267:21733-21737, 1992). Briefly. HA (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) and c-myc
(Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) tags cont~ining initiaeor methionine residues
were introduced into the receptor cDNAs immediately before their initiator methionines by
subcloning the corresponding double-stranded oligonucleotides. Cells were infected with
recombinant baculoviruses at multiplicities of infection ranging from 3-5.
Sf9 Cell Culture
Sf9 cells were m~int;~ine~i at 27 C in serum-supplemented [10% fetal bovine serum (FBS)
v/v] Grace's insect medium (Gibco-BRL) with gentamycin and fungizone. Cells were3 o grown either as monolayers in T flasks or in suspension in spinner bottles supplemented
with pluronic acid to prevent cell taring due to agitation. Cells were infected at log phase
SUBSTITUTE SHEEr (RU~E 2
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at a density of 1 x 10 cells per ml for 48 h.
M~mm~ n Cell Culture
5 CHW and LTK cell lines with and without stably transfected ~2AR were m~int~3ined as
described (34). Cells were grown in Dulbecco's modified eagle medium (DMEM)
supplemented with L-glutamate, 10% FBS, gentamycin and fungizone. Transfected CHW
cells expressed 10-5 pmol receptor/mg protein while transfected LTK cells expressed 200
fmol receptor/mg protein. Stably transfected cell lines were grown in the presence of 150
0 ~g/ml G4 18 .
For transient expression of V2 vasopressin receptors the following procedures were
followed. COS-7 cells were m~int~ine~l in supplemented DMEM as described above.
Genomic DNA for the V2 vasopressin receptor was isolated from nephrogenic diabetes
insipidus (NDI) patients or unaffected individuals, subcloned into a construct cont~ining a
c-myc epitope tag and ligated into a m~mm~ n expression vector, pBC12BI (Cullen, B.R,
Meth. Enzymol., 1~2:684-704, 1987). Using DEAE-dextran, COS-7 cells were transiently
transfected with the expression vector encoding either wildtype V2 vasopressin receptor, a
truncation mutant O-11 or with vector alone for 48 hours.
Membrane Preparation
Membranes were p,epared as follows and washed. Sf9 or m~mm~ n cells were washed
twice with ice-cold PBS. The cells were then disrupted by homogenization with a polytron
in 10 ml of ice-cold buffer cont~ining 5 mM Tris-HCl, pH 7.4, 2 mM EDTA (plus a
protease inhibitor coclctail consisting of S mg/ml leupeptin, 10 mg/ml bem~mi(line and 5
mg/ml soybean trypsin inhibitor). Lysates were centrifuged at 500 x g for 5 minutes at
4 C, the pellets homogenized as before, spun again and the supernatants were pooled. The
supernatant was then centrifuged at 45,000 x g for 20 minutes and the pellets washed twice
3 o in the same buffer. In some cases receptors were then solubilized in 2% digitonin or 0.3 %
N-dodecyl- -D-maltoside and purified by affinity chromatography on alprenolol-sepharose
SUBSTITUTE SHEET (RULE 26
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as or by immunoprecipitation as described below.
Affinity purification of ~2ARs
5 Solubilized receptors were affinity purified by alprenolol-sepharose chromatography as
described (Mouillac, B., et al., J. Biol. Cem., 267:21733-21737, 1992; Shorr, R.G.L., et
al., J. Biol. Chem., 256:5820-5826, 1981). The affinity purified preparations were
concentrated using Cen~liplep and Centricon cartridges (Amicon) and the amount of ~2AR
in each sample was determined in soluble [ I]CYP radioligand binding assays as
0 described (Mouillac, et al., 1981, supra). Purified receptors were desalted on Sephadex G-
50 columns prior to SDS-PAGE.
Immunoprecipitation of ~32ARs
5 Tagged ~2ARs were immunoprecipitated with either a mouse anti-c-myc monoclonalantibody (9E10; Evan, G.I., et al., Mol. Cell. Biol., 5:3610-3616, 1985) or a mouse anti-
hemagglutinin monoclonal antibody (12CA5; Nimar, H.L., et al., Proc. Natl. Acad. Sci.
USA, 80:4949-4953, 1983) as described previously (Mouillac~ et al., 1981, supra).
