Receptor-Galpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.     

Bimodal regulation of cyclic AMP by muscarinic receptors. Involvement of multiple G proteins and different forms of adenylyl cyclase

In membranes of rat olfactory bulb, muscarinic receptor agonists stimulate basal adenylyl cyclase activity . This response is inhibited by a number of muscarinic receptor antagonists with a rank order of potency suggesting the involvement of the M4 muscarinic receptor subtype. The stimulatory effect does not require Ca2+ and occurs independently of activation of phosphoinositide hydrolysis. Pretreatment with pertussis toxin completely prevents the muscarinic stimulation of adenylyl cyclase, indicating the participation of G proteins of the Gi/Go family. Immunological impairment of the G protein, Gs, also reduces the muscarinic response, whereas concomitant activation of Gs-coupled receptors by CRH or VIP results in a synergistic stimulation of adenylyl cyclase activity. Although these data suggest a role for Gs, a body of evidence indicates that the muscarinic receptors do not interact directly with this G protein. Moreover, the Ca2+/calmodulin (Ca2+/CaM)- and forskolin-stimulated enzyme activities are inhibited by muscarinic receptor activation in a pertussis toxin-sensitive manner and with a pharmacological profile similar to that observed for the stimulatory response. These data indicate that in rat olfactory bulb M4 muscarinic receptors exert a bimodal control on cyclic AMP formation through a sequence of events that may involve activation of Gi/Go proteins, synergistic interaction with Gs and differential modulation of Ca2+/CaM-independent and -dependent forms of adenylyl cyclase.     

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.     

Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics

The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel.     

Reconstitution of the muscarinic acetylcholine receptor. Guanine nucleotide-sensitive high affinity binding of agonists to purified muscarinic receptors reconstituted with GTP-binding proteins (Gi and Go)

Muscarinic acetylcholine receptors purified from porcine brain were reconstituted with two kinds of GTP-binding proteins (Gi and Go). The binding of agonists was affected by guanine nucleotides when the receptor was reconstituted with either Gi or Go, but not in the absence of one of the GTP-binding proteins. The displacement curves with agonists for the [3H]quinuclidinyl benzylate [( 3H]QNB) binding were explained by assuming there are two sites with different affinities for a given agonist. The proportion of the high affinity site increased with increasing concentrations of the GTP-binding proteins, and the maximum value represented 50-70% of the total [3H]QNB-binding sites. Reconstitution of the receptor with both Gi and Go did not increase the proportion any further. These results indicate that Gi and Go interact with the same site, which rules out the possibility that there are two kinds of muscarinic receptors, one interacting with Gi and the other with Go. GDP as well as GTP decreased the affinity for the agonists of the muscarinic receptors reconstituted with Gi or Go. The conversion of GDP to GTP during the incubation was less than 1%, indicating that the effect of GDP is not due to its conversion to GTP, and that the binding of either GTP or GDP with the GTP-binding proteins suppresses their interaction with the receptor.     

Different roles for Gi and Go proteins in modulation of adenylyl cyclase type-2 activity

The effect of Gi/o protein-coupled receptors on adenylyl cyclase type 2 (AC2) has been studied in Sf9 insect cells. Stimulation of cells expressing AC2 with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to a twofold stimulation of cAMP synthesis that could be blocked with the protein kinase C inhibitor GF109203X. Activation of a coexpressed alpha2A-adrenoceptor or muscarinic M4 receptor inhibited the stimulation by TPA almost completely in a pertussis toxin-sensitive manner. Activation of Gs proteins switched the response of the alpha2A-adrenoceptor to potentiation of prestimulated AC2 activity. The potentiation, but not the inhibition, could be blocked by a Gbetagamma scavenger. A novel methodological approach, whereby signalling through endogenous G proteins was ablated, was used to assess specific G protein species in the signal pathway. Expression of Go proteins (alphao1 + beta1gamma2) restored both the inhibition and the potentiation, whereas expression of Gi proteins (alphai1 + beta1gamma2) resulted in a potentiation of both the TPA- and the Gs-stimulated AC2 activity. The data presented supports the view of AC2 as a molecular switch and implicates this isoform as a target for Go protein-linked signalling.     

Regulation of cyclic AMP accumulation in lymphoid cells

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

First fatty acylated dipeptides to affect muscarinic receptor ligand binding

Fatty acylated dipeptides homologous to Gi alpha N-termini affect ligand binding to muscarinic acetylcholine receptors. Myristylglycine-serine containing dipeptides decrease antagonist binding at both M1 and M2 muscarinic receptors. Palmitate on the serine analogous to native palmitoylated cysteine affords dipeptide which selectively decreases the number of high affinity agonist binding sites at M2 but not M1 receptor.     

