Regulator of G protein signaling (RGS) proteins are responsible for the rapid turnoff of G protein-coupled receptor signaling pathways. The major mechanism whereby RGS proteins negatively regulate G proteins is via the GTPase activating protein activity of their RGS domain. Structural and mutational analyses have characterized the RGS/G alpha interaction in detail, explaining the molecular mechanisms of the GTPase activating protein activity of RGS proteins. More than 20 RGS proteins have been isolated, and there are indications that specific RGS proteins regulate specific G protein-coupled receptor pathways. This specificity is probably created by a combination of cell type-specific expression, tissue distribution, intracellular localization, posttranslational modifications, and domains other than the RGS domain that link them to other signaling pathways. In this review we discuss what has been learned so far about the role of RGS proteins in regulating G protein-coupled receptor signaling and point out areas that may be fruitful for future research.

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of Gprotein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein G␣ i3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [ 35 S]methionine, two pools of GAIP-a soluble and a membrane-anchored pool-were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membraneanchored GAIP might be palmitoylated. We found that after labeling with [ 3 H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the G␣ i subfamily, G␣ i1 , G␣ i2 , G␣ i3 , G␣ z , and G␣ o , but not with members of other G␣ subfamilies, G␣ s , G␣ q , and G␣ 12/13 . The C terminus of G␣ i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of G␣ i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of G␣ i3 , which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP's possible involvement in membrane trafficking.Using the yeast two-hybrid system, we recently identified GAIP, a human protein that specifically interacts with the heterotrimeric G protein G␣ i3 (1). GAIP is a member of the newly described RGS family (for Regulators of G-protein Signaling) (1-5) whose Ϸ15 members share an Ϸ125-aa homologous core domain and are thought to regulate G-protein signaling. This core domain, now referred to as the RGS domain, is the site of interaction with the G␣ subunit (1). Mutants of two RGS family members, EGL-10 in Caenorhabditis elegans and Sst2 in Saccharomyces cerevisiae, show a delay in egg-laying behavior (3) and desensitization to pheromone (6), respectively. Another family member, RGS4, was shown to inhibit mitogen-activated protein (MAP) kinase activity stimulated through G-protein-coupled receptors (2).The recent demonstration that GAIP, RGS4, and other RGS proteins function as GTPase-activating proteins (GAPs) for G␣ i subunits in vitro (7-9) indicates that these molecules negatively regulate heterotrimeric G proteins by stimulating their intrinsically low GTPase activity, returning them to the inactive GDP-bound state. A number of GAPs have been isolated for the small GTP-binding proteins. The distribution and interaction of rasGAP with ras is particularly well documented (10, 11). To date no information is av...

Monitoring Editor: Suzanne R. Pfeffer RGS-GAIP (G␣-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate G␣ i /G␣ 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 G␣ 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. INTRODUCTIONClassical G protein-mediated signaling pathways are three-component systems consisting of serpentine (seven-transmembrane domain) plasma membrane (PM) 1 receptors, heterotrimeric G proteins composed of ␣, ␤, and ␥ subunits, and an effector, usually an enzyme or an ion channel (Gilman, 1987;Bourne et al., 1990;Neer, 1995;Hamm and Gilchrist, 1996). The newly discovered family of proteins known as RGS proteins (regulators of G protein signaling) constitute a fourth component of these systems (Dohlman and Thorner, 1997;Koelle, 1997;Neer, 1997;Berman and Gilman, 1998). RGS proteins serve as guanosine triphosphatase-activating proteins (GAPs) that accelerate the guanosine triphosphatase activity of G␣i/ G␣q subunits by stabilizing the G␣ subunit in its guanosine triphosphate (GTP)-to-guanosine diphosphate (GDP) transition state , returning them to their inactive GDP-bound form Hunt et al., 1996;Watson et al., 1996), and thereby terminating the G protein signal. The RGS protein family has been implicated in desensitization and negative regulation of heterotrimeric G proteinsignaling pathways in yeast, fungi, and nematodes (Dohlman et al., 1996;Koelle and Horvitz, 1996;Yu et al., 1996). In mammalian cells, RGS proteins have been implicated in the negative regulation of MAP kinase and phosphoinositide-phospholipase C activity and a loss of inhibition of adenylate cyclase activity by G␣i subunits Chatterjee et al., 1997;Huang et al., 1997;Yan et al., 1997). RGS proteins may also regulate cell death as suggested by the finding that A28-RGS14 is transcriptionally activated by the tumor ...

Regulators of G-protein signaling (RGS) proteins are GTPaseactivating proteins (GAPs) that bind to G␣ subunits and attenuate G protein signaling, but where these events occur in the cell is not yet established. Here we investigated, by immunofluorescence labeling and deconvolution analysis, the site at which endogenous G␣-interacting protein (GAIP) (RGS19) binds to G␣i3-YFP and its fate after activation of ␦-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from G␣i3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas G␣i3-YPF and DOR are located in non-clathrin-coated microdomains of the PM. Upon addition of agonist, G␣i3 partially colocalizes with GAIP in CCPs at the PM. When endocytosis is blocked by expression of a dynamin mutant [dyn(K44A)], there is a striking overlap in the distribution of DOR and G␣i3-YFP with GAIP in CCPs. Moreover, G␣i3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and G␣i3 move together into CCPs where G␣i3 and GAIP meet and turn off G protein signaling. Subsequently, G␣i3 returns to non-clathrin-coated microdomains of the PM, GAIP remains stably associated with CCPs, and DOR is internalized via clathrin-coated vesicles. This constitutes a novel mechanism for regulation of G␣ signaling through spatial segregation of a GAP in clathrin-coated pits.