Removal of digitonin and concentration of the solubilized receptor was performed by
2 0 dialysis using Cent~ p cartridges (Amicon) against an ice-cold solution (Buffer A)
cont~ining 100 mM NaCl, 10 mM Tris-HCl pH 7.4, 2 mM EDTA (plus protease inhibitors
described above) until the digitonin concentration was reduced below 0.05~. Purified
9E10 or 12CA5 antibody (1: 1000 dilution) was added to the concentrate and gently agitated
for 2 hours at 4 C. Anti-mouse IgG agarose (Sigma; at an 11: 1 secondary to primary
2 5 antibody molar ratio) and protease inhibitor cocktail were then added. The reaction was
allowed to proceed overnight at 4 C with gentle agitation. The immunoprecipitate was
centrifuged at 12,000 rpm in a microcentrifuge for 10 minutes at 4 C. The pellet was
washed three times in buffer A and ~mally resuspended in 200 ,uL of non-reducing SDS
PAGE loading buffer for 30 minutes, sonicated and centrifuged at 12,000 rpm. The3 o supernatant was then subjected to SDS PAGE and Western blotting as described below.
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Cross-linking of ~2ARs
Ten ml of Sf9 cell suspension (2 x 10 cells/ml) were taken 48 hours post-infection and
either mock-treated with vehicle or treated with 1 mg of the membrane permeant
photoactivatible cross-linking agent BASED (bis [,~-(4 azidosalicylamindo) ethyl]
disulphide; Pierce Chemicals) for 60 minutes at room temperature with gentle agitation.
Membranes were then prepared from cells as described above and resuspended in non-
reducing SDS PAGE sample buffer. Gels were subsequently immunoblotted as described
below.
SDS-PAGE and Western blottin~
Membrane preparations from Sf9 or m~mm~ n cells or in some cases affinity-purified or
immunoprecipitated ~2AR were prepared for non-reducing SDS-PAGE on 10% slab gelsas described previously (I ~emmli, U.K., Nature, 227:680-686, 1970). In the case of the
V2 vasopressin receptors reducing SDS-PAGE was performed. For Western blotting, gels
were transferred to nitrocellulose and blotted with either the mouse anti-c-myc monoclonal
antibody (9E10), the anti-hemagglutinin monoclonal antibody (12CA5) at dilutions of
1:1000 or in the case of m:~mm~ n cells expressing the ~2AR, a polyclonal rabbit anti-
2 o ,~2AR antiserum raised against a peptide from the C-terminal region of the ~ 2AR at a
dilution of 1:2000. Imrnunoblots against the anti-c-myc or anti-HA antibodies were
revealed using a goat anti-mouse ~lk~line phosphatase-coupled second antibody (GIBCO-
BRL) or a chemiluminescent substrate for a horseradish peroxidase coupled secondantibody (Renaissance, NEN Dupont). For the experiments performed using ~ """~ n2 5 cells expressing the ~2AR western blots were developed using a chemiluminescent
substrate for goat anti-rabbit coupled horseradish peroxidase antisera (Sigma). To assess
total immunoreactivity of the various receptor species, blots were scanned by laser
densitometry (Pharmacia-LE~B Ultrascan).
3 o Receptor quantification and adenylyl cyclase assay
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Receptor number was calculated from saturation binding experiments using [ I]
cyanopindolol (CYP) as the radioligand (Bouvier et al., Mol. Pharmacol., 267:7-19,
1994). Briefly, 10 ,uL of a membrane preparation in a total volume of 0.5 mL was labelled
with 250 pmol of [ I]-CYP which is at a near saturating concentration. Non-specific
binding was defined using 10 ~L alprenolol.
Adenylyl cyclase activity was assayed by the method of Salomon et al., (Anal. Biochem.,
58:541-548, 1974). Membranes were prepared and washed as described above. Again 10
,llL of membranes (3-5 ,ug of protein) were used in a total volume of 50 ~L. In some
0 experiments, the peptides or the buffer used to solubilize them were added to the enzyme
assay mix. Enzyme activities were determined in the presence of 1 nM to 100 ,uM
isoproterenol, 100 ~M forskolin or 10 mM NaF. Data were calculated as pmoles cAMP
produced/min/mg protein and were analyzed by least squares regression using SigmaPlot
4.17 (Jandel Scientific).
