The polycystic kidney disease-1 protein, polycystin-1, binds and activates heterotrimeric G-proteins in vitro

Analysis of the C-terminal cytosolic domain of human and mouse polycystin-1 has identified a number of conserved protein motifs, including a 20-amino-acid heterotrimeric G-protein activation sequence. A peptide specific for this sequence was synthesized and shown to activate purified bovine brain heterotrimeric Gi/Go in vitro. To test whether the C-terminal cytosolic domain of polycystin-1 stably binds G-proteins, GST-fusion constructs were used in pull-down and co-immunoprecipitation assays with purified bovine brain Gi/Go and rat brain lysates. This identified a 74-amino-acid minimal binding domain that includes the G-protein activation sequence. This region of polycystin-1, including the G-protein activation peptide and flanking amino acid sequences, is highly conserved in mouse, human, and puffer fish, lending further support to the functional importance of the minimal binding domain. These results suggest that polycystin-1 may function as a heterotrimeric G-protein coupled receptor.     

Receptor-mediated and receptor-independent activation of G-proteins in rat brain membranes

High-affinity GTPase activity intrinsic to G-proteins, which serves as an index of G-protein activation elicited through agonist-stimulated receptors as well as by receptor-independent direct G-protein activators like mastoparan, was measured in rat brain membranes. Receptor-mediated high-affinity GTPase activity was detectable preferentially for the Gi subfamily associated with adenylyl cyclase inhibition mediated by group II metabotropic glutamate, pirenzepine-insensitive muscarinic acetylcholine, GABA(B), adenosine A1, dopamine D2-like (striatum), and serotonin 5-HT1A (hippocampus) receptors. The pharmacological characteristics of such receptor-mediated high-affinity GTPase activities were presented. Mastoparan, a tetradecapeptide from wasp venom which has been shown to directly activate Gi and Go, inhibited low-affinity GTP hydrolyzing activity, probably due to its activating effect on nucleoside diphosphokinase (NDPK). When NDPK activity was inhibited completely by UDP, mastoparan stimulated high-affinity GTPase activity in a concentration-dependent manner. There are many compounds other than mastoparan with apparently diverse structural properties which have been shown to directly activate G-proteins. The relevance and possible participation of receptor-independent mode of G-protein activation for some neuropeptides were discussed.     

Characterization of the leukotriene B4 receptor in porcine leukocytes. Separation and reconstitution with heterotrimeric GTP-binding proteins

Leukotriene B4 (LTB4) is a potent chemoattractant derived from arachidonic acid. When cDNAs for LTB4 receptor (BLT) were cloned it was found that they belong to a guanine nucleotide-binding regulatory protein (G-protein)-coupled receptor superfamily. However, purification of BLT from inflammatory cells and reconstitution with various types of G-proteins have not been successful. In the present study, BLT from porcine leukocytes was solubilized, separated from associated G-proteins by Ricinus communis agglutinin (RCA) 120 chromatography, and reconstituted with several endogenous and exogenous G-proteins, in combination with the fraction which contained endogenous phospholipids and Gbeta gamma. Kinetic studies of LTB4 were performed to determine the association with G-proteins. A partially purified BLT fraction (retained on an RCA120 column) free of G-proteins showed a lower affinity for LTB4 (Kd = 500 nm), but reconstitution of the BLT fraction with a G-protein-rich fraction (flow-through of an RCA column) increased the affinity for LTB4 10-fold (Kd = 50 nm). The partially purified BLT fraction was also reconstituted with exogenous G-proteins such as a heterotrimeric Gi2 purified from bovine brain or recombinant alpha subunits of Gi1, Gi2, Gi3, and Go expressed in Spodoptera frugiperda-9 cells. These increases in LTB4 bindings demonstrate that the BLT of porcine leukocytes can interact with pertussis toxin-sensitive G-proteins in vitro. The method is useful for the purification and reconstitution of other, as yet unisolated, G-protein-coupled receptors.     