Novel signalling events mediated by muscarinic receptor subtypes

The M2 muscarinic acetylcholine receptor (mAChR) activates Gi protein coupled pathways, such as stimulation of G-protein activated inwardly rectifying K channels (GIRKs). Here we report a novel heterologous desensitization of these GIRK currents, which appeared to be specifically induced by M2/M4 mAChR stimulation, but not via adenosine (Ado) and alpha2-adrenergic receptors (AR). This heterologous desensitization reflected an inhibition of the GIRK signalling pathway downstream of G-protein activation. It was mediated in a membrane-delimited fashion via a PTX insensitive GTP dependent pathway and could be competed with exogenous Gbetagamma. The activation of M3 mAChR/Gq coupled receptors potently inhibited GIRK currents similar as M2 mAChR. By monitoring simultaneously the response of A1 adenosine receptor (AdoR) activation on N-type Ca2+ channels and GIRK channels, the stimulation of M3 mAChR was found to cause an inhibition of the Ado response in both effector systems, suggesting that the inhibition occurred at the level of the G-protein common to both effectors. These results indicated that Gq proteins inhibit pathways that are commonly regulated by Gbetagamma proteins.     

G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Fluoroaluminates mimic muscarinic- and oxytocin-receptor-mediated generation of inositol phosphates and contraction in the intact guinea-pig myometrium. Role for a pertussis/cholera-toxin-insensitive G protein.

1. In the intact guinea-pig myometrium, carbachol and oxytocin stimulated a specific receptor-mediated phospholipase C activation, catalysing the breakdown of PtdIns(4,5)P2 with the sequential generation of InsP3, InsP2 and InsP. Stimulation of muscarinic receptors also triggered an inhibition of cyclic AMP accumulation caused by prostacyclin. 2. NaF plus AlCl3 mimicked the effects of carbachol and oxytocin by inducing, in a dose-dependent manner, the generation of all three inositol phosphates as well as uterine contractions. AlCl3 enhanced the fluoride effect, supporting the concept that A1F4- was the active species. Under similar conditions, fluoroaluminates activated the guanine nucleotide regulatory protein Gi, reproducing the inhibitory effect of carbachol on cyclic AMP concentrations. 3. Both carbachol- and oxytocin-mediated increases in inositol phosphates, as well as contractions, were insensitive to pertussis toxin, under conditions where the expression of Gi was totally prevented. Cholera toxin, which activates Gs and enhances cyclic AMP accumulation, failed to affect basal or oxytocin-evoked inositol phosphate generation, but induced a slight, though consistent, attenuation of the muscarinic inositol phosphate response, which was similarly evoked by forskolin. 4. The data provide evidence that, in the myometrium, (a) a G protein mediates the generation of inositol phosphates and the Ca2+-dependent contractile event, (b) the relevant G protein that most probably couples muscarinic and oxytocin receptors to phospholipase C is different from Gi and Gs, the proteins that couple receptors to adenylate cyclase, and (c) cyclic AMP does not seem to control the phosphoinositide cycle, but rather exerts a negative regulation at the muscarinic-receptor level.     

Gi affects the agonist-binding properties of beta-adrenoceptors in the presence of Gs

Pertussis-toxin-catalyzed ADP-ribosylation of Gi in S49 membranes, but not in S49AC- membranes, which lack Gs, induces a threefold reduction of isoproterenol affinity to the beta-adrenoceptors. A similar treatment of turkey erythrocyte membranes, which are devoid of functional Gi, has no effect on beta-agonist affinity to their beta-adrenoceptors. Non-hydrolyzable analogs such as GTP[S] induce a larger decrease in beta-adrenoceptor affinity in S49 cells towards the agonist isoproterenol as compared to pertussis-toxin-catalyzed ADP-ribosylation of Gi. These results suggest that Gi affects beta-adrenoceptor affinity to its agonist and that this interaction requires the presence of Gs. It seems, therefore, that Gi physically interacts with Gs to exert its effects on the receptor and probably on adenylate cyclase as well. Our ability to detect (a) the effect of pertussis-toxin-catalyzed ADP-ribosylation in S49 cells on beta-agonist affinity and (b) the quantitative difference between the effect of pertussis toxin (approx. threefold) and GTP[S] (fivefold to sevenfold) depends on the use of a simple but rigorous method to study in detail the affinity of beta-agonists to their receptors. This method seems to be superior to the analysis of displacement curves as a means to examine receptor-ligand interactions.     

