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In the present work we examined localization and behavior of G protein-coupled receptors (GPCR) in polarized exocrine cells to address the questions of how luminal to basal Ca2+waves can be generated in a receptor-specific manner and whether quantal Ca2+release reflects partial release from a continuous pool or an all-or-none release from a compartmentalized pool. Immunolocalization revealed that expression of GPCRs in polarized cells is not uniform, with high levels of GPCR expression at or near the tight junctions. Measurement of phospholipase Cβ activity and receptor-dependent recruitment and trapping of the box domain of RGS4 in GPCRs complexes indicated autonomous functioning of Gq-coupled receptors in acinar cells. These findings explain the generation of receptor-specific Ca2+waves and why the waves are always initiated at the apical pole. The initiation site of Ca2+wave at the apical pole and the pattern of wave propagation were independent of inositol 1,4,5-trisphosphate concentration. Furthermore, a second Ca2+wave with the same initiation site and pattern was launched by inhibition of sarco/endoplasmic reticulum Ca2+-ATPase pumps of cells continuously stimulated with sub-maximal agonist concentration. By contrast, rapid sequential application of sub-maximal and maximal agonist concentrations to the same cell triggered Ca2+waves with different initiation sites. These findings indicate that signaling specificity in pancreatic acinar cells is aided by polarized expression and autonomous functioning of GPCRs and that quantal Ca2+release is not due to a partial Ca2+release from a continuous pool, but rather, it is due to an all-or-none Ca2+release from a compartmentalized Ca2+pool.

RetGC-1, a member of the membrane guanylyl cyclase family of proteins, is regulated in photoreceptor cells by a Ca2+-binding protein known as GCAP-1. Proper regulation of RetGC-1 is essential in photoreceptor cells for normal light adaptation and recovery to the dark state. In this study we show that cGMP synthesis by RetGC-1 requires dimerization, because critical functions in the catalytic site must be provided by each of the two polypeptide chains of the dimer. We also show that an intact α-helical coiled-coil structure is required to provide dimerization strength for the catalytic domain of RetGC-1. However, the dimerization strength of this domain must be precisely optimized for proper regulation by GCAP-1. We found that Arg838within the dimerization domain establishes the Ca2+sensitivity of RetGC-1 by determining the strength of the coiled-coil interaction. Arg838substitutions dominantly enhance cGMP synthesis even at the highest Ca2+concentrations that occur in normal dark-adapted photoreceptor cells. Molecular dynamics simulations suggest that Arg838substitutions disrupt a small network of salt bridges to allow an abnormal extension of coiled-coil structure. Substitutions at Arg838were first identified by linkage to the retinal degenerative disease, autosomal dominant cone rod dystrophy (adCORD). Consistent with the characteristics of this disease, the Arg838-substituted RetGC-1 mutants exhibit a dominant biochemical phenotype. We propose that accelerated cGMP synthesis in humans with adCORD is the primary cause of cone-rod degeneration.

Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for Giand/or Gqclass G protein α subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP3) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP3but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation-resistant mutant RGS4 is inhibited by PIP3. Calmodulin binds all RGS proteins we tested in a Ca2+-dependent manner but does not directly affect GAP activity. Indeed, Ca2+/calmodulin binds a complex of RGS4 and a transition state analog of Gαi1-GDP-AlF4−. Ca2+/calmodulin reverses PIP3-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca2+/calmodulin competition with PIP3may provide an intracellular mechanism for feedback regulation of Ca2+signaling evoked by G protein-coupled agonists.

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Giand Gqclass α subunits. In the present study, we used knockouts of Gqclass genes in mice to evaluate the potency and selectivity of RGS4 in modulating Ca2+signaling transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+signaling by RGS4 was observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol was about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent inhibitors of Gq-dependent Ca2+signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 was observed in acinar cells from wild type and several single and double Gqclass knockout mice. Thus, these receptors appear to couple Gqclass α subunit isotypes equally. Difference in receptor selectivity of RGS proteins action indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes.

The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway.

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.

Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by Gq and Gi alpha subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via Gq. Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 10(4)-fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates Gq class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by Galphaq and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled Gq signaling.

Mice with deficiencies in one or more Gq class alpha subunit genes were used to examine the role of the alpha subunit in regulating Ca2+ signaling in pancreatic and submandibular gland cells. Western blot analysis showed that these cells express three of the four Gq class subunits, Galphaq, Galpha11, and Galpha14 but not Galpha15. Surprisingly, all parameters of Ca2+ signaling were identical in cells from wild type and four lines of mutant mice: 1) Galpha11-/-, 2) Galpha11-/-/Galpha14-/-, 3) Galpha14-/-/Galpha15-/-, and 4) Galphaq-/-/Galpha15-/-. These parameters included the Kapp for several Gq class coupled receptors, induction of [Ca2+]i oscillations by weak stimulation, and a biphasic [Ca2+]i response by strong stimulation. Furthermore, Ca2+ release from internal stores and Ca2+ entry were not affected in cells from any of the mutant mice. We conclude that Galphaq, Galpha11, and Galpha14 promiscuously couple several receptors (m3 muscarinic, bombesin, cholecystokinin, and alpha1 adrenergic) to effector proteins that activate both Ca2+ release from internal stores and Ca2+ entry.

Activation of the mitogen-activated protein kinase (MAPK) cascade has been widely associated with cell proliferation; previous studies have shown that angiotensin II (AII), acting on 7-transmembrane G protein-coupled receptors, stimulates the MAPK pathway. In this report we investigate whether the MAPK pathway is required for the mitogenic response to AII stimulation of vascular smooth muscle cells derived from the hypertensive rat (SHR-VSM). AII stimulates the phosphorylation of MAPK, as determined by Western blot specific for the tyrosine 204 phosphorylated form of the protein. This MAPK phosphorylation was inhibited by the presence of the inhibitor of MAPK kinase activation, PD 098059. Using a peptide kinase assay shown to measure the p42 and p44 isoforms of MAPK, the stimulated response to AII was inhibited by PD 098059 with an IC50 of 15.6 +/- 1.6 microM. The AII stimulation of [3H]thymidine incorporation was inhibited by PD 098059 with an IC50 of 17.8 +/- 3.1 microM. PD 098059 had no effect on AII-stimulated phospholipase C or phospholipase D (PLD) activity. When the SHR-VSM cells were stimulated with phorbol ester, there was an activation of MAPK similar in size and duration to the response to AII, but there was no significant enhancement of [3H]thymidine incorporation. There was also no activation of PLD by phorbol ester, while AII produced a robust PLD response. Diversion of the product of the PLD reaction by 1-butanol caused a partial loss of the [3H]thymidine response; this did not occur with tertiary butanol, which did not interfere with the PLD reaction. These results show that in these cells the MAPK cascade is required but not sufficient for the mitogenic response to AII, and suggest that the full mitogenic response requires both MAPK in conjunction with other signaling components, one of which is PLD.

Mice with deficiencies in one or more Gq class α subunit genes were used to examine the role of the α subunit in regulating Ca2+signaling in pancreatic and submandibular gland cells. Western blot analysis showed that these cells express three of the four Gq class subunits, Gαq, Gα11, and Gα14 but not Gα15. Surprisingly, all parameters of Ca2+signaling were identical in cells from wild type and four lines of mutant mice: 1) Gα11−/−, 2) Gα11−/−/Gα14−/−, 3) Gα14−/−/Gα15−/−, and 4) Gαq−/−/Gα15−/−. These parameters included the Kappfor several Gq class coupled receptors, induction of [Ca2+]ioscillations by weak stimulation, and a biphasic [Ca2+]iresponse by strong stimulation. Furthermore, Ca2+release from internal stores and Ca2+entry were not affected in cells from any of the mutant mice. We conclude that Gαq, Gα11, and Gα14 promiscuously couple several receptors (m3muscarinic, bombesin, cholecystokinin, and α1 adrenergic) to effector proteins that activate both Ca2+release from internal stores and Ca2+entry.