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 ...

We have identified two processes in the G1 phase of the Saccharomyces cerevisiae cell cycle that are required before nutritionally arrested cells are able to return to proliferative growth. The first process requires protein synthesis and is associated with increased expression of the G1 cycin gene CLN3. This process requires nutrients but is independent of Ras and cyclic AMP (cAMP). The second process requires cAMP. This second process is rapid, is independent of protein synthesis, and produces a rapid induction of START-specific transcripts, including CLNI and CLN2. The ability of a nutritionally arrested cell to respond to cAMP is dependent on completion of the first process, and this is delayed in cells carrying a CLN3 deletion. Mating pheromone blocks the cAMP response but does not alter the process upstream of Ras-cAMP. These results suggest a model linking the Ras-cAMP pathway with regulation of G1 cyclin expression.A fundamental goal in biology is to understand how cells control proliferative growth. In recent years, progress toward this goal has been made in two important areas. The first area involves the identification and study of oncogenes-genes that in many cases encode proteins that carry signals regulating cellular proliferation. Mutations in these genes lead to aberrant signalling, unregulated proliferation, and cancer. The second area involves the discovery of two families of proteins, the cyclins and the cell cycle-dependent protein kinases (CDKs), that are believed to allow cells to pass checkpoints in the cell cycle (26,28). Included among these cell cycle checkpoints is the G1-to-S phase transition that is known as START in the yeast Saccharomyces cerevisiae or as the restriction point in mammals (16,25).In S. cerevisiae, three cyclin genes that affect passage through START have been identified: CLN1, CLN2, and CLN3. The protein products of these genes activate the only member of the CDK family known to exist in S. cerevisiae, encoded by CDC28. Activation of p34CDC28 enables cells to pass the START checkpoint. Cells remain viable after loss of any two of the G1 cyclin genes; however, loss of all three G1 cyclin genes leaves the cells arrested permanently at START, an effect similar to that produced by loss of the CDC28 kinase (29). In contrast, activating mutations in any of the G, cyclin genes, or the overexpression of any of these genes, results in small cells with an abbreviated or absent G, phase (8). These and other results suggest a pathway in which three redundant cyclins associate with and activate the p34CDC28 kinase in order to carry cells through START.Although the three mitotic cyclins appear to serve redundant functions, there are distinct differences between them. CLN1, CLN2, and CLN3 all show sequence homologies with the mitotic cyclins (7, 22, 32); however, CLNI and CLN2 show much greater similarity to each other than to CLN3. CLN3 also stands apart in its expression pattern. While CLNI and CLN2 expression peaks dramatically at the Ga/S boundary, the level of CLN3 message r...

Previous studies have shown that lysine- and arginine-rich proteins can enhance the activity of tyrosine and serine/threonine protein kinases. However, the kinetics and mechanism of this activation are not fully understood. Therefore we investigated the ability of poly(amino acids) and the arginine-rich protein, protamine, to alter the kinetic properties of epidermal growth factor (EGF) receptor protein-tyrosine kinase activity using immunoaffinity-purified receptor isolated from human epidermoid carcinoma (A431) cells. Poly(L-lysine), poly(L-arginine) and protamine stimulated EGF receptor kinase activity by 3-5-fold at non-saturating doses of ATP and peptide substrate, while poly(L-glutamate) had no effect. Initial kinetic studies demonstrated an increase in the maximum velocity and a decrease in the apparent Km for the peptide substrate angiotensin II in the presence of the basic effectors. Further analysis of the kinetic mechanism by product inhibition revealed that protamine altered the pattern of ADP inhibition towards the peptide substrate but not towards ATP. The change was indicative of the receptor's ability to form an enzyme-angiotensin II-ADP ternary complex in the presence of protamine but not in its absence. In addition, the basic effectors had a substantially decreased influence on the kinase activity of a C-terminally truncated form of the EGF receptor. Thus the changes in kinase activity may be partially mediated by the C-terminal region of the receptor, which contains the sites of receptor self-phosphorylation. These results suggest that the basic domains of proteins can interact with the EGF receptor to induce changes in its kinetic properties, especially with regard to reactant recognition and binding.