Reconstitution reveals additional roles for N- and C-terminal domains of g(alpha) in muscarinic receptor coupling

The molecular basis for selectivity of M1 and M2 muscarinic receptor coupling to heterotrimeric G proteins has been studied using receptors expressed in Sf9 cell membranes and reconstituted with purified chimeric G(alpha) subunits containing different regions of Gi1alpha and Gq(alpha). The abilities of G protein heterotrimers containing chimeric alpha subunits to stabilize the high-affinity state of the receptors for agonist and to undergo receptor stimulated guanine nucleotide exchange was compared with G protein heterotrimers containing either native Gi1alpha or Gq(alpha). The data confirm the importance of the proper context of the C-terminus of Galpha by demonstrating that the C-terminus of Gi1alpha, when placed in the context of Gq(alpha), prevents coupling to muscarinic M1 receptors, while the C-terminus of Gq(alpha), when placed in the context of Gi1alpha, prevents coupling to muscarinic M2 receptors. However, C-terminal amino acids of Gq(alpha) placed in the context of Gi1alpha were not sufficient to allow M1 receptor coupling, nor were C-terminal amino acids of Gi1alpha placed in the context of Gq(alpha) sufficient for M2 receptor coupling. The unique six amino acid N-terminal extension of Gq(alpha) when added to the N-terminus of Gi1alpha neither prevented M2 receptor coupling nor permitted M1 receptor coupling. A Gi1alpha-based chimera containing both N- and C-terminal regions of Gq(alpha) gained the ability to productively couple M1 receptors suggesting that the proper context of both N- and C-termini is required for muscarinic receptor coupling.     

Detection of G protein-activator regions in M4 subtype muscarinic, cholinergic, and alpha 2-adrenergic receptors based upon characteristics in primary structure.

The 14-residue region Arg2410-Lys2423 of the human insulin-like growth factor II receptor possesses the ability to stimulate Gi, the activity being dependent on two structural characteristics: (i) at least two basic residues at the N-terminal side and (ii) the C-terminal motif, B-B-X-B or B-B-X-X-B (where B is a basic residue and X is a non-basic residue). The regions satisfying (i) and (ii) with 10 less than or equal to residue length less than or equal to 26 were located in all of the third inner loops and some of the other intracellular domains of the Gi-coupled M4 sub-type muscarinic cholinergic receptor (M4AChR) and the alpha 2-adrenergic receptor (alpha 2AR). Both the second inner loop 130-147 and the C-terminal portion of the third inner loop 382-400 (MIII) of human M4AChR had the ability to stimulate G proteins with the order Gi approximately Go greater than Gs, but only MIII could activate Gi/Go at nanomolar concentrations. In contrast, the N-terminal portion of the third inner loop 218-228 of human alpha 2AR-C10 activated Gi, Go, and Gs at micromolar concentrations with equal potency, whereas the further C-terminal portion of the third inner loop 301-313 of this receptor lacked the ability to activate any G protein. Among these active regions, only MIII indicated Mg(2+)-dependent Gi-stimulating function. Therefore, the search for the regions satisfying (i) and (ii) was useful to localize the G protein-activating activity of Gi-coupled receptors in limited regions, which were not always in the C-terminal portions of the third intracellular loops and activated G proteins in various modes of actions.     

Interactions Between Phospholipase C-Coupled and N-Methyl-D-Aspartate Receptors in Cultured Cerebellar Granule Cells: Protein Kinase C Mediated Inhibition of N-Methyl-D-Aspartate Responses

The N-methyl-D-aspartate (NMDA) receptor of rat cerebellar granule cells in primary culture is inhibited by phospholipase C-coupled receptor activation. In the absence of ionotropic agonist, cells modulate their cytoplasmic free Ca2+, [Ca2+]c, in response to stimulation of M3 muscarinic receptors, metabotropic glutamate receptors, and endothelin receptors by the respective agonists carbachol, trans-1-amino-1,3-cyclopentanedicarboxylic acid, and endothelin-1. The response is consistent with the ability of phospholipase C-coupled receptors to release a pool of intracellular Ca2+ and induce a subsequent Ca2+ entry into the cell; both of these responses can be abolished by discharge of internal Ca2+ stores with low concentrations of ionomycin or thapsigargin. In the case of cells stimulated with NMDA, the [Ca2+]c response to the phospholipase C-coupled agonists is complex and agonist dependent; however, in the presence of ionomycin each agonist produces a partial inhibition of the NMDA component of the [Ca2+]c signal. This inhibition can be mimicked by the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate. It is concluded that NMDA receptors on cerebellar granule cells are inhibited by phospholipase C-coupled muscarinic M3, glutamatergic, and endothelin receptors via activation of protein kinase C.     

