GPCR kinase knockout cells reveal the impact of individual GRKs on arrestin binding and GPCR regulation.

G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor-arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of ß-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with ß-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and ß-arrestin mutants, we present evidence for differential receptor-ß-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and ß-arrestin complex formation.

Superagonism at G protein-coupled receptors and beyond.

Ligands targeting GPCRs can be categorized according to their intrinsic efficacy to trigger a specific, receptor-mediated response. A ligand endowed with the same level of efficacy as the endogenous agonist can be classified as a full agonist, whereas a compound that displays greater efficacy, that is, higher receptor signalling output than the endogenous agonist, can be called a superagonist. Subsequent to GPCR activation, an intracellular signalling cascade is set in motion, which may generate substantial amplification of the signal. This may obscure superagonism in pharmacological assays and, therefore, the definition of superagonism necessitates a combination of operational approaches, reduction of spare receptors or estimation of receptor activation close to the receptor level to quantify relative agonist efficacies in a particular system. The first part of this review will compare GPCR superagonism with superagonism in the field of immunology, where this term is well established. In the second part, known GPCR superagonists will be reviewed. Then, the experimental and analytical challenges in the deconvolution of GPCR superagonism will be addressed. Finally, the potential benefit of superagonism is discussed. The molecular mechanisms behind GPCR superagonism are not completely understood. However, crystallography shows that agonist binding alone is not sufficient for a fully active receptor state and that binding of the G protein is at least equally important. Accordingly, the emerging number of reported superagonists implies that ligand-induced receptor conformations more active than the ones stabilized by the endogenous agonist are indeed feasible. Superagonists may have therapeutic potential when receptor function is impaired or to induce negative feedback mechanisms. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Heteromerization of GPR55 and cannabinoid CB2 receptors modulates signalling.

BACKGROUND AND PURPOSE: Heteromerization of GPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer-selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositol GPCR 55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB2 receptor) in human neutrophils, we investigated the possible heteromerization of CB2 receptors with GPR55. EXPERIMENTAL APPROACH: The direct interaction of human GPR55 and CB2 receptors heterologously expressed in HEK293 cells was assessed by co-immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross-talk on signalling was investigated at downstream levels by label-free real-time methods (Epic dynamic mass redistribution and CellKey impedance assays), ERK1/2-MAPK activation and gene reporter assays. KEY RESULTS: GPR55 and CB2 receptors co-localized on the surface of HEK293 cells, co-precipitated in membrane extracts and formed heteromers in living HEK293 cells. Whereas heteromerization led to a reduction in GPR55-mediated activation of transcription factors (nuclear factor of activated T-cells, NF-κB and cAMP response element), ERK1/2-MAPK activation was potentiated in the presence of CB2 receptors. CB2 receptor-mediated signalling was also affected by co-expression with GPR55. Label-free assays confirmed cross-talk between the two receptors. CONCLUSIONS AND IMPLICATIONS: Heteromers, unique signalling units, form in HEK293 cells expressing GPR55 and CB2 receptors. The signalling by agonists of either receptor was governed (i) by the presence or absence of the partner receptors (with the consequent formation of heteromers) and (ii) by the activation state of the partner receptor.

Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor.

BACKGROUND AND PURPOSE: Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such 'superagonism' has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a 'superagonist'. EXPERIMENTAL APPROACH: Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. KEY RESULTS: In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi /Gs signalling competence. In the orthosteric loss-of-function mutant M2 -Y104(3.33) A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. 'Superagonism' is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure-signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that 'superagonism' of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. CONCLUSION AND IMPLICATIONS: Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR 'superagonism' is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators.

Pro-fibrotic processes in human lung fibroblasts are driven by an autocrine/paracrine endothelinergic system.

BACKGROUND AND PURPOSE: Since endothelin (ET) may act as pro-fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro-fibrotic ET effects were studied in human lung fibroblasts. EXPERIMENTAL APPROACH: MRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by elisa. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [(3) H]-thymidine was determined as measure of proliferation and that of [(3) H]-proline for collagen synthesis. Phospho-p42/44 MAP kinase was determined by Western blot. KEY RESULTS: ET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-ß caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin. CONCLUSIONS AND IMPLICATIONS: hLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.

The ß2-subtype of adrenoceptors mediates inhibition of pro-fibrotic events in human lung fibroblasts.

