PTH stimulates PLCbeta and PLCgamma isoenzymes in rat enterocytes: influence of ageing

We previously reported that in rat duodenal cells (enterocytes), parathyroid hormone (PTH [1-34]: PTH) stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP(3)) and diacylglycerol (DAG) and that this mechanism is severely altered in old animals. In the present study, we show that PTH [1-34]-dependent IP(3) release in young rats was blocked to a great extent by an antibody against guanine nucleotide binding protein Galphaq/11, indicating that the hormone activates a beta isoform of PLC coupled to the alpha subunit of Gq/11. In addition, PTH rapidly (within 30 s, with maximal effects at 1 min) stimulated tyrosine phosphorylation of PLCgamma in a dose-dependent fashion (10(-10)-10(-7) M). The hormone response was specific as PTH [7-34] was without effects. The tyrosine kinase inhibitors, genistein (100 microM) and herbimycin (2 microM), suppressed PTH-dependent PLCgamma tyrosine phosphorylation. Stimulation of PLCgamma tyrosine phosphorylation by PTH [1-34] greatly decreased with ageing. PP1 (10 microM), a specific inhibitor of the Src family of tyrosine kinases, completely abolished PLCgamma phosphorylation. The hormone-induced Src tyrosine dephosphorylation, a major mechanism of Src activation, an effect that was blunted in old animals. These results indicate that in rat enterocytes PTH generates IP(3) mainly through G-protein-coupled PLCbeta and stimulates PLCgamma phosphorylation via the nonreceptor tyrosine kinase Src. Impairment of PTH activation of both PLC isoforms upon ageing may result in abnormal hormone regulation of cell Ca(2+) and proliferation in the duodenum.     

Membrane-Bound and cytosolic forms of heterotrimeric G proteins in young and adult rat myocardium: influence of neonatal hypo- and hyperthyroidism.

Membrane and cytosolic fractions prepared from ventricular myocardium of young (21-day-old) hypo- or hyperthyroid rats and adult (84-day-old) previously hypo- or hyperthyroid rats were analyzed by immunoblotting with specific anti-G-protein antibodies for the relative content of Gs alpha, Gi alpha/Go alpha, Gq alpha/G11 alpha, and G beta. All tested G protein subunits were present not only in myocardial membranes but were at least partially distributed in the cytosol, except for Go alpha2, and G11 alpha. Cytosolic forms of the individual G proteins represented about 5-60% of total cellular amounts of these proteins. The long (Gs alpha-L) isoform of Gs alpha prevailed over the short (Gs alpha-S) isoform in both crude myocardial membranes and cytosol. The Gs alpha-L/Gs alpha-S ratio in membranes as well as in cytosol increased during maturation due to a substantial increase in Gs alpha-L. Interestingly, whereas the amount of membrane-bound Gi alpha/Go alpha and Gq alpha/G11 alpha proteins tend to lower during postnatal development, cytosolic forms of these G proteins mostly rise. Neonatal hypothyroidism reduced the amount of myocardial Gs alpha and increased that of Gi alpha/Go alpha proteins. By contrast, neonatal hyperthyroidism increased expression of Gs alpha and decreased that of Gi alpha and G11 alpha in young myocardium. Changes in G protein content induced by neonatal hypo- and hyperthyroidism in young rat myocardium were restored in adulthood. Alterations in the membrane-cytosol balance of G protein subunits associated with maturation or induced by altered thyroid status indicate physiological importance of cytosolic forms of these proteins in the rat myocardium.     