Telomerase and proliferative activity in placenta from women with and without fetal growth restriction

-Natriuretic peptides suppress adrenergic neurotransmission by a mechanism sensitive to pertussis toxin, suggesting that GTP-binding proteins are involved in the response. The major GTP-binding proteins present in the pheochromocytoma (PC12) cells used in this report are Goalpha and Gialpha2. We tested the hypothesis that the more abundant GTP-binding protein, Goalpha, mediates natriuretic peptide effects in PC12 cells by selectively ablating Goalpha from the cells with antisense oligodeoxynucleotides. The results indicate that a selective ablation of Goalpha with this technique eliminated C-type natriuretic peptide (CNP) effects and suppressed dopamine efflux evoked by a depolarizing stimulus. However, the activation of guanylyl cyclase (GC) by CNP was sustained after the Goalpha ablation. Further, Nomega-nitro-L-arginine methyl ester suppressed evoked dopamine efflux equally in the presence and absence of Goalpha. These results suggest that CNP attenuates evoked catecholamine efflux from PC12 cells by a mechanism requiring Goalpha but independent of GC activation.     

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin

The gamma subunits of trimeric G-proteins (gamma1, gamma2, gamma5, and gamma7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure beta [HIT-T15] cells. Of these, GTPgammaS significantly stimulated the carboxyl methylation selectively of gamma2 and gamma5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists--a stimulatory concentration of glucose or a depolarizing concentration of K+--resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of gamma subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from beta cells), also stimulated the carboxyl methylation of gamma subunits in intact HIT cells. Stimulatory effects of glucose or K+ were not demonstrable after removal of extracellular Ca2+ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca2+, implying a mechanism of action that is different from glucose or K+ but also suggesting that dissociation of the alphabetagamma trimer is conducive to gamma subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K+ or mastoparan without altering the rise in intracellular calcium induced by glucose or K+. Glucose-induced carboxyl methylation of gamma2 and gamma5 isoforms was vitiated by coprovision of any of three structurally different cyclooxygenase inhibitors. Conversely, exogenous PGE2, which activates Gi and Go in HIT cells and which thereby would dissociate alpha from beta(gamma), stimulated the carboxyl methylation of gamma2 and gamma5 isoforms and reversed the inhibition of glucose-stimulated carboxyl methylation of gamma subunits elicited by cyclooxygenase inhibitors. These data indicate that gamma subunits of trimeric G-proteins undergo a glucose- and calcium-regulated methylation-demethylation cycle in insulin-secreting cells, findings that may imply an important role in beta cell function. Furthermore, this is the first example of the regulation of the posttranslational modification of G-protein gamma subunits via nonreceptor-mediated activation mechanisms, which are apparently dependent on calcium influx and the consequent activation of phospholipases releasing arachidonic acid.     

Localization of G-proteins in macrophages and E. coli during phagocytosis

Heterotrimeric GTP-binding proteins (G-proteins) have been shown to play an important role in cellular signalling. However, G-protein involvement in the intracellular spreading of bacterial pathogens is still poorly understood. In this study, antibodies, that recognize G-protein alpha-subunits (anti-G alpha), were used to investigate the localization of G-proteins in the macrophage-like cell line P388D1 and E. coli, also in their L-forms, during phagocytosis. In E. coli, anti-G alpha-binding sites were detected preferably in the cell wall and septa of the whole bacterial forms as well as in the cytoplasm of L-forms. Western blotting of bacterial lysates demonstrated protein bands with positive immunoreaction to antibodies against Gs alpha, Gi alpha, and Gcommon alpha with a higher affinity to the antibody against Gs alpha. Immunoreaction with the anti-Gs alpha-antibody was markedly higher in pathogenic strains of E. coli. Because of the conserved structure in all GTP-binding proteins which seem to derive from a single primordial protein involved in signal transduction mechanisms, it is reasonable to assume that some anti-Ga-positive proteins in E. coli might be related to G-proteins of higher organisms. A putative candidate for bacterial G-proteins seems to be a 36 kDa protein. Enhancement in G-protein immunostaining in the cytoplasm of macrophages around the internalized bacteria testifies to the involvement of G-proteins in mediation of endocytosis responses of phagocytes.     

Selective activation of Galphao by D2L dopamine receptors in NS20Y neuroblastoma cells

D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.     