Analysis of results
Immunoblotting of c-myc epitope tagged ,~2AR expressed in Sf9 cells with the anti-c-myc
antibody consistently revealed the presence of molecular species corresponding to the
2 o anticipated monomeric receptor (43-50 kDa) in Sfg cells (Mouillac, et al., 1981, supra) as
well as higher molecular weight forms. In particular, a prominent band was detected at an
a~palell~ molecular weight corresponding to twice that of the monomer (85-95 kDa)
suggesting the existence of an SDS-resistant dimeric species of the receptor. In some
membrane preparations discrete bands which could represent even higher order structures
of the ,~2AR can also be detected (Figure 1, lane 1). The dimer, which was readily
observed in membrane preparations, was also detected in digitonin-solubilized receptors
(lane 2) and following affinity purification of receptors on alprenolol-sepharose (lane 3).
As shown in lanes 4 and 5, when whole cells expressing the ~ 2AR were treated with the
membrane permeant cross-linking agent BASED, the dimer to monomer ratio as assessed
3 o by immllnoblotting was increased by two-fold. This suggests that the dimer is already
present before cell fractionation and that crosslinking stabilizes this form of the receptor.
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therefore, the dimeric species does not represent an artifact of membrane preparation or
solubilization. Identical results were obtained when membranes were solubilized with
0.3% N-dodecyl-~-D-maltoside instead of digitonin (data not shown)
5 In order to demonstrate that the higher molecular weight species observed in this study
corresponded to a specific ,~2AR homodimer, we devised a differential co-
immunoprecipitation strategy using c-myc and hemagglutinin (HA) epitope tagging.Human ~2ARs bearing either of these tags were co-expressed in Sf9 cells. The receptors
were then immllnoprecipitated with the anti-HA or anti-c-myc antibodies, subjected to SDS
0 PAGE and blotted with one or the other antibody. In the results shown in Figure 2 the
anti-HA mAb was used to blot receptors immunoprecipitated with either the anti-HA mAb
or the anti-c-myc rnAb. As seen in lane ., blotting of the anti-HA irnmunoprecipitate
revealed both the 45 kDa and the 90 kDa forms of the receptor. The ~ 2AR could also be
detected by the anti-HA mAb in the c-myc immllnoprecipitate of co-expressed receptors but
5 the dimer then represented the predominant form (lane 1). This in~lic~3tec that the two
molecular species (HA-tagged and c-myc-tagged ,~2ARs) were co-immunoprecipitated as
part of a complex which is stable in SDS, consistent with the higher molecular weight form
being a ~2AR homodimer. Similar but complementary results are obtained when co-
expressed receptors are immunoprecipitated with either anti-c-myc or anti-HA antibodies
2 o and then irr~nunoblotted with the anti-c-myc or anti-HA antibodies and then imrnunoblotted
with the anti-c-myc antibody (data not shown). The specificity of the mAbs is illustrated
by the absence of cross-reactivity in cells expressing one tagged receptor species only
(Figure 2 lanes 3-6). The occurrence of intermolecular interactions appears to be receptor-
specific. Indeed, although dimers of c-nZyc tagged M, muscarinic receptor could be
2 5 detected in Sf9-derived membranes expressing this receptor (data not shown and see
Debburman, S.K., et al., Mol. Pharmacol., 47:224-233, 1995) no co-immunoprecipitation
with the HA-tagged ~2AR was detected when the two receptors were co-expressed (Figure
2, lanes 7,8).
3 o V2 vasopressin receptors are also dimeric.
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The vasopressin receptor is critical for regulation of water retention in the kidney.
Recently, several mutations of this receptor have been linked to congenital nephrogenic
diabetes insipidus (NDI, Bichet, D.G., et al., Am J. Hum. Genet., 55:278-286, 1994). In
another approach to demonstrate GPCR dimer formation, transient expression of both
wildtype and a truncated form of the V2 vasopressin receptor in COS-7 cells was studied.