Signaling via histamine receptors in cat duodenal smooth muscle cells

Histamine produced concentration-dependent contractions in cat duodenal smooth muscle cells that were obtained by enzymatic digestion of smooth muscle with collagenase F. Pyrilamine, an H1 receptor antagonist, inhibited the contractile response while famotidine, an H2 receptor antagonist, augmented it. In cells with selectively preserved H1 receptors, produced by pretreatment with pyrilamine followed by inactivation of all unprotected receptors with N-ethylmaleimide, histamine-induced contraction was significantly augmented as compared with control cells. Pertussis toxin (PTX) had no effect on contraction, suggesting that the H1 receptor is coupled to a PTX-insensitive G protein. Gi2, Gi3, Go, Gs, and Gq subunits were present in cat duodenum, and histamine-induced contraction was inhibited by Gq antibody after cell permeabilization. Neomycin, a PLC inhibitor, inhibited the histamine-induced cell contraction, but not rhoCMB, a PLD inhibitor, or DEDA, a PLA2 inhibitor. Heparin, an IP3 receptor inhibitor, inhibited contraction whereas chelerythrine, a PKC inhibitor, had no effect. We conclude that histamine-induced contraction in cat duodenal smooth muscle cells is mediated by H1 receptors coupled to a PTX-insensitive Gq protein and results in activation of phosphatidylinositol-specific phospholipase C (PI-PLC).     

Physicochemical modulation of agonist-induced [35s]GTPgammaS binding: implications for coexistence of multiple functional conformations of dopamine D1-like receptors

Dopamine agonist-stimulated [35S]GTPgammaS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins Gi, Gs, or Gq. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D2-like/Gi), SKF38393 (D1-like/Gq, D1-like/Gs), SKF85174 (D1-like/Gs), and SKF83959 (D1-like/Gq). Dopamine and SKF38393 significantly stimulated [35S]GTPgammaS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D1-like receptor conformations that mediate agonist stimulation of Gs/adenylylcyclase may be structurally different from those that mediate Gq/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates Gs/adenylylcyclase to one that favors Gq/PLC signaling.     

Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors

Pathways of transduction employed by receptors for sphingosine 1-phosphate (S1P) are identified by the nature of second messengers and/or downstream targets regulated and, more formally, by direct assays of heterotrimeric G protein activation. The different methods generally agree. S1P1 couples to members of the Gi family, apparently selectively, although reported pertussis toxin (PTX)-insensitive actions make categorical statements regarding exclusivity difficult. S1P2 and S1P3 couple to members of the Gi, Gq, and G12/13 families. S1P4 couples to Gi and possibly G12/13, while S1P5 couples to Gi and G12/13 but not to Gq. In virtually all circumstances, coupling of S1P receptors to Gi is reflected in PTX-sensitive inhibition of adenylyl cyclase, activation of extracellular-regulated kinases (ERKs), and, depending on the cell, activation of phospholipase C (PLC). Coupling to Gq is reflected in PTX-insensitive activation of phospholipase C. Coupling to G12/13 is reflected in activation of Rho and subsequent activation of serum response factor (SRF). Specific linkages have been verified in almost all instances by receptor-promoted [35S]GTPgammaS/GDP exchange on identified G proteins.     

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.     

Somatotropin has no effect on the quantity of guanine nucleotide binding proteins Gqalpha/G11alpha in goat adipose tissue in vivo

The decapeptide QLNLKEYNLV corresponding to the C-terminus of Gq/G11alpha guanine nucleotide-binding proteins (G-proteins) was synthesized by the solid-phase method and conjugated to keyhole limpet hemocyanin. The rabbits were immunized with these conjugates and an antiserum that reacted specifically with the alpha subunit of Gq/G11 proteins was used in this study. The antiserum exhibited no cross-reactivity with the alpha subunits of stimulatory (Gs) or inhibitory (Gi) G-proteins associated with adenylate cyclase. Immunoblots with the antiserum showed that it specifically recognized the Gq/G11 alpha-proteins in cholate extracts of adipose tissue membranes of goats. Treatment of young castrated male goats with bST had no effect on the quantity of Gq/G11 alpha subunits in adipose tissue and the results thus obtained did not support the idea that the bST signal in adipose tissue is transmitted via Gq/G11 alpha-proteins.     

Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11.

