GIPC recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling

Pleiotropic G proteins are essential for the action of hormones and neurotransmitters and are activated by stimulation of G protein-coupled receptors (GPCR), which initiates heterotrimer dissociation of the G protein, exchange of GDP for GTP on its Galpha subunit and activation of effector proteins. Regulator of G protein signaling (RGS) proteins regulate this cascade and can be recruited to the membrane upon GPCR activation. Direct functional interaction between RGS and GPCR has been hypothesized. We show that recruitment of GAIP (RGS19) by the dopamine D2 receptor (D2R), a GPCR, required the scaffold protein GIPC (GAIP-interacting protein, C terminus) and that all three were coexpressed in neurons and neuroendocrine cells. Dynamic translocation of GAIP to the plasma membrane and coassembly in a protein complex in which GIPC was a required component was dictated by D2R activation and physical interactions. In addition, two different D2R-mediated responses were regulated by the GTPase activity of GAIP at the level of the G protein coupling in a GIPC-dependent manner. Since GIPC exclusively interacted with GAIP and selectively with subsets of GPCR, this mechanism may serve to sort GPCR signaling in cells that usually express a large repertoire of GPCRs, G proteins, and RGS.     

Unique isoform of Galpha -interacting protein (RGS-GAIP) selectively discriminates between two Go-mediated pathways that inhibit Ca2+ channels

Regulators of G-protein signaling (RGS) proteins constitute a large family of GTPase-activating proteins for heterotrimeric G proteins. More than 20 RGS genes have been identified in mammals. One of these, the Galpha-interacting protein (GAIP), preferentially interacts with members of the G(i)/G(o) subfamily of G proteins in mammalian cells, but its selectivity among members of this subfamily in vitro is limited. Here we report the cloning and functional characterization of a unique cDNA isoform of GAIP, derived from embryonic chicken dorsal root ganglion neurons. Chick GAIP is composed of 199 amino acids, organized into a conserved RGS domain (85% identical to human GAIP), and a unique, short N terminus (only 41% identical, 50% homologous to known mammalian orthologues). Consistent with this unique primary structure, chick GAIP has physiological properties that distinguish it from mammalian GAIPs. We have explored the selectivity of chick GAIP in electrophysiological assays of two G(o)-mediated forms of Ca(2+) channel inhibition produced by gamma-aminobutyric acid in chick dorsal root ganglion neurons, voltage-independent inhibition (mediated by G(o)alpha) and voltage-dependent inhibition (mediated by G(o)betagamma). Dialyzing recombinant chick GAIP in these cells selectively reduced voltage-independent inhibition without affecting voltage-dependent inhibition. Mammalian GAIP, tested under identical conditions in previous studies, demonstrated no selectivity between these two inhibitory processes; thus, our results suggest that the functional specificity of chick GAIP is likely to be determined by its unique N terminus.     

Solution structure of human GAIP (Galpha interacting protein): a regulator of G protein signaling.

The solution structure of the human protein GAIP (Galpha interacting protein), a regulator of G protein signaling, has been determined by NMR techniques. Dipolar couplings of the oriented protein in two different liquid crystal media have been used in the structure calculation. The solution structure of GAIP is compared to the crystal structure of an homologous protein from rat (RGS4) complexed to the alpha-subunit of a G protein. Some of RGS4 residues involved in the Galpha-RGS binding interface have similar orientations in GAIP (free form), indicating that upon binding these residues do not suffer conformational rearrangements, and therefore, their role does not seem to be restricted to Galpha interaction but also to RGS folding and stability. We suggest that other structural differences between the two proteins may be related to the process of binding as well as to a distinct efficiency in their respective GTPase activating function.     

Endogenous RGS proteins attenuate Galpha(i)-mediated lysophosphatidic acid signaling pathways in ovarian cancer cells

Lysophosphatidic acid is a bioactive phospholipid that is produced by and stimulates ovarian cancer cells, promoting proliferation, migration, invasion, and survival. Effects of LPA are mediated by cell surface G-protein coupled receptors (GPCRs) that activate multiple heterotrimeric G-proteins. G-proteins are deactivated by Regulator of G-protein Signaling (RGS) proteins. This led us to hypothesize that RGS proteins may regulate G-protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared LPA activity in SKOV-3 ovarian cancer cells expressing G(i) subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS wild-type (RGSwt) counterparts. Both forms of the G-protein contained a point mutation rendering them insensitive to inhibition with pertussis toxin, and cells were treated with pertussis toxin prior to experiments to eliminate endogenous G(i/o) signaling. The potency and efficacy of LPA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi G(i) proteins as compared to RGSwt G(i). We further showed that LPA signaling that is subject to RGS regulation terminates much faster than signaling thru RGS insensitive G-proteins. Finally, LPA-stimulated SKOV-3 cell migration, as measured in a wound-induced migration assay, was enhanced in cells expressing Galpha(i2) RGSi as compared to cells expressing Galpha(i2) RGSwt, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate this LPA effect. These data establish RGS proteins as novel regulators of LPA signaling in ovarian cancer cells.     

