HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 43, 42578-42587, October 24, 2003

This Article Abstract Full Text (PDF) All Versions of this Article: 278/43/42578    most recent M306165200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carrillo, J. J. Articles by Milligan, G. Search for Related Content PubMed PubMed Citation Articles by Carrillo, J. J. Articles by Milligan, G. Social Bookmarking                      What's this?

Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins^*

Juan J. Carrillo, John Pediani, and Graeme Milligan

From the Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom

Received for publication, June 11, 2003 , and in revised form, August 4, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The histamine H1 receptor and the _1b-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca²⁺] via activation of G_q/G₁₁. Assessed by co-immunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homo-dimers. The addition of the G protein G₁₁ to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5'-3-O-([³⁵S]thio)triphosphate ([³⁵S]GTPS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG₁₁ into the fusions. Mutation of a highly conserved leucine in intracellular loop 2 of each receptor also eliminated agonist function but not binding. Co-expression of the two non-functional but complementary fusion constructs reconstituted agonist-mediated binding of [³⁵S]GTPS in membranes of HEK293 cells and elevation of [Ca²⁺]_i in mouse embryo fibroblasts lacking both G_q and G₁₁. Co-expression of the histamine H1 receptor- and the _1b-adrenoreceptor-G₁₁ fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G₁₁ with increasing amounts of L151D_1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [³⁵S]GTPS binding. Co-expression of the _1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the _1b-adrenoreceptor and G₁₁ did not result in agonist activation of the G protein but did indicate a role for transmembrane helix 1 in dimerization. These data demonstrate that dimers of these class A receptors function via trans-activation of associated G proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The concept that G protein-coupled receptors (GPCRs)¹ exist as dimers or higher order oligomers has moved rapidly from hypothesis to being widely accepted (1-4). A range of approaches has contributed to this understanding. This includes the ability to co-immunoprecipitate differentially epitope-tagged forms of a GPCR from cells in which they are co-expressed, and, in intact cells, the application of a number of resonance energy transfer-based techniques. However, the role of dimerization in function and the mechanisms then responsible for initiation of signal transduction by the dimer have been more recalcitrant to analysis. Significant progress in this area has recently been achieved for the class C GPCRs. These contain both a long extracellular N-terminal domain, to which agonist ligands bind, and the prototypic seven transmembrane (TM) helix bundle architecture that is the common feature of all GPCR families (for review, see Ref. 5). The functional -aminobutyric acid, type b receptor is a hetero-dimer of two distinct gene products in which trafficking to the plasma membrane requires interaction between the partner polypeptides (6-9). This indicates that a key role of dimerization is achieving appropriate cellular localization. This is also true for the class A rhodopsin-like GPCRs because non-functional, truncated splice variants can restrict plasma membrane delivery of full-length GPCRs and, thus, limit their function (10-12). Chimeric class C GPCRs consisting of the extracellular domain of one GPCR and the TM and intracellular elements of a second, closely related GPCR or in which the intracellular loops of dimer partners are exchanged have provided strong evidence that the mechanism of action of the dimer involves trans-activation (13-14); that is, ligand binding to one element of the dimer results in activation of G protein produced by the other GPCR within the dimer. Again, the -aminobutyric acid, type b receptor has been particularly informative in this regard as only one of the two gene products that forms the dimer is able to bind the agonist -aminobutyric acid. Equivalent systems are not available for the majority of class A GPCRs. However, for the luteinizing hormone receptor, which also has a long N-terminal domain that binds the ligand, partial reconstitution of function has been achieved by co-expression of distinct pairs of mutants (15-16). Furthermore, in recent studies co-expression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescued hormone-activated cAMP production (17).

Fusion proteins in which G protein subunits are linked in-frame to the C terminus of a GPCR have become widely used tools to study the details of information transfer between these proteins (18-19). Because these can be considered as bi-functional proteins containing the sequence and function of both GPCR and G protein they can be utilized to generate contrasting pairs of non-functional mutants. Herein we generate and analyze fusion proteins incorporating either wild type GPCRs or mutants unable to activate G proteins and wild type or mutant G proteins unable to be activated by GPCRs. We show that co-expression of pairs of these non-functional mutant fusion proteins is complementary and results in the generation of functional dimers. This reconstitution can be both easily quantitated in membrane preparations and monitored in single intact cells. Studies of hetero-dimeric GPCR pairs support a mechanism in which agonists at aminergic class A GPCRs mediate trans-activation of the GPCR-associated G proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—A fibroblast cell line (EF88) (20-22) derived from a combined G_q/G₁₁ double knockout mouse (23-24) was the gift of Dr. M. I. Simon, California Institute of Technology, Pasadena CA. All materials for tissue culture were supplied by Invitrogen. [³H]Prazosin (80 Ci/mmol), [³H]mepyramine (20 Ci/mmol), and [³⁵S]GTPS (1250 Ci/mmol) were from PerkinElmer Life Sciences. Oligonucleotides were purchased from Cruachem (Glasgow, Strathclyde, UK). Reagents for time-resolved fluorescence resonance energy transfer (tr-FRET) were from PerkinElmer Life Sciences. Receptor ligands were purchased from RBI (Gillingham, Kent, UK). Production and characterization of the anti-G_q/G₁₁ antiserum CQ was described by Mitchell et al. (25). All other chemicals were from Sigma and were of the highest grade available.