Presenilins upregulate functional K+ channel currents in mammalian cells

Mutations in presenilin 1 (PS-1) and presenilin 2 (PS-2) have been linked to early onset, autosomal dominant Alzheimer's disease. Neither the normal function(s) of the presenilins nor their role(s) in mediating the devastating neurological and pathological changes associated with Alzheimer's Disease, however, are well understood. The results of the experiments described here demonstrate that expression of wild-type PS-1 or PS-2 increases outward K+ current densities in HEK-293 cells relative to untransfected or mock-transfected cells. Western blot analysis reveals that there is a marked increase in full-length, rather than processed, presenilins in transiently transfected HEK-293 cells, suggesting that full-length PS-1 (or PS-2) underlies the observed increases in outward K+ current densities. Consistent with this hypothesis, EXpression of an N-terminal proteolytic fragment of PS-1 is without effects on the membrane properties of HEK-293 cells. Mean outward K+ current densities are also shown to be increased in HEK-293 cells expressing the exon 9 splice site PS-1 mutation (deltaex9/PS-1), a mutant that does not undergo proteolytic processing. In HEK- 293 cells transiently transfected with a missense (G209V) PS-1 mutant, however, mean K+ current densities were not significantly different from controls. Expression of wild-type PS-1 in neonatal rat ventricular myocytes also results in increased outward K+ currents, whereas no detectable effects on membrane currents were seen in PS-1-transfected COS-7 cells. These results suggest that the presenilins do not actually form K+ channels, but rather that these proteins upregulate functional K+ channel expression either directly by associating with K+ channel pore-forming subunits or indirectly by increasing the synthesis, assembly, and/or transport of these subunits. The observation that PS-1 and PS-2 are highly expressed in neurons, localized to the endoplasmic reticulum, suggests that the presenilins could regulate neuronal K+ channel expression; mutations in PS-1/PS-2 would then be expected to result in profound changes in neuronal excitability and contribute to the cognitive decline commonly associated with Alzheimer's Disease.     

Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism

The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation. In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.     

Pituitary adenylate cyclase activating polypeptide induces multiple signaling pathways in rat peritoneal mast cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is a high-affinity ligand for at least two types of G-protein coupled receptors, the PACAP type 1 and type 2 receptor. In this study it is demonstrated that the C-terminal PACAP-fragment PACAP(6-27) stimulates serotonin release from rat peritoneal mast cells with higher potency (EC50: 0.2 vs. 2.0 microM) than the PACAP receptor ligand PACAP(1-27). PACAP-induced degranulation of rat peritoneal mast cells was abolished by pertussis toxin and by benzalkonium chloride (IC50: 9.1 microg/ml) indicating the involvement of heterotrimeric G-proteins of the Gi-type. The PACAP effect was also reduced by inhibitors of the phosphatidylinositol specific phospholipase C ((U73122), IC50: 4 microM; (ET-18-O-CH3), IC50: 18 microM), by D609, a specific inhibitor of the phosphatidylcholine specific phospholipase C (IC50: 41 microM), by the protein kinase C-inhibitor staurosporine (IC50: 0.6 microM) and by the lipoxygenase inhibitor nordihydroguaiaretic acid (NGDA) but not by indomethacin. It is concluded that PACAP peptides stimulate secretion in rat peritoneal mast cells in a PACAP receptor-independent manner, probably via direct activation of heterotrimeric G-proteins of the Gi-type; these G-proteins may lead to a sequential activation of different signaling cascades (see above), which may converge at the level of one or more staurosporine-sensitive protein kinase.     