RGS17/RGSZ2 and the RZ/A family of regulators of G-protein signaling

Regulators of G-protein signaling (RGS proteins) comprise over 20 different proteins that have been classified into subfamilies on the basis of structural homology. The RZ/A family includes RGSZ2/RGS17 (the most recently discovered member of this family), GAIP/RGS19, RGSZ1/RGS20, and the RGSZ1 variant Ret-RGS. The RGS proteins are GTPase activating proteins (GAPs) that turn off G-proteins and thus negatively regulate the signaling of G-protein coupled receptors (GPCRs). In addition, some RZ/A family RGS proteins are able to modify signaling through interactions with adapter proteins (such as GIPC and GIPN). The RZ/A proteins have a simple structure that includes a conserved amino-terminal cysteine string motif, RGS box and short carboxyl-terminal, which confer GAP activity (RGS box) and the ability to undergo covalent modification and interact with other proteins (amino-terminal). This review focuses on RGS17 and its RZ/A sibling proteins and discusses the similarities and differences among these proteins in terms of their palmitoylation, phosphorylation, intracellular localization and interactions with GPCRs and adapter proteins. The specificity of these RGS protein for different Galpha proteins and receptors, and the consequences for signaling are discussed. The tissue and brain distribution, and the evolving understanding of the roles of this family of RGS proteins in receptor signaling and brain function are highlighted.     

RGS7 and RGS8 differentially accelerate G protein-mediated modulation of K+ currents

The recently discovered family of RGS (regulators of G protein signaling) proteins acts as GTPase activating proteins which bind to alpha subunits of heterotrimeric G proteins. We previously showed that a brain-specific RGS, RGS8 speeds up the activation and deactivation kinetics of the G protein-coupled inward rectifier K+ channel (GIRK) upon receptor stimulation (Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529). Here we report the isolation of a full-length rat cDNA of another brain-specific RGS, RGS7. In situ hybridization study revealed that RGS7 mRNA is predominantly expressed in Golgi cells within granule cell layer of cerebellar cortex. We observed that RGS7 recombinant protein binds preferentially to Galphao, Galphai3, and Galphaz. When co-expressed with GIRK1/2 in Xenopus oocytes, RGS7 and RGS8 differentially accelerate G protein-mediated modulation of GIRK. RGS7 clearly accelerated activation of GIRK current similarly with RGS8 but the acceleration effect of deactivation was significantly weaker than that of RGS8. These acceleration properties of RGS proteins may play important roles in the rapid regulation of neuronal excitability and the cellular responses to short-lived stimulations.     

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells

Regulators of G protein signaling (RGS proteins) serve as GTPase activating proteins for the signal transducing Gα subunits. RGS19, also known as Gα-interacting protein (GAIP), has been shown to subserve other functions such as the regulation of macroautophagy and growth factor signaling. We have recently demonstrated that the expression of RGS19 in human embryonic kidney (HEK) 293 cells resulted in the disruption of serum-induced mitogenic response along the classical Ras/Raf/MEK/ERK pathway. Here, we further examined the effect of RGS19 expression on the stress-activated protein kinases (SAPKs). Both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) became non-responsive to serum in 293/RGS19 cells, yet the two SAPKs responded to UV irradiation or osmotic stress induced by sorbitol. Kinases upstream of JNK and p38 MAPK, including MKK3/6, MKK4, and MLK3, also failed to respond to serum stimulation in 293/RGS19 cells. Serum-induced activation of the small GTPases Rac1 and Cdc42 was similarly suppressed in these cells. Our results indicate that elevated expression of RGS19 can severely disrupt the regulation of MAPKs by small GTPases.     

Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.     

Amino-terminal Cysteine Residues Differentially Influence RGS4 Protein Plasma Membrane Targeting, Intracellular Trafficking, and Function

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.     

Two chimeric regulators of G-protein signaling (RGS) proteins differentially modulate soybean heterotrimeric G-protein cycle

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1-4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1-4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks.     

RGS-GAIP, a GTPase-activating Protein for Gαi Heterotrimeric G Proteins, Is Located on Clathrin-coated Vesicles

RGS-GAIP (Gα-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Gα_i/Gα_q-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Gα_i3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.     