The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alpha beta gamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (Gp) was suggested to mediate receptor regulation. The required Gp protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the Gq subfamily (alpha q and alpha 11) which regulate the activity of the beta 1 isoenzyme of PLC. Gq and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common Gq/11 alpha-subunit carboxyterminal sequence was generated and shown to react with unique 42-kilodalton Gq alpha and 43-kilodalton G11 alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alpha T3-1 pituitary cells. The Gq/11 alpha peptide antibody was shown to immunodeplete the Gp activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta 1. In addition, the immunoglobulin G fraction of Gq/11 alpha peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alpha T3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a Gp protein(s) which corresponds to Gq, G11 or both.     

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.     

Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.     

The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2.

Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2.     

Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release

Recently we have shown that the metabotropic glutamate 5 (mGlu5) receptor can be expressed on nuclear membranes of heterologous cells or endogenously on striatal neurons where it can mediate nuclear Ca2+ changes. Here, pharmacological, optical, and genetic techniques were used to show that upon activation, nuclear mGlu5 receptors generate nuclear inositol 1,4,5-trisphosphate (IP3) in situ. Specifically, expression of an mGlu5 F767S mutant in HEK293 cells that blocks Gq/11 coupling or introduction of a dominant negative Galphaq construct in striatal neurons prevented nuclear Ca2+ changes following receptor activation. These data indicate that nuclear mGlu5 receptors couple to Gq/11 to mobilize nuclear Ca2+. Nuclear mGlu5-mediated Ca2+ responses could also be blocked by the phospholipase C (PLC) inhibitor, U73122, the phosphatidylinositol (PI) PLC inhibitor 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3), or by using small interfering RNA targeted against PLCbeta1 demonstrating that PI-PLC is involved. Direct assessment of inositol phosphate production using a PIP2/IP3 "biosensor" revealed for the first time that IP3 can be generated in the nucleus following activation of nuclear mGlu5 receptors. Finally, both IP3 and ryanodine receptor blockers prevented nuclear mGlu5-mediated increases in intranuclear Ca2+. Collectively, this study shows that like plasma membrane receptors, activated nuclear mGlu5 receptors couple to Gq/11 and PLC to generate IP3-mediated release of Ca2+ from Ca2+-release channels in the nucleus. Thus the nucleus can function as an autonomous organelle independent of signals originating in the cytoplasm, and nuclear mGlu5 receptors play a dynamic role in mobilizing Ca2+ in a specific, localized fashion.     

胃动素和胃泌素引起大鼠胃窦平滑肌细胞收缩的信号转导通路

应用大鼠游离胃窦平滑肌细胞,观察胃动素和胃泌素对胃窦平滑肌细胞收缩作用的胞内信号转导通路。结果显示：⑴胃动素和胃泌素对胃窦平滑肌细胞均有收缩作用;⑵Ｇａｉ－３抗体可抑制胃动素和胃泌素加强胃窦平滑肌细胞的收缩,胃动素、胃泌明显增加Ｇａｉ－３抗体与「＾３５Ｓ」ＣＴＰγＳ的结合;⑶磷脂酶抑制剂Ｕ－７３１２２、三磷酸肌醇受体拮抗剂肝素可抑制胃坳素和胃泌素引起的胃窦平滑肌细胞的收缩。结果表明：胃坳素和胃泌表     

Comparative analysis of the efficacy of A1 adenosine receptor activation of Gi/o alpha G proteins following coexpression of receptor and G protein and expression of A1 adenosine receptor-Gi/o alpha fusion proteins