Neuronal soma-dendritic and prejunctional M1-M4 receptors in gastrointestinal and genitourinary smooth muscle

A variety of neurons in gastrointestinal and genitourinary smooth muscle express muscarinic auto- as well as heteroreceptors. These receptors are found on the soma and dendrites of many cholinergic, sympathetic and NANC neurons and on axon terminals. A given neuron may contain both excitatory and inhibitory presynaptic muscarinic receptors. The subtypes involved are species- and tissue-dependent, and neuronal M1 to M4 receptors have been shown to be expressed in smooth muscle tissues. In this study, the ability of several selective muscarinic receptor antagonists to inhibit the effect of arecaidine propargyl ester (APE) on prejunctional muscarinic receptors on sympathetic nerve endings in the rabbit anococcygeus muscle (RAM) was investigated to characterise the receptor subtype involved. Electrical field stimulation (EFS) resulted in a release of noradrenaline (NA) eliciting monophasic contractions due to stimulation of postjunctional alpha1-adrenoceptors. The selective muscarinic agonist APE did not reduce contractions to exogenous NA, but caused a concentration-related and N-methylatropine-sensitive inhibition of neurogenic responses. All muscarinic antagonists investigated failed to affect the EFS-induced contractions, but shifted the concentration-response curve of APE to the right in a parallel and surmountable fashion. Schild analysis yielded regression lines of unit slope, indicating competitive antagonism. The following rank order of antagonist potencies (pA2 values) was found: tripitramine (9.10) > AQ-RA 741 (8.26) > or = himbacine (8.04) > or = (S)-dimethindene (7.69) > pirenzepine (6.46) > or = p-F-HHSiD (6.27). A comparison of the pA2 values determined in the present study with literature binding and functional affinities obtained at native or recombinant M1 to M5 receptors strongly suggests that NA release from sympathetic nerve endings in RAM is inhibited by activation of prejunctional muscarinic M2 receptors.     

Closely related G-protein-coupled receptors use multiple and distinct domains on G-protein alpha-subunits for selective coupling

The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric Galpha subunits, comprised of various regions of Gi1alpha, Gtalpha, and Gqalpha, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between Gi1alpha and Gtalpha sequences, the 5-HT1A and 5-HT1B serotonin and M2 muscarinic receptors can couple with Gi1 but not Gt. It is possible to distinguish domains that eliminate coupling and are defined as "critical," from those that impair coupling and are defined as "important." Domains within the N terminus, alpha4-helix, and alpha4-helix-alpha4/beta6-loop of Gi1alpha are involved in 5-HT and M2 receptor interactions. Chimeric Gi1alpha/Gqalpha subunits verify the critical role of the Galpha C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC50 for interactions with Gi1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration.     

Functional domains essential for Gs activity in prostaglandin EP2 and EP3 receptors

The interaction of cell surface hormone receptors with heterotrimeric G proteins is crucial for hormonal actions. The domains of the receptor, which interact with and activate G protein, have been extensively studied. However, precise molecular mechanisms underlying regulation of the receptor-induced G protein activation are still poorly understood. Prostaglandin E(2) (PGE(2)) receptors comprise of four subtypes, EP1, EP2, EP3 and EP4. Among them, EP2 and EP4 couple to Gs and EP3 to Gi. To assess the functional domains essential for Gs activation in prostanoid receptors, EP2, EP3beta and each intracellular loop- (IC-) interchanged EP2/EP3 chimeras were tested for agonist binding and functional responses. In EP2 receptor, substitution of IC1 or IC3 resulted in loss of binding activity, while substitution of IC2, N- (IC2N) or C-terminal half region of IC2 (IC2C) had no effects on the binding activity. Wild-type EP2 and IC2C-substituted EP2 showed agonist-induced Gs activity, but IC2- and IC2N-substituted EP2 failed to elicit Gs activity upon agonist stimulation. On the other hand, in EP3 receptor substitution of IC1 resulted in loss of PGE(2) binding, while substitution of IC2, IC3, IC2N or IC2C had no effects on binding activity. Wild-type EP3beta, IC3- or IC2C-substituted EP3 failed to show Gs activity upon agonist stimulation, but IC2- or IC2N-substituted EP3 chimera showed agonist-dependent Gs activity. These results indicated that the second intracellular loop of the EP2 plays an essential role in activation of Gs.     

Pronounced conformational changes following agonist activation of the M(3) muscarinic acetylcholine receptor

The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M(3) receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M(3) receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M(3) receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs.