Thomas M. Wilkie

GAIP and RGS4 Are GTPase-Activating Proteins for the Gi Subfamily of G Protein α Subunits

A novel class of regulators of G protein signaling (RGS) proteins has been identified recently. Genetic evidence suggests that RGS proteins inhibit G protein-mediated signaling at the level of the receptor-G protein interaction or the G protein alpha subunit itself. We have found that two RGS family members, GAIP and RGS4, are GTPase-activating proteins (GAPs), accelerating the rate of GTP hydrolysis by Gi alpha 1 at least 40-fold. All Gi subfamily members assayed were substrates for these GAPs; Gs alpha was not. RGS4 activates the GTPase activity of certain Gi alpha 1 mutants (e.g., R178C), but not others (e.g., Q204L). The GAP activity of RGS proteins is consistent with their proposed role as negative regulators of G protein-mediated signaling.

RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Giand Gq class α subunits. In the present study, we used knockouts of Gq class 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 Gq class knockout mice. Thus, these receptors appear to couple Gq class 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.

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase

Stimulation of the T lymphocyte antigen receptor‐CD3 complex (TCR‐CD3) causes T cell activation by a process associated with increased phosphatidylinositol‐specific phospholipase C (PI‐PLC) activity. Evidence exists suggesting that GTP‐binding (G) proteins, particularly the pertussis toxin (PT)‐sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR‐CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte‐specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL‐2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck‐PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT‐sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.

Differential involvement of Gα12 and Gα13 in receptor-mediated stress fiber formation.

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 Gαq/Gα11-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 Gαq/Gα11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1α receptors was dependent on Gq/G11proteins. 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 Gα13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Gα12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Gαq and Gα11 and that this involves either a Gα12or a Gα13/EGF receptor-mediated pathway.

Embryonic cardiomyocyte hypoplasia and craniofacial defects in Gαq. Gα11‐mutant mice

Heterotrimeric G proteins of the Gq class have been implicated in signaling pathways regulating cardiac growth under physiological and pathological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed Gαq class genes, Gαq and Gα11, demonstrate that at least two active alleles of these genes are required for extrauterine life. Mice carrying only one intact allele [Gαq(−/+);Gα11(−/−) or Gαq(−/−);Gα11(−/+)] died shortly after birth. These mutants showed a high incidence of cardiac malformation. In addition, Gαq(−/−);Gα11(−/+) newborns suffered from craniofacial defects. Mice lacking both Gαq and Gα11 [Gαq(−/−);Gα11(−/−)] died at embryonic day 11 due to cardiomyocyte hypoplasia. These data demonstrate overlap in Gαq and Gα11 gene functions and indicate that the Gq class of G proteins plays a crucial role in cardiac growth and development.

Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues.

Regulators of G-protein signaling (RGS) play a critical role in G-protein-coupled receptor signaling in mammalian cells. RGS proteins are GTPase-accelerating proteins (GAPs) for alpha subunits of heterotrimeric G proteins of the Gi and Gq class. RGS GAPs can modulate the frequency and duration of G-protein signaling and may constitute a new family of therapeutic targets. Identifying the tissue distribution and cellular localization of RGS proteins has been hindered by the lack of effective antibodies for immunodetection. The measurement of mRNA levels for RGS proteins, however, can provide insight into their tissue specificity and regulation. This article describes the use of a highly sensitive and rapid method for measuring RGS mRNA in mouse tissues. This quantitative real-time polymerase chain reaction method is established for the 19 reported mouse RGS genes and is used to study the tissue distribution of the R4 family of RGS genes and the diurnal regulation of RGS16 in mouse liver.

RGS Proteins Provide Biochemical Control of Agonist-Evoked [Ca2+]i Oscillations

Agonist-evoked [Ca2+]i oscillations have been considered a biophysical phenomenon reflecting the regulation of the IP3 receptor by [Ca2+]i. Here we show that [Ca2+]i oscillations are a biochemical phenomenon emanating from regulation of Ca2+ signaling by the regulators of G protein signaling (RGS) proteins. [Ca2+]i oscillations evoked by G protein-coupled receptors require the action of RGS proteins. Inhibition of endogenous RGS protein action disrupted agonist-evoked [Ca2+]i oscillations by a stepwise conversion to a sustained response. Based on these findings and the effect of mutant RGS proteins and anti-RGS protein antibodies on Ca2+ signaling, we propose that RGS proteins within the G protein-coupled receptor complexes provide a biochemical control of [Ca2+]i oscillations.

Differentially Regulated Expression of Endogenous RGS4 and RGS7

Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27–28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30–40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short half-life. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction.

ALTERNATE COUPLING OF RECEPTORS TO GS AND GI IN PANCREATIC AND SUBMANDIBULAR GLAND CELLS

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 Gqclass 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 Cβ 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 β-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gsand Gi. This inference was based on the inhibitory action of antibodies specific for Gαs, Gαi, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of β-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 Gα15 −/− 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.

New Roles for Gα and RGS Proteins: Communication Continues despite Pulling Sisters Apart

Large G protein alpha subunits and their attendant regulators of G-protein signaling (RGS) proteins control both intercellular signaling and asymmetric cell divisions by distinct pathways. The classical pathway, found throughout higher eukaryotic organisms, mediates intercellular communication via hormone binding to G-protein-coupled receptors (GPCRs). Recent studies have led to the discovery of GPCR-independent activation of Galpha subunits by the guanine nucleotide exchange factor RIC-8 in both asymmetric cell division and synaptic vesicle priming in metazoan organisms. Protein-protein interactions and protein function in each pathway are driven through the cycle of GTP binding and hydrolysis by the Galpha subunit. This review builds a conceptual framework for understanding RIC-8-mediated pathways by comparison with the mechanism of classical G-protein activation and inhibition in GPCR signaling.