HEK293T cells were transiently transfected to express either the human A1 adenosine receptor together with pertussis toxin-resistant cysteine-to-glycine forms of the alpha subunits of Gi1 (C351G), Gi2 (C352G), and Gi3 (C351G) and wild-type Go1alpha or fusion proteins comprising the A1 adenosine receptor and these Gi/o G proteins to compare A1 adenosine receptor agonist-mediated activation of these Gi family G proteins upon coexpression of individual Gi/o G proteins and receptor versus expression as receptor-G protein fusion proteins. Addition of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) to membranes of pertussis toxin-treated cells resulted in a concentration-dependent stimulation of [35S]GTPgammaS binding with comparable amounts of NECA required to produce half-maximal stimulation following transfection of A1 adenosine receptor and Gi/o G proteins either as fusion proteins or as separate polypeptides. However, the magnitude of agonist-mediated activation of GTPgammaS binding was greatly enhanced by expressing the A1 adenosine receptor and Gi family G proteins from chimaeric open reading frames. This observation was consistent following the study of more than 40 agonists. No preferential activation of any G protein was observed with more than 40 A1 receptor agonists following cotransfection of receptor with G protein or transfection of receptor-G protein fusion proteins. These studies demonstrate the utility of using fusion proteins to study receptor-G protein interaction, show that the A1 adenosine receptor couples equally well to the Gi/o G proteins Gi1alpha, G i2alpha, Gi3alpha, and Go1alpha, and demonstrate that for a range of agonists there is no selectivity for activation of any particular A1 adenosine receptor-Gi/o G protein combination.     

The alpha 2B adrenergic receptor of undifferentiated neuroblastoma x glioma hybrid NG108-15 cells, interacts directly with the guanine nucleotide binding protein, Gi2

In membranes of undifferentiated neuroblastoma x glioma hybrid cell line NG108-15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 microM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of GTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the alpha subunit of the G-protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Go alpha Gi3 alpha or Gs alpha or from non-immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the alpha 2B adrenergic receptor of NG108-15 cells with Gi2.     

Involvement of G-protein βγ subunits on the influence of inhibitory α2-autoreceptors on the angiotensin AT1-receptor modulation of noradrenaline release in the rat vas deferens

The influence of alpha2-autoreceptors on the facilitation of [3H]-noradrenaline release mediated by angiotensin II was studied in prostatic portions of rat vas deferens preincubated with [3H]-noradrenaline. Angiotensin II enhanced tritium overflow evoked by trains of 100 pulses at 8 Hz, an effect that was attenuated by the AT1-receptor antagonist losartan (0.3-1 microM), at concentrations suggesting the involvement of the AT1B subtype. The effect of angiotensin II was also attenuated by inhibition of phospholipase C (PLC) and protein kinase C (PKC) indicating that prejunctional AT1-receptors are coupled to the PLC-PKC pathway. Angiotensin II (0.3-100 nM) enhanced tritium overflow more markedly, up to 64%, under conditions that favor alpha2-autoinhibition, observed when stimulation consisted of 100 pulses at 8 Hz, than under poor alpha2-autoinhibition conditions, only up to 14%, observed when alpha2-adrenoceptors were blocked with yohimbine (1 microM) or when stimulation consisted of 20 pulses at 50 Hz. Activation of PKC with 12-myristate 13-acetate (PMA, 0.1-3 microM) also enhanced tritium overflow more markedly under strong alpha2-autoinhibition conditions. Inhibition of Gi/o-proteins with pertussis toxin (8 microg/ml) or blockade of Gbetagamma subunits with the anti-betagamma peptide MPS-Phos (30 microM) attenuated the effects of angiotensin II and PMA. The results indicate that activation of AT1-receptors coupled to the PLC-PKC pathway enhances noradrenaline release, an effect that is markedly favoured by an ongoing activation of alpha2-autoreceptors. Interaction between alpha2-adrenoceptors and AT1-receptors seems to involve the betagamma subunits released from the Gi/o-proteins coupled to alpha2-adrenoceptors and protein kinase C activated by AT1-receptors.     