Construction of Fusion Proteins—Production and subcloning of wild type and mutated _1b-adrenoreceptor-G₁₁ fusion proteins was performed as described in Carrillo et al. (26). Production and subcloning of the human histamine H₁ receptor-G₁₁ fusion proteins was performed in two separate stages. In the first step, using the N-terminal primer 5'-GATACTGGGCTATCCAAGCTTATGAGCCTCCCCAATTCCTC-3', a HindIII restriction site (underlined) was introduced by PCR upstream of the coding sequence of the human histamine H₁ receptor. Using a C-terminal primer 5'-AAGGAAAAAAGCGGCCGCTGGAGCGAATATGCAGAATTCTCT-3', a three amino acid spacer (Ser-Gly-Arg) and a NotI restriction site were introduced immediately upstream of the stop codon. Similarly, the mouse G₁₁ sequence was amplified by PCR using the N-terminal primer 5'-AAGGAAAAAAGCGGCCGCATGACTCTGGAGTCCATGATGGC-3' and the C-terminal primer 5'-ATGAAACCGCTCGAGTCACACCAGGTTGTACTCCTTCAG-3'. This introduced NotI and XhoI restriction sites flanking the G₁₁-coding sequence. In the second step, the amplified receptor fragment was digested with HindIII/NotI, and the G₁₁ fragment was digested with NotI/XhoI. These fragments were purified and ligated into pcDNA 3 vector (Invitrogen) previously digested with HindIII/XhoI. The choice of intracellular loop 2 Leu to Asp mutants was based on the studies of Greasley et al. (27). For co-immunoprecipitation and tr-FRET studies, c-Myc (EQKLISEEDL) or FLAG (DYKDDDDK) epitopes were introduced immediately after the N-terminal methionine. Each construct was fully sequenced before its expression and analysis. Construction of the c-Myc-NtTM1_1b-G₁₁ fusion protein was also carried out in two stages. Using the N-terminal primer 5'-TTTCCCAAGCTTATGGAACAAAAACTTATTTCTGAAGAAGATCTGAATCCCGATCTGGACACC-3' a HindIII restriction site (underlined) followed by an ATG codon and the c-Myc tag sequence (italics) was introduced by PCR upstream of the coding sequence of the _1b-adrenoreceptor. Using a C-terminal primer 5'-AATCGGGGTACCGGTGGGCGTCCGCAGGTGCCGAAT-3', a two-amino acid spacer (Lys-Leu) and a KpnI restriction site (underlined) were introduced immediately after the codon for the 80th amino acid of the hamster _1b-adrenoreceptor. In the second stage the resulting PCR fragment was digested with HindIII/KpnI. Next, the _1b-adrenoreceptor element of the _1b-adrenoreceptor-G₁₁ fusion protein was removed by digestion with HindIII/KpnI, and the digested PCR fragment was ligated into the resulting vector. Thus, the generated fusion protein contained a c-Myc tag linked in-frame with the first 80 amino acids of the _1b adrenoreceptor and G₁₁.

Transient Transfection of HEK293 Cells—HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.292 g/liter L-glutamine and 10% (v/v) newborn calf serum at 37 °C in a 5% CO₂-humidified atmosphere. Cells were grown to 60-80% confluency before transient transfection in 60-mm dishes. Transfection was performed using LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions.

[³⁵S]GTPS Binding—[³⁵S]GTPS binding experiments were initiated by the addition of membranes containing defined amounts of the fusion constructs (see "Results" for details) to an assay buffer (20 mM HEPES (pH 7.4), 3 mM MgCl₂, 100 mM NaCl, 1 µM guanosine 5'-diphosphate, 0.2 mM ascorbic acid, 50 nCi of [³⁵S]GTPS) containing the indicated concentrations of receptor ligands. Nonspecific binding was determined in the same conditions but in the presence of 100 µM GTPS. Reactions were incubated for 15 min at 30 °C and were terminated by the addition of 0.5 ml of ice-cold buffer containing 20 mM HEPES (pH 7.4), 3 mM MgCl₂, and 100 mM NaCl. The samples were centrifuged at 16,000 x g for 15 min at 4 °C, and the resulting pellets were resuspended in solubilization buffer (100 mM Tris, 200 mM NaCl, 1 mM EDTA, 1.25% Nonidet P-40) plus 0.2% sodium dodecyl sulfate. Samples were pre-cleared with Pansorbin (Calbiochem) followed by immunoprecipitation with CQ antiserum (25). Finally, the immunocomplexes were washed twice with solubilization buffer, and bound [³⁵S]GTPS was measured by liquid scintillation spectrometry.

[³H]Ligand Binding Studies—[³H]Prazosin binding studies, monitoring expression of the _1b-adrenoreceptor-containing constructs, were performed as in Carrillo et al. (26). [³H]Mepyramine binding assays, monitoring expression of the histamine H1 receptor-containing constructs were initiated by the addition of 8 µg of cell membranes to an assay buffer (50 mM Tris-HCl, 100 mM NaCl, 3 mM MgCl₂ (pH 7.4) containing [³H]mepyramine (0.1-10 nM). Nonspecific binding was determined in the presence of 100 µM triprolidine. Reactions were incubated for 30 min at 25 °C, and bound ligand was separated from free ligand by vacuum filtration through GF/B filters. The filters were washed twice with assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.

[Ca²⁺]_i Imaging—EF88 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and L-glutamine (1 mM) in a 95% air and 5% CO₂ atmosphere at 37 °C. A portion of the cells harvested during trypsinization were plated onto glass coverslips, and after a 24-h growth period they were transfected using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. After 3 h cells were washed twice with Opti-MEM I and then cultured in Dulbecco's modified Eagle's medium growth medium for a further 24 h. A total of 3 µg of pCDNA3 containing the relevant cDNA species was used to transfect each coverslip. Transfected EF88 cells were loaded with the Ca²⁺-sensitive dye Fura-2 by incubation (15-20 min, 37 °C) under reduced light in Dulbecco's modified Eagle's growth medium containing the dye's membrane-permeant acetoxymethyl ester form (1.5 µM). Details of the imaging studies and their analysis have been provided previously (28).

GPCR Co-immunoprecipitation Studies—Co-immunoprecipitation studies using FLAG- and c-Myc-tagged forms of the _1b-adrenoreceptor and histamine H1 receptor constructs were performed as in McVey et al. (29) and Ramsay et al. (30). In the studies with the histamine H1 receptor, 30 units/ml of endoglycosidase F were added. Time-resolved fluorescence resonance energy transfer was performed on intact HEK293 cells using Eu³⁺-labeled anti-c-Myc antibodies and allophycocyanin-labeled anti-FLAG antibodies as described in McVey et al. (29).