Both monomeric (appx. 64-69 kDa) and dimeric (appx. 120-135 kDa) forms of the
wildtype human V2 vasopressin receptor were detected when expressed in COS-7 cells
(Figure 3, lane 1). A mutant form of the V2 receptor truncated in the C-terminal tail at
residue 33y (Ol l, isolated from a patient with congenital nephrogenic diabetes insipidus
(Bichet, D.G. et al., supra, 1994) was also capable of forming dimers when expressed in
COS-7 cells (Figure 3, lane 2). Indeed, the Ol l V2 receptor was detected as approx. 55-
58 kD and appx. 89-100 kDa species consistent with the idea that higher molecular weight
form represents a homodimer. These results confirm by a different approach that G
protein-coupled receptors can form SDS-resistant dimers when expressed in m~mm~ n
cells.
Modulation of ~2AR dimerization by TM VI peptide
As shown in Figure 4a the addition of the TM VI peptide substantially reduced the amount
2 ~ of ~2AR dimer detected in Sf9 membranes in a time-dependent fashion (Figure 4a, lanes 1-
4). In this experiment the relative amount of receptor dimer was gradually reduced from
54% at time zero to 17% after 30 minutes of treatment with TM VI peptide. When results
of three such experiments were averaged, the TM Vl peptide was found to reduce the
relative amount of dimer by 69% after 30 minutes (Figure 4b). A control hydrophobic
2 s peptide (from transmembrane domain VII from the D2 dopamine receptor) at maximal
concentration had no effect on the relative amount of dimer detected. (Figure 4b). This
does not appear to result from a non-specific hydrophobic interaction since the unrelated
dopamine receptor TM VlI peptide was without effect. To address the importance of the
glycine and leucine residues identified above, a second control peptide corresponding to
3 o TM VI of the ~2AR with Gly 276, Gly 280 and Leu 284 replaced by alanine residues (TM
VI Ala) was synthesized. Although this peptide slightly decreased the amount of dimer its
26
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effect was very modest compared with that of the TM VI peptide (Figure 4b) thus
suggesting that these three residues may be a part of the interface between two receptor
monomers. One mecl ~ni~m which could explain the effect of the TM VI peptide is that it
may interact with monomeric ~2AR thus preventing it from interacting with a second
5 receptor monomer.
The effect of the TM VI peptide on dimer formation was also detected using purified ~ 2AR
preparations and was shown to be dose-dependent. As seen in Figure 5a, increasing
concentrations of the TM VI peptide led to a gradual reduction in the amount of dimer.
0 This was accompanied by a concomitant increase in the level of the monomer such that the
proportion of the dimer decreased from a control level of 43.1 + 4.3~c to a final level of
12.6 + 3.2% (Figure 5A~ lanes l - 8; Figure SB) The D~ receptor TM VII control peptide
had no effect on receptor dimerization (Figure 5A~ compare lanes 9 and l0) similar to the
results shown using membrane preparations (Figure 4B). We also noted a modest but
5 reproducible upward shift in the apparent molecular weight of the monomer resulting in a
widening of the band as the concentration of peptide was increased (Figure SB, inset).
This suggests that as proposed above the peptide forms a stable complex with the receptor
monomer thus mimicking receptor-receptor interactions.
2 o Functional consequences of receptor dimerization
The functional signi~lr~nce for receptor dimerization is suggested by the inhibitory action
of the TM VI peptide on receptor-stimul~3ted adenylyl cyclase activity. As shown in Figure
6A, the addition of TM VI peptide to membrane preparations at a concentration of 0.15
2 5 ,ug/~l significantly reduced isoproterenol-stim~ te~ adenylyl cyclase activity (p < 0.05). In
contrast, neither the peptide solubilization buffer (data not shown) nor control peptides
(TM VI-Ala or TM VII of the D, dopamine receptor) had significant effects on
isoproterenol-stimulated adenylyl cyclase activity.
3 o The effect of the peptide was receptor-specific as it had no effect on either NaF-mPdi~t-od
or forskolin-m~ te~ adenylyl cyclase stimulation (Figure 6B). Notably, the ligand-
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independent basal adenylyl cyclase activity was slightly inhibited by the TM VI peptide
suggesting that it may effect the spontaneous activity of the receptor as well. Indeed,
spontaneous receptor activity is in large part responsible for the ligand-independent
adenylyl cyclase activity observed in both Sf9 and m~mm~ n cells expressing the ,~ 2AR
(Chiciac, P., et al., Mol. Pharmacol. 45:490-499, 1994). A receptor-dependent effect is
also supported by the fact that the TM VI peptide was without effect on basal cyclase
activity in Sf9 cells which were infected with the wildtype baculovirus (data not shown).