Dimers of class A G protein-coupled receptors function via agonist-mediated trans-activation of associated G proteins

The histamine H1 receptor and the alpha1b-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca2+] via activation of Gq/G11. Assessed by co-immunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homo-dimers. The addition of the G protein G11alpha to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5'-3-O-([35S]thio)triphosphate ([35S]GTPgammaS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG11alpha into the fusions. Mutation of a highly conserved leucine in intracellular loop 2 of each receptor also eliminated agonist function but not binding. Co-expression of the two non-functional but complementary fusion constructs reconstituted agonist-mediated binding of [35S]GTPgammaS in membranes of HEK293 cells and elevation of [Ca2+]i in mouse embryo fibroblasts lacking both Gq and G11. Co-expression of the histamine H1 receptor- and the alpha1b-adrenoreceptor-G11alpha fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G11alpha with increasing amounts of L151Dalpha1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [35S]GTPgammaS binding. Co-expression of the alpha1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the alpha1b-adrenoreceptor and G11alpha did not result in agonist activation of the G protein but did indicate a role for transmembrane helix 1 in dimerization. These data demonstrate that dimers of these class A receptors function via trans-activation of associated G proteins.     

Mediation of growth factor induced DNA synthesis and calcium mobilization by Gq and Gi2

A newly identified subclass of the heterotrimeric GTP binding regulatory protein family, Gq, has been found to be expressed in a diverse range of cell types. We investigated the potential role of this protein in growth factor signal transduction pathways and its potential relationship to the function of other G alpha subclasses. Recent biochemical studies have suggested that Gq regulates the beta 1 isozyme of phospholipase C (PLC beta 1), an effector for some growth factors. By microinjection of inhibitory antibodies specific to distinct G alpha subunits into living cells, we have determined that G alpha q transduces bradykinin- and thrombin-stimulated intracellular calcium transients which are likely to be mediated by PLC beta 1. Moreover, we found that G alpha q function is required for the mitogenic action of both of these growth factors. These results indicate that both thrombin and bradykinin utilize Gq to couple to increases in intracellular calcium, and that Gq is a necessary component of the mitogenic action of these factors. While microinjection of antibodies against G alpha i2 did not abolish calcium transients stimulated by either of these factors, such microinjection prevented DNA synthesis in response to thrombin but not to bradykinin. These data suggest that thrombin- induced mitogenesis requires both Gq and Gi2, whereas bradykinin needs only the former. Thus, different growth factors operating upon the same cell type use overlapping yet distinct sets of G alpha subtypes in mitogenic signal transduction pathways. The direct identification of the coupling of both a pertussis toxin sensitive and insensitive G protein subtype in the mitogenic pathways utilized by thrombin offers an in vivo biochemical clarification of previous results obtained by pharmacologic studies.     

The C-terminus of Gi family G-proteins as a determinant of 5-HT(1A) receptor coupling

Using a universal signaling assay employing G-protein chimeras comprising the C-terminal five amino acids of Gi1/2, Gi3, Go, and Gz fused to Gq, the calcium mobilizing G-protein, we explored the role of the C-terminus of Gi family G-proteins as a determinant for 5-HT(1A) receptor functional coupling. Co-expression of the 5-HT(1A) receptor with each of the Gq/Gi family chimeras resulted in a concentration-dependent increase in calcium upon addition of 5-HT, although the coupling efficiency differed dramatically. Gq/Gi3 resulted in the most efficient coupling based on both potency and relative maximum response to 5-HT. Gq/Go also produced efficient coupling in terms of relative 5-HT efficacy (76% of the Gq/Gi3 maximum response), although 5-HT exhibited 4-fold lower agonist potency, and Gq/Gz and Gq/Gi1/2 conferred poor functional coupling. Agonist potencies and relative efficacies determined for a number of 5-HT(1A) receptor agonists using Gq/Gi3 coupling were significantly weaker than those described previously for coupling through the native G-protein. These results indicate the C-terminus of Gi3 as an important determinant for coupling to the 5-HT(1A) receptor, while the reduced functional agonist activities suggest additional motifs participate in receptor/G-protein coupling.