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by ß-adrenoceptor activation. We aimed, first, to characterise the expression pattern of ß-adrenoceptor subtypes in human lung fibroblasts and, second, to probe ß-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding ß(2)-adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for ß(1)- and ß(3)-adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed ß-agonist-induced G(s)-signalling. Proliferation of MRC-5 cells (determined by [(3)H]-thymidine incorporation) was significantly inhibited by ß-agonists including the ß(2)-selective agonist formoterol (-logIC(50), 10.2) and olodaterol (-logIC(50), 10.6). Formoterol's effect was insensitive to ß(1)-antagonism (GCP 20712, 3 µM), but sensitive to ß(2)-antagonism (ICI 118,551; apparent, pA (2), 9.6). Collagen synthesis in MRC-5 cells (determined by [(3)H]-proline incorporation) was inhibited by ß-agonists including formoterol (-logIC(50), 10.0) and olodaterol (-logIC(50), 10.3) in a ß(2)-blocker-sensitive manner. α-Smooth muscle actin, a marker of myo-fibroblast differentiation, was down-regulated at the mRNA and the protein level by about 50% following 24 and 48 h exposure to 1 nM formoterol, a maximally active concentration. In conclusion, human lung fibroblasts exclusively express ß(2)-adrenoceptors and these mediate inhibition of various markers of pro-fibrotic cellular activity. Under clinical conditions, anti-fibrotic actions may accompany the therapeutic effect of long-term ß(2)-agonist treatment of bronchial asthma and COPD.

A novel antagonist of CRTH2 blocks eosinophil release from bone marrow, chemotaxis and respiratory burst.

BACKGROUND: Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) has been revealed to be a novel receptor for prostaglandin (PG) D(2), which is a major mast cell product released during the allergic response. The aim of this study was to analyze the effects of a newly developed small molecule antagonist of CRTH2, Cay10471, on eosinophil function with respect to recruitment, respiratory burst and degranulation. METHODS: Chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils were determined using microBoyden chambers. Eosinophil release from bone marrow was investigated in the in situ perfused guinea pig hind limb preparation. Respiratory burst and degranulation were measured by flow cytometry. RESULTS: Cay10471 bound with high affinity to recombinant human and guinea pig CRTH2, but not DP, receptors. The antagonist prevented the PGD(2)-induced release of eosinophils from guinea pig bone marrow, and inhibited the chemotaxis of guinea pig bone marrow eosinophils and human peripheral blood eosinophils. Pretreatment with PGD(2) primed eosinophils for chemotaxis towards eotaxin, and this effect was prevented by Cay10471. In contrast, PGD(2) inhibited the C5a-induced up-regulation of CD63, a cellular marker of degranulation, in a Cay10471-sensitive manner. Finally, Cay10471 abolished the respiratory burst of eosinophils upon stimulation by PGD(2). CONCLUSION: These data further emphasize the importance of CRTH2 in eosinophil function and show that Cay10471 is a highly potent and selective antagonist of PGD(2)-induced eosinophil responses. Cay10471 might hence be a useful compound for the treatment of allergic diseases.

Is Galpha16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors?

Identification of natural ligands for orphan G-protein-coupled receptors will help expand the boundaries of physiology and pharmacology. Powerful approaches are needed that can pair biologically active ligands with their corresponding receptors. Many attempts have been made to set up universal screening schemes such that receptor activation by its cognate ligand is transduced into a common intracellular signal that is amenable to high-throughput screening analysis. One possibility that achieves such a 'universal assay' takes advantage of the promiscuous nature of the G-protein subunit Galpha16. However, a truly critical look at Galpha16 is still required. In this article, the strengths, weaknesses, problems and pitfalls that are associated with the use of Galpha16 will be discussed, and suggestions of how problems might be overcome with an optimized universal G-protein system will be proposed.

Muscarinic allosteric modulation: M2/M3 subtype selectivity of gallamine is independent of G-protein coupling specificity.

Among the five subtypes of muscarinic acetylcholine receptors, the sensitivity towards allosteric modulation is generally higher in M2 and M4 receptors that preferentially couple to inhibitory G-proteins of the Gi/o type than in M1, M3, and M5 that preferentially couple to stimulatory G-proteins such as Gq/11. We aimed to check whether the high allosteric sensitivity of the M2 receptor compared to M3 is related to the differential G-protein coupling preference. As the third intracellular loop (i3) is known to be the major determinant in receptor G-protein coupling specificity, we used wild-type M2 and M3 receptors and the related chimeric constructs with exchanged i3-loops, i.e., M2 containing M3-i3 (M2/M3-i3) and M3 containing M2-i3 (M3/M2-i3). The allosteric effect of the archetypal modulator gallamine on the dissociation and the equilibrium binding of [3H]N-methylscopolamine ([3H]NMS) was measured in membranes of mouse A9L cells stably expressing the wild-type and the chimeric receptors (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C). The dissociation of [3H]NMS was monophasic under all conditions studied. Control values of t 1/2 were (means +/- SEM, n = 4-7): M2: 3.8 +/- 0.2 min, M2/M3-i3: 4.8 +/- 0.3 min, M3:43.3 +/- 4.2 min, M3/M2-i3: 41.1 +/- 3.6 min. At M2 receptors, 0.2 microM gallamine allosterically reduced the apparent rate constant of dissociation k-1 to 51 +/- 5% of the control value (n = 5). At M2/M3-i3 the allosteric potency of gallamine was not significantly changed (0.2 microM gallamine --> k-1 = 61 +/- 4%, n = 7). At M3, a 20-fold higher concentration was required for an equieffective allosteric action (10 microM gallamine --> k-1 = 51 +/- 5%, n = 5). The potency of gallamine at M3/M2-i3 was not increased compared with M3 receptors (10 microM gallamine --> k-1 = 73 +/- 2%, n = 4) but even significantly diminished. [3H]NMS equilibrium binding experiments revealed that neither the binding constants of gallamine at free receptor subtypes (pKA,M2: 7.57 +/- 0.04, n = 4; pKA,M3: 5.56 +/- 0.13, n = 3) nor the factors of negative cooperativity with [3H]NMS (alphaM2 = 31 +/- 1, alphaM3 = 3 +/- 0.4) were affected by the exchanged i3-loops (pKA,M2/M3-i3: 7.65 +/- 0.03, pKA,M3/M2-i2: 5.35 +/- 0.24, alphaM2/M3-i3= 30 +/- 2, alphaM3/M2-i2 = 3 +/- 0.7). In conclusion, the different sensitivities of M2 and M3 receptors towards allosteric modulation by gallamine are not related to the G-protein coupling specificity of the receptors.

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast.

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice.

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.

Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis.

To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.

Structure-activity relationships in a series of bisquaternary bisphthalimidine derivatives modulating the muscarinic M(2)-receptor allosterically.

Hexane-bisammonium-type compounds containing lateral phthalimide moieties are well-established ligands of the common allosteric binding site of muscarinic M(2) receptors. Previous structure-activity relationships (SAR) revealed two positively charged centers and two lateral phthalimide moieties in a defined arrangement to be essential of a high allosteric potency. The purpose of this study was to replace one carbonyl group of the phthalimides with hydrogens, hydroxy, alkoxy, phenyl, benzyl, and benzylidene groups in order to check the influence of these substituents on the allosteric activity in antagonist-linked receptors. The analysis of the quantitative SAR indicated that a high allosteric potency is related to a certain amount of rigidity as well as polarizibility and the ability to form hydrophobic interactions.

Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice.

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions of the central and peripheral nervous system. Primarily because of the lack of receptor subtype-selective ligands, the precise physiological roles of the individual muscarinic receptor subtypes remain to be elucidated. Interestingly, the M(4) receptor subtype is expressed abundantly in the striatum and various other forebrain regions. To study its potential role in the regulation of locomotor activity and other central functions, we used gene-targeting technology to create mice that lack functional M(4) receptors. Pharmacologic analysis of M(4) receptor-deficient mice indicated that M(4) receptors are not required for muscarinic receptor-mediated analgesia, tremor, hypothermia, and salivation. Strikingly, M(4) receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses (as compared with their wild-type littermates) after activation of D1 dopamine receptors. These results indicate that M(4) receptors exert inhibitory control on D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are coexpressed at high levels. Our findings offer new perspectives for the treatment of Parkinson's disease and other movement disorders that are characterized by an imbalance between muscarinic cholinergic and dopaminergic neurotransmission.

Structure-function analysis of muscarinic receptors and their associated G proteins.