Also consistent with a receptor-specific action of the peptide is the observation that D,
dopamine receptor-stim~ tecl adenylyl cyclase activity was not significantly affected by the
TM VI peptide (Figure 6C). As was the case for the inhibition of dimerization, the
inhibitory action of the TM VI peptide on receptor-mediated adenylyl cyclase activity was
dose-dependent (Figure 6C). It should be noted that the peptide IC50 values for the
inhibition of dimer formation are very similar (2.14 + 0.05 ,uM and 3.~ + 0.04 ,uM,
respectively) thus suggesting that receptor dimerization may be an important step in
~2AR-mediated sign~lling. Although our data suggest a role for dimerization in receptor
activity, one cannot exclude the possibility that the effect of the TM VI peptide is not
directly due to an effect on the monomer:dimer e~uilibrium. Still, these results clearly
show that this domain of the receptor is important in moc~ ring ~ 2AR signal transduction.
Furthermore, the peptide represents a novel pharmacological tool for the study of receptor
2 0 activity.
The effect of TM VI peptide on adenylyl cyclase stim~ tion does not result from a loss of
receptor sites as neither the affinity or the maximum number of binding sites for I CYP
were affected (KD= 1.8 + 0.5 x 10 and BmaS = 16.5 + 2 pmol/mg protein for
untreated membranes compared with KD=4.2 + 1.5 x 10 and BmaX = 21.3 + 4.5
pmollmg protein for TM VI peptide treated membranes, n = 3 for both determinations).
Effect of the TM VI peptide on GPCR in ~ n cells
3 o In this study, ~2AR dimers were observed in CHW cells stably transfected with the
receptor (Figure 8A, inset) by immunoblotting with a polyclonal anti-~2AR antisera.
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Similar to our observations in Sf9 cells, the TM VI peptide also reduced the amount of
,~2AR dimer cletected in membrane derived from CHW cells (Figure 8A, inset lane 2).
This peptide also reduced basal and isoproterenol-stim~ ted adenylyl cyclase activity in
these cells while leaving forskolin- and NaF-mediated st;mul~tion unaffected (Figure 8A).
5 Similar fin-ling~ were also obtained with LTK- cells expressing as little as 200 fmol of
~2AR/mg protein (Figure 8b). These results taken together suggest a similar functional
signifir~nce for ~2AR dimerization in m~mm~ n cells as in Sf9 cells.
The results presented here demonstrate that both human ,~2AR and V2 vasopressin
0 receptors can form SDS-resistant homodimers. For the ,~2AR, the relative amount of
dimer can be altered by a peptide derived from TM VI and by receptor ligands suggesting
that under basal conditions there appears to be a dynamic equilibrium between monomeric
and dimeric species of receptors. The data also suggest that shifting the equilibrium away
from the dimeric form of the receptor interferes with the ability of the ~ 2AR to
5 productively interact with its sign~llin~ pathway.
Application of Assay to other GPCRs
Higher molecular weight species have been detected in both m~mm~ n and Sf9 expression
2 o systems for many GPCRs. These include the V2 vasopressin receptor (see discussion
above - this study, Figure 3), platelet activating factor receptor, metabotropic glut~m~te
receptor, substance P receptor, neurokinin-2 receptor, the C5a anaphylaxotoxin receptor,
glucagon receptor, the dopamine D, receptor, D, receptor, the SHTlB receptor, the M;
muscarinic receptor and the M3 muscarinic receptor (see Hebert, T.E et al., J. Biol. Chem.
2 5 accepted, 1996, and references therein) . Thus, GPCR-peptides and peptidomimetic
compounds could be designed for these receptors that would function to as demonstrated in
these examples to selectively prevent or disrupt the functional aggregation of these
receptors, thereby ~ttenll~ting receptor activity.
3 o From the foregoing description, one skilled in the art can easily ascertain the essential
characteristics of this invention, and without departing from the spirit and scope thereof,
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can make various changes and modifications of the invention to adapt it to various usages
and conditions. Consequently, such changes and modifications are properly, equitably, and
"in~enrle~" to be, within the full range of equivalence of the following claims.
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