Miscellaneous—All experiments were performed on at least three independent occasions. Data are presented as the means ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have previously generated a fusion protein between the _1b-adrenoreceptor and the subunit of G₁₁ that binds both agonist and antagonist ligands including [³H]prazosin (26). The addition of the agonist phenylephrine to membranes of HEK293 cells transfected to express this construct resulted in a large stimulation of the binding of [³⁵S]GTPS, monitored after the assay by immunoprecipitation using an antiserum against the C-terminal decapeptide of G₁₁ (Fig. 1A). Introduction of a G208AG₁₁ mutant into the fusion protein essentially eliminated phenylephrine stimulation of [³⁵S]GTPS binding when membranes expressing equal amounts of this construct were used (Fig. 1A), because this form of the G protein is unable to release bound GDP. However, this mutation did not alter the binding properties of either [³H]prazosin or phenylephrine (Fig. 1, B and C, Table I). Previous studies show that mutation of hydrophobic amino acids in intracellular loop 2 of the _1b-adrenoreceptor can eliminate agonist-mediated signal transduction (27). We, thus, generated a fusion protein between L151D _1b-adrenoreceptor and G₁₁. This also bound both [³H]prazosin and phenylephrine as the wild type fusion protein (Fig. 1, B and C, Table I), but phenylephrine was again unable to stimulate binding of [³⁵S]GTPS (Fig. 1A). Co-expression of the two non-functional mutants reconstituted phenylephrine-mediated binding of [³⁵S]GTPS (Fig. 1A), and when the membrane amounts employed contained twice as many [³H]prazosin binding sites as used for each individual construct, the level of agonist-mediated [³⁵S]GTPS was 70% as great as when employing the wild type fusion construct (Fig. 1A). Reconstitution of function required co-expression of the two mutant fusions. When the two constructs were expressed in separate cell populations and either the cells were mixed before membrane preparation or membranes were prepared individually and then combined before assay, no agonist-stimulated binding of [³⁵S]GTPS was observed (Fig. 1D). Such results are consistent with the hypothesis that GPCR dimerization is required for agonist function. Furthermore, within the dimer, one GPCR element must activate the G protein physically linked to the partner GPCR. To extend this concept an equivalent set of experiments was performed using fusions between the histamine H1 receptor and G₁₁. The basic results were the same. The fusion containing wild type forms of both the GPCR and G protein produced a large stimulation of [³⁵S]GTPS binding in the presence of the agonist histamine (Fig. 2). This was absent upon separate expression of either a histamine H1 receptor-G208AG₁₁ fusion protein or a fusion between L133D histamine H1 receptor and wild type G₁₁. Co-expression of these two mutants reconstituted agonist activation of the G protein (Fig. 2). Again, after co-expression of the two mutants, membranes expressing a 2-fold higher number of ³H-labeled antagonist binding sites produced nearly as high a level of [³⁵S]GTPS binding upon the addition of histamine as the wild type histamine H1 receptor-G₁₁ fusion protein expressed in isolation.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Pairs of distinct non-functional mutants of 1b-adrenoreceptor-G11 fusion proteins reconstitute function. A, membranes of HEK293 cells expressing 40 (1-4) or 80 (5) fmol of various 1b-adrenoreceptor-G11 fusion proteins were used to measure the binding of [35S]GTPS in the absence (open bars) or presence (filled bars) of 10 µM phenylephrine. 1, wild type 1b-adrenoreceptor-G11; 2, 1b-adrenoreceptor-G208AG11; 3, L151D1b-adrenoreceptor-G11; 4 and 5, 1b-adrenoreceptor-G208AG11 + L151D1b-adrenoreceptor-G11. B and C, mutation of L151D in the 1b-adrenoreceptor or G208A in G11 does not modify the binding of agonists or antagonists. Membranes expressing the wild type 1b-adrenoreceptor-G11 (squares), the L151D1b-adrenoreceptor-G11 (circles), or the wild type 1b-adrenoreceptor-G208AG11 (triangles) fusion proteins were used to measure the binding of varying concentrations of [3H]prazosin (B) or the ability of varying concentrations of phenylephrine to compete with 1 nM [3H]prazosin for binding (C). D, L151D1b-adrenoreceptor-G11 and 1b-adrenoreceptor-G208AG11 reconstitute function only when they are co-expressed. The binding of [35S]GTPS in the absence (open bars) or presence (filled bars) of 10 µM phenylephrine was measured in HEK293 cell membranes in which L151D1b-adrenoreceptor-G11 and 1b-adrenoreceptor-G208AG11 were co-expressed (1) or in which the two constructs were expressed in separate cell populations that were mixed before membrane preparation (2) or from which membranes were made separately and then mixed before assay (3).

View this table: [in this window] [in a new window]   TABLE I The binding affinity of agonist and antagonists ligands to 1b-adrenoceptor- and histamine H1 receptor-G11 fusion proteins is unaffected by the addition of N-terminal epitope tags or the introduction of specific mutations. Data are presented as means ± S.E. from a minimum of three separate experiments

View larger version (11K): [in this window] [in a new window]   FIG. 2. Pairs of distinct non-functional mutants of histamine H1 receptor-G11 fusion proteins also reconstitute function. Membranes of HEK293 cells expressing 25 (1-4) or 50 (5) fmol of various histamine H1 receptor-G11 fusion proteins were used to measure the binding of [35S]GTPS in the absence (open bars) or presence (filled bars) of 1 mM histamine. 1, wild type histamine H1 receptor-G11; 2, L133D histamine H1 receptor-G11; 3, histamine H1 receptor-G208AG11; 4 and 5, histamine H1 receptor-G208AG11 + L133D histamine H1 receptor-G11.

As an extension to these studies we attempted to monitor dimerization and functional reconstitution in a single cell. To do so we employed Ca²⁺ imaging using EF88 cells. These are a line of mouse embryo fibroblasts that derive from a G_q/G₁₁ double knock-out mouse (20-21). They, thus, require expression of both a functional GPCR and a functional Ca²⁺-mobilizing G protein to produce elevation of intracellular [Ca²⁺] (28, 31). Upon introduction of fusions between wild type forms of either the histamine H1 receptor or the _1b-adrenoreceptor, and G₁₁ appropriate agonists produced elevation of intracellular [Ca²⁺] (Fig. 3). This occurred only in positively transfected cells. Because EF88 cells are recalcitrant to transfection, we co-transfected with enhanced green fluorescent protein (GFP) to allow visualization of the positively transfected cells. Only those cells that were positive for GFP responded to agonist ligands (Fig. 3A). For both the histamine H1 receptor and the _1b-adrenoreceptor the fusions containing either the non-agonist-responsive GPCR or the G protein mutant failed to elevate intracellular [Ca²⁺] (Fig. 3, B and C). However, co-expression of the pairs of complementary fusions again resulted in effective signal generation (Fig. 3, B and C).