Opiate tolerance and dependence: receptors, G-proteins, and antiopiates

Despite the existence of a large body of information on the subject, the mechanisms of opiate tolerance and dependence are not yet fully understood. Although the traditional mechanisms of receptor down-regulation and desensitization seem to play a role, they cannot entirely explain the phenomena of tolerance and dependence. Therefore, other mechanisms, such as the presence of antiopiate systems and the coupling of opiate receptors to alternative G-proteins, should be considered. A further complication of studies of opiate tolerance and dependence is the multiplicity of endogenous opiate receptors and peptides. This review will focus on the endogenous opioid system--peptides, receptors, and coupling of receptors to intracellular signaling via G-proteins--in the context of their roles in tolerance and dependence. Opioid peptides include the recently discovered endomorphins and those encoded by three known genes--pro-opiomelanocortin, pro-enkephalin, and pro-dynorphin. They bind to three types of receptors--mu, delta, and kappa. Each of the receptor types is further divided into multiple subtypes. These receptors are widely known to be coupled to G-proteins of the Gi and Go subtypes, but an increasing body of results suggests coupling to other G-proteins, such as Gs. The coupling of opiate receptors to Gs, in particular, has implications for tolerance and dependence. Alterations at the receptor and transduction level have been the focus of many studies of opiate tolerance and dependence. In these studies, both receptor down-regulation and desensitization have been demonstrated in vivo and in vitro. Receptor down-regulation has been more easily observed in vitro, especially in response to morphine, a phenomenon which suggests that some factor which is missing in vitro prevents receptors from down-regulating in vivo and may play a critical role in tolerance and dependence. We suggest that antiopiate peptides may operate in vivo in this capacity, and we outline the evidence for the antiopiate properties of three peptides: neuropeptide FF, orphanin FQ/nociceptin, and Tyr-W-MIF-1. In addition, we provide new results suggesting that Tyr-W-MIF-1 may act as an antiopiate at the cellular level by inhibiting basal G-protein activation, in contrast to the activation of G-proteins by opiate agonists.     

Concomitant chemoradiotherapy of cancer of the esophagus

Many cell membrane bound receptors communicate with the inside of the cell through guanine nucleotide binding proteins (G-proteins). This holds also for olfactory receptor neurons, which respond to odorants with G-protein mediated increases in the concentration of cyclic adenosine 3', 5'-monophosphate (cAMP) and/or inositol 1,4,5-triphosphate (InsP3). These substances regulate the ionic conductivity of the wall of the cilia. We have studied a similar system, namely G-protein coupled alpha 2-adrenoceptors, present for example in the cells of certain fish scales. These receptors react on, catecholamines and the G-protein mediates a decrease in cAMP, which causes an aggregation of pigment containing granulas to the middle of the cells. The light transmission of the cell increases due to this aggregation. This simple physiological response has been used in a sensitive biosensor for noradrenaline and for pertussis toxin that is based on isolated fish scales from cuckoo wrasse (Labrus ossifagus). The results were obtained with a simple photometer. Measurements can be performed also on single isolated melanophores. The main purpose of this contribution is, however, to point out that G-protein coupled receptors together with a simple physiological response form a principle for biosensing, which could also be an interesting alternative for odour sensing.     

Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors m1-m4

We have studied muscarinic agonist stimulated [35S]GTP gamma S binding and [gamma 32P]GTP hydrolysis (GTPase) in membranes from CHO cells stably transfected with human muscarinic m1-m4 receptors. 'Full' agonists were at least 10-fold more potent at m2 & m4 receptors than at m1 & m3. This pattern was less marked with 'partial' agonists, which had a greater maximal effect at m2 & m4 than at m1 & m3. McN-A343 uniquely was more potent and efficacious at m4 than at m2 receptors. Antagonist affinity constants were estimated by fitting the data from inhibition curves directly to the Schild model. Antagonist affinity estimates were very similar to those measured earlier in binding studies using animal tissues, and confirmed a small degree of m4 selectivity for tropicamide and secoverine. The receptor subtypes activated more than one G-protein subtype; m2 & m4 receptors activated only pertussis (PTX) sensitive G-proteins, while m1 & m3 coupled to both PTX sensitive and insensitive G-proteins. Acetylcholine (ACh) was more potent in stimulating guanine nucleotide exchange in PTX-treated m1 cells than in controls.     

Modulation of bradykinin receptor ligand binding affinity and its coupled G-proteins by nitric oxide