Each member of the muscarinic receptor family (M1-M5) can interact only with a limited subset of the many structurally closely related heterotrimeric G proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level, we have used the G(i/0)-coupled M2 and the Gq/11-coupled M3 muscarinic receptors as model systems. We developed a genetic strategy involving the coexpression of wild type or mutant muscarinic receptors with hybrid or mutant G protein alpha subunits to identify specific, functionally relevant receptor/G protein contact sites. This approach led to the identification of N- and C-terminal amino acids on alpha(q) and alpha(i) that are critical for maintaining proper receptor/G protein coupling. Moreover, several receptor sites were identified that are likely to be contacted by these functionally critical G alpha residues. To gain deeper insight into muscarinic receptor structure, we recently developed a cysteine disulfide cross-linking strategy, using the M3 muscarinic receptor as a model system. Among other structural modifications, this approach involves the removal of most native cysteine residues by site-directed mutagenesis, the insertion of three factor Xa cleavage sites into the third intracellular loop, and systematic 'reintroduction' of pairs of cysteine residues. Following treatment of receptor-containing membrane preparations with factor Xa and oxidizing agents, disulfide cross-linked products can be identified by immunoprecipitation and immunoblotting studies. This approach should greatly advance our knowledge of the molecular architecture of muscarinic and other G protein-coupled receptors.

Bisquaternary ligands of the common allosteric site of M2 acetylcholine receptors: search for the minimum essential distances between the pharmacophoric elements.

Structurally diverse molecules, such as alcuronium, gallamine, and tubocurarine as well as W84 and WDUO, are known to interact allosterically with ligand binding to muscarinic M2 acetylcholine receptors. Preliminary molecular modeling studies revealed two positive charges in the middle and two lateral aromatic areas to be essential elements of a high allosteric potency. To find out the optimum distances between these pharmacophoric elements, a systematic variation of the spacer in the series of W84, WDUO, and IWDUO compounds was performed. The allosteric reduction of the rate of dissociation of the antagonist [3H]-N-methylscopolamine from porcine heart M2 receptors served as a test system. The minimal essential distance between the positive charges was found to be 10 A. The length of the peripheral spacers connecting the positive charge and the lateral aromatic moiety appears to depend on the chemical functionality; the peripheral spacers have to be long and flexible enough to position the aromatic skeletons in the spatial neighborhood of the alkane middle chain: in the case of an oxime ether containing peripheral spacer, six atoms are required, and in the case of an alkane chain, four carbon atoms are necessary to adopt the pharmacophoric S-shape conformation.

Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice.

Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.

Structure-function analysis of muscarinic acetylcholine receptors.

The structural basis underlying the G protein coupling selectivity of different muscarinic receptor subtypes was analyzed by using a combined molecular genetic/biochemical approach. These studies led to the identification of key residues on the receptors as well as the associated G proteins that are critically involved in determining proper receptor/G protein recognition. Mutational analysis of the m3 muscarinic receptor showed that most native cysteine residues are not required for productive receptor/G protein coupling. The putative extracellular disulfide bond was found to be essential for efficient trafficking of the receptor protein to the cell surface but not for receptor-mediated G protein activation.

Functional characterization of a series of mutant G protein alphaq subunits displaying promiscuous receptor coupling properties.

The N termini of two G protein alpha subunits, alphaq and alpha11, differ from those of other alpha subunits in that they display a unique, highly conserved six-amino acid extension (MTLESI(M)). We recently showed that an alphaq deletion mutant lacking these six amino acids (in contrast to wild type alphaq) was able to couple to several different Gs- and Gi/o-coupled receptors, apparently due to promiscuous receptor/G protein coupling (Kostenis, E., Degtyarev, M. Y., Conklin, B. R., and Wess, J. (1997) J. Biol. Chem. 272, 19107-19110). To study which specific amino acids within the N-terminal segment of alphaq/11 are critical for constraining the receptor coupling selectivity of these subunits, this region of alphaq was subjected to systematic deletion and alanine scanning mutagenesis. All mutant alphaq constructs (or wild type alphaq as a control) were coexpressed (in COS-7 cells) with the m2 muscarinic or the D2 dopamine receptors, two prototypical Gi/o-coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Surprisingly, all 14 mutant G proteins studied (but not wild type alphaq) gained the ability to productively interact with the two Gi/o-linked receptors. Similar results were obtained when we examined the ability of selected mutant alphaq subunits to couple to the Gs-coupled beta2-adrenergic receptor. Additional experiments indicated that the functional promiscuity displayed by all investigated mutant alphaq constructs was not due to overexpression (as compared with wild type alphaq), lack of palmitoylation, or initiation of translation at a downstream ATG codon (codon seven). These data are consistent with the notion that the six-amino acid extension characteristic for alphaq/11 subunits forms a tightly folded protein subdomain that is critical for regulating the receptor coupling selectivity of these subunits.