View larger version (25K): [in this window] [in a new window]   FIG. 3. GPCR dimerization and functional reconstitution in single cells. EF88 cells were transfected to express GPCR-G11 fusion proteins, and GFP and the ability of agonist ligands to elevate intracellular Ca2+ were monitored. A, only positively transfected cells respond to agonist. Cells were co-transfected with the wild type histamine H1 receptor-G11 fusion and GFP. In the field shown only a single cell expressed GFP (left). Basal (center) and 1 mM histamine (right)-stimulated Ca2+ was then monitored in these cells. The warmer color represents elevated [Ca2+]. B, EF88 cells were transfected with GFP and histamine H1 receptor-G11 (black, n = 6), histamine H1 receptor-G208AG11 (blue, n = 10), L133D histamine H1 receptor-G11 (green, n = 12), and both histamine H1 receptor-G208AG11 and L133D histamine H1 receptor-G11 (red, n = 8). The response of GFP-positive cells to 1 mM histamine was then measured over time. n = the number of individual cells quantitated. C, EF88 cells were transfected with GFP and 1b-adrenoreceptor-G11 (black, n = 8), 1b-adrenoreceptor-G208AG11 (blue, n = 10), L151D 1b-adrenoreceptor-G11 (green, n = 8), and both 1b-adrenoreceptor-G208AG11 and L151D 1b-adrenoreceptor-G11 (red, n = 12). The response of GFP-positive cells to 10 µM phenylephrine (Phe) was then measured over time. n = the number of individual cells quantitated.

We also wished to demonstrate directly the ability of both the isolated GPCRs and the GPCR-G protein fusions to form dimers. Constructs were N-terminally epitope-tagged with either the c-Myc or FLAG tags. After co-expression in HEK293 cells of both tagged forms of the _1b-adrenoreceptor, but not their separate expression, followed by cell mixing, immunoprecipitation with anti-FLAG antibodies resulted in the presence of anti-c-Myc immunoreactivity in the precipitate (Fig. 4A). SDS-PAGE demonstrated the presence of bands identified by the c-Myc antibody of apparent size 53 and 110 kDa, consistent with monomeric and dimeric forms of the _1b-adrenoreceptor. Anti-c-Myc immunoreactivity was also observed near the top of the gel, and this may represent either a higher order oligomer or aggregated protein (Fig. 4A). When equivalent experiments were performed with the _1b-adrenoreceptor-G₁₁ fusion protein similar results were obtained except that the anti-c-Myc-reactive bands were now of apparent mass 95 and 190kDa, consistent with the anticipated size of monomeric and dimeric forms of this fusion protein (Fig. 4A). Similar results were obtained for FLAG- and c-Myc-tagged forms of the histamine H1 receptor (Fig. 4B). The monomeric form of the isolated receptor migrated as a 50-kDa polypeptide with the dimeric form migrating as anticipated for a polypeptide of some 100 kDa (Fig. 4B). Again, as with the _1b-adrenoreceptor, a series of higher molecular mass species were also detected. When using the histamine H1 receptor-G₁₁ fusion protein both the monomeric and dimeric species were also easily detected (Fig. 4B).

View larger version (31K): [in this window] [in a new window]   FIG. 4. Co-immunoprecipitation of differentially epitope-tagged forms of both GPCRs and GPCR-G protein fusions. A, 1b-adrenoreceptor (AR) constructs. B, histamine H1 receptor constructs. HEK293 cells were mock-transfected (control) or transfected to express either FLAG (flag), c-Myc (myc), or a combination (flag + myc) of both epitope-tagged forms of the isolated GPCRs or GPCR-G protein fusions. Cells expressing either FLAG- or c-Myc-tagged forms were also mixed (mix). Samples were immunoprecipitated with anti-FLAG antibody, and these precipitates were resolved by SDS-PAGE and immunoblotted with anti-c-Myc antibodies.

A series of issues have been raised about the meaning and validity of GPCR dimerization data that rely exclusively on co-immunoprecipitation (3, 32). We, thus, monitored dimerization of both the isolated _1b-adrenoreceptor and the _1b-adrenoreceptor-G₁₁ fusion protein in intact HEK293 cells using tr-FRET. When co-expressing c-Myc- and FLAG-tagged forms of either the isolated GPCR or the fusion protein, a clear energy transfer signal was obtained in intact cells upon the addition of a combination of Eu³⁺-labeled anti-c-Myc antibodies as energy donor and allophycocyanin-labeled anti-FLAG antibodies as energy acceptor (Fig. 5A). An energy transfer signal was not obtained when the tagged forms of the GPCR constructs were expressed in separate populations of cells that were mixed before the addition of the antibodies. Equivalent results were obtained in HEK293 cells expressing N-terminal c-Myc- and FLAG-tagged forms of both the histamine H1 receptor and the histamine H1 receptor-G₁₁ fusion protein (Fig. 5B).

View larger version (14K): [in this window] [in a new window]   FIG. 5. tr-FRET demonstrates cell surface oligomers of both GPCRs and GPCR-G protein fusions. HEK293 cells were transfected to express c-Myc- and FLAG-tagged forms of either the 1b-adrenoreceptor (AR) or the 1b-adrenoreceptor-G11 fusion protein (A) or the histamine H1 receptor (H1R) or the histamine H1 receptor-G11 fusion protein (B). Cells were either co-transfected with the c-Myc- and FLAG-tagged cDNAs (filled bars) or separate pools of cells that were subsequently mixed (open bars) were transfected to express either the c-Myc- or FLAG-tagged forms. A combination of Eu3+-labeled anti-c-Myc- and allophycocyanin-labeled anti-FLAG antibodies were added, and tr-FRET was measured.

To examine the possibility of hetero-dimerization between the histamine H1 receptor and the _1b-adrenoreceptor and the mechanism of G protein activation by GPCR dimers we co-expressed a FLAG-tagged form of the histamine H1 receptor and the c-Myc-tagged form of the _1b-adrenoreceptor. After immunoprecipitation with anti-FLAG antibodies and SDS-PAGE, c-Myc immunoreactivity was detected in polypeptides of apparent molecular mass 50 and 100 kDa, consistent with the immunoprecipitation of histamine H1 receptor-_1b-adrenoreceptor hetero-dimers that are only partially separated by the electrophoresis conditions employed (Fig. 6A). tr-FRET studies after co-expression of the FLAG-tagged form of the histamine H1 receptor and the c-Myc-tagged form of the _1b-adrenoreceptor confirmed the presence of histamine H1 receptor/_1b-adrenoreceptor hetero-dimers at the cell surface (Fig. 6B), although the absolute level of the signal indicated that these hetero-dimers formed less efficiently than the corresponding homo-dimer pairs (see the y axis of Figs. 5, A and B, compared with Fig. 6B). As in the homo-dimer studies, no tr-FRET signal was observed when separate cell populations expressing each of these receptors were mixed before analysis (Fig. 6B).

View larger version (18K): [in this window] [in a new window]   FIG. 6. The 1b-adrenoreceptor and the histamine H1 receptor can form hetero-dimeric complexes. A, a FLAG-tagged form of the histamine H1 receptor (flag H1) and a c-Myc-tagged form of the 1b-adrenoreceptor (myc 1b) were expressed either individually or together (flag + myc) in HEK293 cells. Cells expressing the two constructs individually were also mixed before analysis. Samples were immunoprecipitated with anti-FLAG and, after SDS-PAGE and transfer, immunoblotted with anti-c-Myc-antibodies. B, cells either co-expressing FLAG H1 and c-Myc 1b (filled bars) or separate populations of cells expressing either of the two constructs that were then mixed were treated with a combination of Eu3+-labeled anti-c-Myc and allophycocyanin-labeled anti-FLAG antibodies, and tr-FRET was measured.

When L133D histamine H1 receptor-G₁₁ was co-expressed in EF88 cells with _1b-adrenoreceptor-G208AG₁₁, phenylephrine was able to elevate intracellular [Ca²⁺], but histamine was not (Fig. 7A). This can only occur if the _1b-adrenoreceptor activates the G protein physically linked to the L133D histamine H1 receptor. When the protocol was reversed by co-expression of L151D_1b-adrenoreceptor-G₁₁ and histamine H1 receptor-G208AG₁₁, histamine now caused elevation of intracellular [Ca²⁺], but phenylephrine did not (Fig. 7B). To extend this type of analysis the histamine H1 receptor-G₁₁ fusion was co-expressed with the isolated L151D _1b-adrenoreceptor that is unable to activate G protein and, thus, stimulate binding of [³⁵S]GTPS. Histamine stimulation of [³⁵S]GTPS binding was significantly reduced in comparison to membranes expressing the same level of only the histamine H1 receptor-G₁₁ fusion (Fig. 8A). Such data are consistent with the L151D _1b-adrenoreceptor generating inactive hetero-dimers with histamine H1 receptor-G₁₁ and indicate that the histamine H1 receptor in the hetero-dimer does not activate the G protein physically associated with it. The remaining signal produced by histamine in the co-transfection reflects that some functional histamine H1 receptor-G₁₁ homo-dimer is still formed in the presence of L151D _1b-adrenoreceptor. Indeed, when we co-expressed histamine H1 receptor-G₁₁ with increasing amounts of L151D _1b-adrenoreceptor cDNA, the ability of histamine to cause [³⁵S]GTPS binding in membranes expressing the same number of histamine H1 receptor binding sites decreased as levels of L151D _1b-adrenoreceptor cDNA were increased (Fig. 8A). Similar results were obtained after co-transfection of L151D _1b-adrenoreceptor with the histamine H1 receptor-G₁₁ in EF88 cells. Histamine stimulation of intracellular [Ca²⁺] was reduced markedly (Fig. 8B).

View larger version (31K): [in this window] [in a new window]   FIG. 7. Agonist function in hetero-dimeric complexes proceeds via trans-activation of receptor-associated G proteins. EF88 cells were transfected to co-express L151D 1b-adrenoreceptor-G11 + histamine H1 receptor-G208AG11 (A) or L133D histamine H1 receptor-G11 and 1b-adrenoreceptor-G208AG11 (B). The effect of 10 µM phenylephrine (Phe) or 1 mM histamine (Hist) on cellular [Ca2+]i was then assessed. In both panels, co-transfection with GFP demonstrated that only the cell in the field that responded to agonist was transfected.

View larger version (13K): [in this window] [in a new window]   FIG. 8. Co-expression of an inactive form of the 1b-adrenoreceptor suppresses signaling by a histamine H1 receptor-G11 fusion protein. HEK293 cells were transfected to express the histamine H1 receptor-G11 fusion protein and with increasing amounts of cDNA encoding the isolated, inactive L151D 1b-adrenoreceptor. A, membranes from these cells were used to measure expression of the histamine H1 receptor-G11 fusion protein, and amounts containing 25 fmol of specific [3H]mepyramine binding sites were used to measure basal and 1 mM histamine-stimulated [35S]GTPS binding. B, EF88 cells were transfected to express the histamine H1 receptor-G11 fusion protein (1) or to co-express this with L151D 1b-adrenoreceptor. The ability of 1 mM histamine to elevate cellular [Ca2+]i was then assessed. Data represent the means ± S.E. n = 6.

Co-expression of two distinct GPCRs must result in the presence of the respective homo-dimers as well as providing the potential for hetero-dimer formation. We wished to ensure that the reconstitution of Ca²⁺ signaling observed upon co-expression of L133D histamine H1 receptor-G₁₁ with _1b-adrenoreceptor-G208AG₁₁ did not reflect that only _1b-adrenoreceptor and histamine H1 receptor homo-dimers were present and that the _1b-adrenoreceptor-G208AG₁₁ homo-dimers were simply able to contact and activate G₁₁ linked to L133D histamine H1 receptor-G₁₁ homo-dimers. To enhance the levels of appropriately membrane-targeted G protein we generated a construct in which G₁₁ was linked to the C terminus of a c-Myc-tagged form of the N-terminal and first TM region of the _1b-adrenoreceptor (c-Myc-Nt-TM1_1b-G₁₁). This was transfected into HEK293 cells. Immunoblots of membrane fractions clearly demonstrated its expression as a doublet of 53 and 47 kDa whether detection was via anti-c-Myc (Fig. 9A) or anti-G protein antisera (data not shown). Based on immunodetection by the anti-c-Myc antibody, levels of c-Myc-Nt-TM1_1b-G₁₁ were significantly greater than those of the c-Myc-_1b-adrenoreceptor-G₁₁ fusion protein (Fig. 9A). [³⁵S]GTPS binding assays, at the end of which the c-Myc-Nt-TM1_1b-G₁₁ construct was immunoprecipitated with anti-c-Myc antibodies, confirmed this construct did not bind [³⁵S]GTPS in response to phenylephrine (Fig. 9B). Parallel experiments showed that the anti-c-Myc antibodies did capture phenylephrine-stimulated binding of [³⁵S]GTPS to the full-length c-Myc-tagged _1b-adrenoreceptor-G₁₁ fusion protein (Fig. 9B). However, co-expression of c-Myc-Nt-TM1_1b-G₁₁ with the isolated _1b-adrenoreceptor equally did not result in significant stimulation of [³⁵S]GTPS binding in anti-c-Myc immunoprecipitates (Fig. 9B), and this was also true when c-Myc-Nt-TM1_1b-G₁₁ was co-expressed with the _1b-adrenoreceptor-G208AG₁₁ fusion protein (Fig. 9B). Thus, simply increasing the concentration of membrane-associated G protein did not allow the _1b-adrenoreceptor or _1b-adrenoreceptor fusion protein homo-dimers to activate this G protein. It was possible that co-expression of c-Myc-Nt-TM1_1b-G₁₁ with forms of the _1b-adrenoreceptor did not result in their physical proximity and that this might account for the lack of activation of the membrane-tethered G₁₁ by phenylephrine. To assess this we co-expressed c-Myc-Nt-TM1_1b-G₁₁ with the FLAG-tagged _1b-adrenoreceptor-G₁₁ fusion protein. This pairing produced a strong tr-FRET signal after the addition of a combination of Eu³⁺-labeled anti-c-Myc and allophycocyanin-labeled anti-FLAG antibodies (Fig. 9C). This, again, was not observed when the two constructs were expressed in different cell populations that were mixed before analysis (Fig. 9C). These data demonstrate the proximity of co-expressed c-Myc-Nt-TM1_1b-G₁₁ and _1b-adrenoreceptor-G₁₁. Certain other equivalent _1b-adrenoreceptor TM-G₁₁ constructs did not behave in this fashion,² and these data are, thus, consistent with the concept that TM1 of the _1b-adrenoreceptor may be a contact interface for receptor homo-dimerization. Overall the data from co-expression of the pairs of inactive histamine H1 receptor- and _1b-adrenoreceptor-G protein fusions argue strongly that function must result from trans-activation of the G proteins within the dimers.

View larger version (12K): [in this window] [in a new window]   FIG. 9. Provision of excess membrane targeted G11 does not account for the reconstitution of function in cells expressing pairs of non-functional mutants. HEK293 cells were transfected to express the c-Myc-tagged 1b-adrenoreceptor-G11 fusion protein (1), G11 linked to the C terminus of a c-Myc-tagged form of the N-terminal and first transmembrane region of the 1b-adrenoreceptor (2), both the 1b-adrenoreceptor and the c-Myc-Nt-TM11b-G11 construct (3), or c-Myc-Nt-TM11b-G11 and the 1b-adrenoreceptor-G208AG11 fusion protein (4). A, membrane samples were resolved by SDS-PAGE and immunoblotted with anti-c-Myc antibodies. B, basal (open bars) and 10 µM phenylephrine stimulation (filled bars) of binding of [35S]GTPS recovered in anti-c-Myc immunoprecipitates. C, HEK293 cells were transfected to express either c-Myc-Nt-TM11b-G11 or the FLAG 1b-adrenoreceptor-G11 fusion protein (open bar), or these constructs were co-transfected (filled bar). tr-FRET was then measured as in Figs. 5 and 6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
That GPCRs can exist as dimers is now widely accepted (1-4). However, the basis and importance of this for function has been less easy to establish. Early studies on the ₂-adrenoreceptor indicated that a peptide corresponding to TMVI of the receptor was able to prevent dimerization and limit agonist stimulation of adenylyl cyclase activity (33). The general applicability of these observations, however, remains to be exemplified. Indeed, although recent studies on the leukotriene B₄ BLT1 receptor also support a key role for TMVI in homo-dimer formation (34), studies on the dopamine D2 receptor suggest a key role for TMIV (35). Furthermore, application of atomic force microscopy to the study of rhodopsin in native disc membranes (36-37) suggests that inter-dimeric contacts may involve TM segments IV and V. Distinct approaches provide evidence of roles for TM1 and TM2 in the stabilization of dimers of the yeast ste2 receptor (38) and of TM segments I, II, and/or IV in the complement C5a receptor (39). This range of results is fascinating, and combinations of direct experimentation and bio-informatic approaches (40-41) are likely to be required to provide understanding. Given the variation in potential orientation and organization between GPCRs, it is thus interesting that in experiments described herein, we have also provided evidence consistent with a role for TM1 in the dimerization of this receptor (Fig. 9C). As well as homo-dimers, many pairs of GPCRs are able to form hetero-dimers (1-4), and there is evidence that this results in production of distinct pharmacology and alterations in function (42-47). Although yet to be tested directly, there are suggestions that in such hetero-dimers, the individual GPCRs may contribute different elements to the dimer interface (48). We have demonstrated the capability of the _1b-adrenoreceptor and the histamine H1 receptor to form hetero-dimers. It is, thus, of importance to note that both of these GPCRs are expressed in arterial smooth muscle cells (49-50) and may, thus, form hetero-dimers in vivo.

Although class C is the smallest family of GPCRs, studies on the importance of dimerization and the mechanisms of signal transduction within the dimer have advanced most rapidly in this group (5). This reflects both that the -aminobutyric acid, type b receptor is an obligate hetero-dimer between two distinct but related GPCR gene products and that all members of this family have a large N-terminal extracellular domain that is responsible for binding of agonist ligands. It has, thus, been relatively easy to generate chimeric class C receptors that mix and match the extracellular and transmembrane and intracellular domains of individual GPCRs. The rhodopsin-like class A GPCRs comprise greater than 80% of the entire family present in the human genome (51-52). However, few class A GPCRs have structural features to facilitate such an approach, and GPCRs with aminergic agonists bind the ligands deep within the cleft produced by the architecture of the seven TM helices. By employing fusion proteins between both the _1b-adrenoreceptor and the histamine H1 receptor with the G protein G₁₁, we now show that these GPCRs dimerize and that this is not compromised by the addition of the G protein to the C-terminal tail of the GPCR. Equally, by employing tr-FRET to detect GPCR dimers in intact cells we were able to demonstrate the presence of these complexes at the cell surface. This also was not compromised by the addition of the G protein sequence to the C-terminal tail of the GPCRs. Furthermore, by introducing mutations that prevent agonist activation of the G protein into either the GPCR or the G protein, we produced pairs of distinct, non-functional fusion proteins that were able to restore agonist-mediated function when co-expressed. Many of the studies utilized combinations of epitope tagging and/or mutagenesis of the fusion proteins. As such, it was important to confirm that these modifications did not cause substantial alterations in the binding of ligands compared with the parental fusion proteins. We demonstrated that the binding of both agonist and antagonists ligands was not markedly different for any of the modified constructs (Table I), and these values were similar to those of the corresponding wild type GPCRs. Functional reconstitution was monitored in two ways. First, agonists were able to produce elevation of intracellular [Ca²⁺] in EF88 cells only after co-expression of two mutants that were each non-functional in isolation. EF88 cells lack expression of phospholipase C-coupled G proteins, and thus, it is necessary to introduce both a suitable GPCR and G protein into these cells to generate a Ca²⁺ signal (31). This assay had the obvious benefit that Ca²⁺ imaging allowed us to monitor functional dimerization in single cells and in the absence of excess G protein. However, although [Ca²⁺]_i can be calculated from such studies, this is an amplified signal and, thus, not ideal for quantitation of the effectiveness of dimerization. One of the earliest steps that can be measured in the signal transduction cascade is agonist-induced guanine nucleotide exchange on the G protein. This can be monitored conveniently by the binding of [³⁵S]GTPS. Historically, the characteristics of guanine nucleotide exchange by different G protein families had meant that this assay was only suitable for the pertussis toxin-sensitive members of the G_i-subfamily (53). However, recent addition of end of assay immunocapture steps has allowed it to be adapted to also measure activation of the G_s and G_q family G proteins (53). In all the [³⁵S]GTPS binding assays we initially measured the level of expression of each of the GPCR-G protein fusions by using saturation ³H-labeled antagonist binding studies. This allowed us to add membrane amounts containing defined quantities of the constructs to the assays. We have previously demonstrated that there is a linear increase in agonist-stimulated [³⁵S]GTPS binding with the addition of increasing amounts of a GPCR-G₁₁ fusion protein (31). When co-expressing the histamine H1 receptor-G208AG₁₁ and L133D histamine H1 receptor-G₁₁ fusion proteins, it required the presence of twice the number of ³H-labeled antagonist binding sites to generate approximately the same amount of agonist-stimulated [³⁵S]GTPS binding as when only the wild type histamine H1 receptor-G₁₁ fusion protein was expressed. This provides supporting evidence that the functional element is a dimer. If the functional histamine H1 receptor is a dimer, then stochastically, when co-expressing the two non-functional mutant fusions, half of the dimers produced should be non-functional because they will represent homo-interactions between either of the non-functional forms, i.e. histamine H1 receptor-G208AG₁₁ or L133D histamine H1 receptor-G₁₁. Only 50% of the dimers would be expected to be hetero-dimers containing one copy of histamine H1 receptor-G208AG₁₁ and one of L133D histamine H1 receptor-G₁₁ and, thus, be functional. These studies are also consistent with the idea that aminergic class A GPCRs function via trans-activation of their associated G proteins. The copy of the G protein in the dimer that can be activated is linked to the non-functional form of the GPCR, whereas the functional form of the GPCR is associated with non-functional G protein. Further studies, however, were required to provide greater support for this mechanism. To do so we took advantage of the known capacity of structurally related GPCRs to form hetero-dimers. Initial studies demonstrated that when co-expressed the histamine H1 receptor and the _1b-adrenoreceptor could be co-immunoprecipitated. Furthermore, co-expression in EF88 cells of L133D histamine H1 receptor-G₁₁ and _1b-adrenoreceptor-G208AG₁₁ resulted in phenylephrine but not histamine-mediated elevation of [Ca²⁺]_i. This can only occur if the _1b-adrenoreceptor activates the G protein physically linked to the inactive histamine H1 receptor (Fig. 10). When the experiment was reversed such that the inactive _1b-adrenoreceptor was linked to the wild type G protein and the wild type histamine H1 receptor was linked to the mutant G protein, histamine was functional, but phenylephrine was not. We extended this idea by examining the effectiveness of histamine to stimulate binding of [³⁵S]GTPS when the histamine H1 receptor fusion protein was co-expressed with increasing amounts of the isolated but inactive L151D _1b-adrenoreceptor. The effect of histamine was reduced. Such information is consistent with the concept than increasing levels of a histamine H1 receptor-G₁₁-L151D _1b-adrenoreceptor hetero-dimer reduces the amounts of the histamine H1 receptor-G₁₁ homo-dimer and that histamine binding to the hetero-dimer is unable to activate the G protein that is physically associated with the histamine H1 receptor. In this situation phenylephrine was inactive because L151D _1b-adrenoreceptor is unable to stimulate any G protein. A number of reports have indicated that GPCR-G protein fusions can interact with and activate endogenously expressed G proteins as well as the G protein element of the fusion (54-55). However, in these studies the GPCR-G protein fusions were expressed at very high levels that are within the range in which nonspecific "bystander" (56) effects have been reported due to physical proximity and crowding in the membrane. Use of EF88 cells eliminated the possibility of interaction with endogenous G proteins as they do not express G_q or G₁₁, and thus, effects have to reflect activation of the fused G proteins. Moreover, after introduction of the G208A mutation into the G protein element of the fusions, agonist stimulation of [³⁵S]GTPS binding in membranes of transfected HEK293 cells was virtually abolished. This indicates that at the level of expression achieved, there was virtually no activation of endogenous G_q or G₁₁ in HEK293 cells even though both are expressed (25). In the hetero-dimerization experiments in HEK293 cells, excess G protein is introduced in a 1:1 molar ratio with the second GPCR due to the 1:1 stoichiometry of GPCR and G protein defined by the fusion. To assess if the results could be ascribed simply to the presence of the extra G protein we provided extra G protein via an alternate strategy. To do so we generated a form of G₁₁ linked to the N terminus and first TM region of the _1b-adrenoreceptor. Equivalent constructs for other G proteins have been employed previously (55, 57-58), and it has been suggested that the link to a TM helix provides the G protein in a particularly effective orientation for activation (55). Although this construct could be expressed to markedly higher levels than the _1b-adrenoreceptor-G₁₁ fusions, the G protein was not activated by phenylephrine whether expressed alone or in combination with either the isolated _1b-adrenoreceptor or an _1b-adrenoreceptor-G₁₁ fusion. As discussed above, tr-FRET analysis confirmed the physical proximity of the _1b-adrenoreceptor-G₁₁ fusion and G protein linked to the TM1-containing construct after their co-expression. These studies provided further evidence that the reconstitution of signal with co-expression of non-functional pairs of GPCR-G protein fusions must reflect an internal trans-activation within the reconstituted dimer and are also consistent with a key role of TM1 as a dimer interface in the _1b-adrenoreceptor.

View larger version (28K): [in this window] [in a new window]   FIG. 10. Only hetero-dimers of GPCR-G protein fusion proteins should reconstitute function. Co-expression of a wild type (white) 1b-adrenoreceptor linked to a mutant (gray) G208AG11 and a mutant (gray) L133D histamine H1 receptor linked to wild type (white) G11 will result in the presence of each of 1b-adrenoreceptor and histamine H1 receptor homo-dimers and 1b-adrenoreceptor-histamine H1 receptor hetero-dimers. Only the hetero-dimers will be functional. In the example shown agonist at the 1b-adrenoreceptor (Phe) should generate a signal, but agonist at the histamine H1 receptor (Hist) will not. Demonstration of this principal is shown in Fig. 7A. The reverse combination of constructs results in the histamine H1 receptor element of the hetero-dimer being functional but not the 1b-adrenoreceptor element. This is demonstrated in Fig. 7B. In the diagram dimer formation is indicated to result from a linear packing arrangement. Other possible models have been described and discussed (40). PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Davidson Bldg., University of Glasgow, Glasgow G12 8QQ, Scotland, UK. Tel.: 44-141-330-5557; Fax: 44-141-330-4620; E-mail: g.milligan{at}bio.gla.ac.uk.

¹ The abbreviations used are: GPCR, G protein-coupled receptor; TM, transmembrane; tr-FRET, time-resolved fluorescence resonance energy transfer; GTPS, guanosine 5'-3-O-(thio)triphosphate; GFP, green fluorescent protein.

² J. J. Carrillo and G. Milligan, manuscript in preparation.

	ACKNOWLEDGMENTS

 
These studies were supported by the Medical Research Council, the Biotechnology and Biosciences Research Council, and the Wellcome Trust.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Bouvier, M. (2001) Nat. Rev. Neurosci. 2, 274-286[CrossRef][Medline] [Order article via Infotrieve]
2. Devi, L. A. (2001) Trends Pharmacol. Sci. 22, 532-537[CrossRef][Medline] [Order article via Infotrieve]
3. Milligan, G. (2001) J. Cell Sci. 114, 1265-1271[Abstract]
4. George, S. R., O'Dowd, B. F., and Lee, S. P. (2002) Nat. Rev. Drug Discov. 1, 808-820[CrossRef][Medline] [Order article via Infotrieve]
5. Parmentier, M. L., Prezeau, L., Bockaert, J., and Pin, J. P. (2002) Trends Pharmacol. Sci. 23, 268-274[CrossRef][Medline] [Order article via Infotrieve]
6. White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M., and Marshall, F. H. (1998) Nature 396, 679-682[CrossRef][Medline] [Order article via Infotrieve]
7. Jones, K. A., Borowsky, B., Tamm, J. A., Craig, D. A., Durkin, M. M., Dai, M., Yao, W. J., Johnson, M., Gunwaldsen, C., Huang, L. Y., Tang, C., Shen, Q., Salon, J. A., Morse, K., Laz, T., Smith, K. E., Nagarathnam, D., Noble, S. A., Branchek, T. A., and Gerald, C. (1998) Nature 396, 674-679[CrossRef][Medline] [Order article via Infotrieve]
8. Kaupmann, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W., Beck, P., Mosbacher, J., Bischoff, S., Kulik, A., Shigemoto, R., Karschin, A., and Bettler, B. (1998) Nature 396, 683-687[CrossRef][Medline] [Order article via Infotrieve]
9. Robbins, M. J., Calver, A. R., Filippov, A. K., Hirst, W. D., Russell, R. B., Wood, M. D., Nasir, S., Couve, A., Brown, D. A., Moss, S. J., and Pangalos, M. N. (2001) J. Neurosci. 21, 8043-8052[Abstract/Free Full Text]
10. Grosse, R., Schoneberg, T., Schultz, G., and Gudermann, T. (1997) Mol. Endocrinol. 11, 1305-1318[Abstract/Free Full Text]
11. Lee, S. P., O'Dowd, B. F., Ng, G. Y. K., Varghese, G., Akil, H., Mansour, A., Nguyen, T., and George, S. R. (2000) Mol. Pharmacol. 58, 120-128