To determine whether nitric oxide (NO) can modulate bradykinin (BK) signaling pathways, we treated endothelial cells with an NO donor, S-nitrosoglutathione (GSNO), to determine its effect(s) on G-proteins (Gi and Gq) that are coupled to the type II kinin (BK2) receptor. Radioligand binding assays and Western analyses showed that GSNO (10-500 microM, 0-72 h) did not alter the expression of BK2 receptor, Gi, or Gq. However, GSNO caused a 6-fold increase in basal cGMP production and decreased high affinity BK bindings sites and GTPase activity by 74 and 85%, respectively. The cGMP analogue, dibutyryl-cGMP, also inhibited BK-stimulated GTPase activity by 74% suggesting that some of the effects of NO may be mediated through activation of guanylyl cyclase. The NO synthase inhibitor, Nomega-monomethyl-L-arginine, inhibited endogenous NO synthase activity and cGMP production by 91 and 76%, respectively, but increased BK-stimulated GTPase activity by 61%. To determine which G-proteins are affected by NO, we performed GTP binding assays with [35S]GTPgammaS followed by immunoprecipitation with specific G-protein antisera. Both GSNO and dibutyryl-cGMP increased basal G-protein GTP binding activities by 18-26%. However, GSNO decreased BK-stimulated Galphai2, Galphai3, and Galphaq/11 GTP binding activity by 93, 61, and 90%, respectively, whereas epinephrine-stimulated Galphas GTP binding activity was unaffected. These results suggest that NO can modulate BK signaling pathways by selectively inhibiting G-proteins of the Gi and Gq family.     

Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells

Binding data point to the coexistence of three endothelin receptors (ET-R) in rat heart myocytes. Induction of phosphoinositide hydrolysis in this preparation by endothelins (ET-1 and ET-3) or sarafotoxins (SRTX-b and SRTX-c) was demonstrated by measurement of labeled inositol phosphate generation. Pertussis toxin (PT) enhanced the induction of phosphoinositide hydrolysis by all four peptides. The process seems to be mediated by at least two heterotrimeric G-proteins, the one sensitive and the other insensitive to PT. Measurement of GTPase activity induced in rat myocytes clearly indicates for the first time the direct functional coupling between ET-R and a G-protein. These GTPase activity experiments provide evidence that phosphoinositide hydrolysis is stimulated via functional coupling between the endothelin receptor of the ETA-R subtype and a PT-insensitive G-protein, Gq/11. The involvement of PT-sensitive G-proteins, i.e. Gi-like and/or Go-like proteins, in the signal transduction pathways of ETs and SRTXs is discussed.     

The alpha subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.     

The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins

Metabotropic glutamate receptors (mGluR) share no sequence homology with any other G-protein-coupled receptors (GPCRs). The characterization of their G-protein coupling domains will therefore help define the general rules for receptor-G-protein interaction. To this end, the intracellular domains of mGluR3 and mGluR1, receptors coupled negatively to adenylyl cyclase and positively to phospholipase C, respectively, were systematically exchanged. The ability of these chimeric receptors to induce Ca2+ signals were examined in Xenopus oocytes and HER 293 cells. The chimeric receptors that still possessed the second intracellular loop (i2) of these proteins were targeted correctly to the plasma membrane. Consistent Ca2+ signals could be recorded only with chimeric mGluR3 receptors that contains i2 and at least one other intracellular domains of mGluR3 have to be replaced by their mGluR1 equivalent to produce optimal coupling to G protein. These observations indicate that i2 of mGluR1 is a critical element in determining the transduction mechanism of this receptor. These results suggest that i2 of mGluRs may play a role similar to i3 of most other GPCRs in the specificity of coupling to the G-proteins. Moreover, as in many other GPCRs, our data revealed cooperation between the different mGluR intracellular domains to control efficient coupling to G-proteins.     

Characterization of a Goalpha mutant that binds xanthine nucleotides

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goalpha mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goalpha subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoalphaD273N/Q205L (GoalphaX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoalphaX (GoalphaD273N/Q205L) formed heterotrimers with betagamma complexes only in the presence of xanthine diphosphate (XDP), and the binding to betagamma was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoalphaX protein underwent a conformational change similar to that of the activated wild-type Goalpha. In transfected COS-7 cells, we demonstrate that the interaction between GoalphaX and betagamma occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein alpha subunit.     

Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz

We have demonstrated previously that D1 dopamine receptors are coupled to both Gs alpha and Go alpha. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both Gs alpha and cholera toxin-insensitive G proteins. The interaction between D5 receptors and Gs alpha was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to Go alpha or Gi alpha. D5 receptors were also not coupled to Gq alpha and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF(-)4-sensitive, N-ethylmaleimide-resistant G protein. Anti-Gz alpha caused immunoprecipitation of 24.2 +/- 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and Gz alpha. The coupling to Gz alpha was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gz alpha.