Acute stress responsive RGS proteins in the mouse brain

Regulator of G-protein signaling (RGS) proteins play an important role in G-protein coupled receptor (GPCR) signaling and the activity of some GPCRs is modulated via RGS protein levels during stress response. The aim of this study was to investigate changes in RGS protein mRNA expressions in the mouse brain after 2h restraint stress. The mRNA level of 19 RGS proteins was analyzed using real-time PCR in six brain regions, which included the prefrontal cortex, amygdala, hippocampus, hypothalamus, striatum, and pituitary gland, from control and stressed mouse. We found that the level of mRNA of each RGS varied according to brain region and that two to eight RGS proteins exhibited changes in mRNA levels in each brain region by restraint stress. It was also revealed that RGS4 protein amount was consistent with mRNA level, indicating RGS4 protein may have regulatory roles in the acute stress response.     

A finer tuning of G-protein signaling through regulated control of RGS proteins

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for various Gα subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate the magnitude and duration of G-protein-coupled receptor signaling and are often referred to as fine tuners of G-protein signaling. Increasing evidence suggests that RGS proteins themselves are regulated through multiple mechanisms, which may provide an even finer tuning of G-protein signaling and crosstalk between G-protein-coupled receptors and other signaling pathways. This review summarizes the current data on the control of RGS function through regulated expression, intracellular localization, and covalent modification of RGS proteins, as related to cell function and the pathogenesis of diseases.     

Altered expression and function of regulator of G-protein signaling-17 (RGS17) in hepatocellular carcinoma

Guanine nucleotide regulatory proteins (G-proteins) are central to normal hepatocyte function and are implicated in hepatic disease initiation and progression. Regulators of G-protein signaling (RGS) are critical to defining G-protein-dependent signal fidelity, yet the role of RGS proteins in the liver is poorly defined. The aims of this study were to determine RGS17 expression in normal and transformed hepatic tissue and cells, and address the function of RGS17 in hepatic tumorgenicity. RGS17 expression was determined in human and rat HCC tissue and cell lines. Molecular approaches were used to alter RGS17 expression in HCC cells, effects on cell function measured, and RGS17 association with specific Gα-subunits determined. Using these approaches RGS17 mRNA, but not protein, was detectable in human and rat HCC tissue and cells. Conversely, RGS17 mRNA was not detected in normal tissue, isolated hepatocytes, or non-tumorigenic hepatic cells. Subsequent studies using transfected cells demonstrated that RGS17 proteins were not post-translationally modified in HCC cells, and RGS17 expression is governed by protein degradation and not via miRNAs. Notwithstanding inherently low RGS17 protein levels, altering RGS17 expression profoundly affected HCC cell mitogenesis and migration. Analysis of RGS17-G-protein interaction demonstrated RGS17 associates with both Giα- and Gqα-subunits in HCC cells of human and rat origin. In conclusion, these data demonstrate that, despite difficulties in measuring endogenous RGS protein expression, RGS17 is differentially expressed in HCC and plays a central role in regulating transformed hepatocyte tumorgenicity.     

GAIP, a Galpha i-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking

Proteins of theregulators of G protein signaling (RGS) family bind to G subunits todownregulate their signaling in a variety of systems. G-interactingprotein (GAIP) is a mammalian RGS protein that shows high affinity forthe activated state of G_i-3, aprotein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP inepithelial cells and to investigate its potential role in the regulation of membrane trafficking.LLC-PK₁ cells were stably transfected with a c-myc-tagged GAIPcDNA. In the transfected and untransfected cells, GAIP was found in thecytosol and on cell membranes. Immunogold labeling showed thatmembrane-bound GAIP was localized on budding vesicles around Golgistacks. When an in vitro assay was used to generate vesicles fromisolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgivesicles. Finally, the constitutive trafficking and secretion ofsulfated proteoglycans was measured in cell lines overexpressing GAIP.We show evidence for GAIP regulation of secretory trafficking beforethe level of the trans-Golgi networkbut not in post-Golgi secretion. The location and functional effects ofGAIP overlap only partially with those ofG_i-3 and suggest multiple roles for GAIP in epithelial cells.     

Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues

Regulator of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for Galpha subunits and negatively regulate G protein-coupled receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N-terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N-terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Galphai coupled cannibinoid receptor-1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Galphaq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Galphaq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.     

Regulators of G protein signaling proteins as determinants of the rate of desensitization of presynaptic calcium channels.

Norepinephrine inhibits omega-conotoxin GVIA-sensitive presynaptic Ca2+ channels in chick dorsal root ganglion neurons through two pathways, one mediated by Go and the other by Gi. These pathways desensitize at different rates. We have found that recombinant Galpha interacting protein (GAIP) and regulators of G protein signaling (RGS)4 selectively accelerate the rate of desensitization of Go- and Gi-mediated pathways, respectively. Blockade of endogenous RGS proteins using antibodies raised against Galpha interacting protein and RGS4 slows the rate of desensitization of these pathways in a selective manner. These results demonstrate that different RGS proteins may interact with Gi and Go selectively, giving rise to distinct time courses of transmitter-mediated effects.