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GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5′ coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 µg·mg of platelet protein-1 ) and contained substantial amounts of Rab32 (0.11 µg·mg protein-1 ). Little Rab31 was present (0.005 µg·mg protein-1 ). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28-kDa Rab32 was replaced by a 52-kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S -transferase (GST)-fusion proteins, did not bind [α-32 P]GTP on nitrocellulose blots but did bind [35 S]GTP[S] in a Mg2+ -dependent manner. Binding of [35 S]GTP[S] was optimal with 5 µm Mg2+ free and was markedly inhibited by higher Mg2+ concentrations in the case of GST–Rab31 but not GST–Rab32. Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations... [ABSTRACT FROM AUTHOR]

Gi3, a member of the Gi family of heterotrimeric GTP-binding proteins, regulates vesicle trafficking along both the constitutive and regulated pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activation of Gi3 provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. & Sagi-Eisenberg, R. (1993) Science 262, 1569-1572]. Such activation can be achieved in patch-clamped or streptolysin-O (SLO)-permeabilized mast cells by a combination of Ca2+ and nonhydrolyzable analogs of GTP. In contrast, Ca2+-activated exocytosis in intact cells is Gi3 independent. We show here that overexpression of a GTPase-deficient mutant (Gαi3Q204L), but not of the wild-type form of Gαi3, in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mucosal mast cells, resulted in marked potentiation of exocytosis and release of arachidonic acid in intact cells activated by a Ca2+ ionophore alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, exocytosis and arachidonic acid release stimulated by aggregation of the cell surface receptors for immunoglobulin E (IgE) were unaffected. These results strongly suggest that the intracellular receptor, responsible for the activation of Gi3, is a low-affinity Ca2+-binding protein that can only be activated during Ca2+ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca2+-activated and Gi3-mediated signaling pathway requires the blocking of Gi3 GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi3. [ABSTRACT FROM AUTHOR]

Gi3, a member of the Gi family of heterotrimeric GTP-binding proteins, regulates vesicle trafficking along both the constitutive and regulated pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activation of Gi3 provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. & Sagi-Eisenberg, R. (1993) Science 262, 1569-1572]. Such activation can be achieved in patch-clamped or streptolysin-O (SLO)-permeabilized mast cells by a combination of Ca2+ and nonhydrolyzable analogs of GTP. In contrast, Ca2+-activated exocytosis in intact cells is Gi3 independent. We show here that overexpression of a GTPase-deficient mutant (Gαi3Q204L), but not of the wild-type form of Gαi3, in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mucosal mast cells, resulted in marked potentiation of exocytosis and release of arachidonic acid in intact cells activated by a Ca2+ ionophore alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, exocytosis and arachidonic acid release stimulated by aggregation of the cell surface receptors for immunoglobulin E (IgE) were unaffected. These results strongly suggest that the intracellular receptor, responsible for the activation of Gi3, is a low-affinity Ca2+-binding protein that can only be activated during Ca2+ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca2+-activated and Gi3-mediated signaling pathway requires the blocking of Gi3 GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi3. [ABSTRACT FROM AUTHOR]

The mononaeric GTP-binding protein Rab3a controls exocytosis in neuroendocrine and neuronal cells. Like other members of the Rab family, Rab3a is posttranslationally modified by the addition of hydrophobic geranylgeranyl groups to its C-terminus. The geranylgeranylation reaction is catalysed by the heterotrimeric geranylgeranyl transferase II. We describe the cDNA cloning of the β-subunit of human geranylgeranyl transferase II by means of the yeast two-hybrid system. The human enzyme, which is 49% and 96% similar to yeast and rat isoforms, respectively, can complement the β-subunit deficiency in the yeast strain ANY119. Furthermore, by means of the two-hybrid system and in vitro geranylgeranylation reactions with purified recombinant rat geranylgeranyl transferase II, we have characterized Rab3a domains implicated in the interaction with geranylgeranyl transferase II. We find that the N-terminus. the effector loop, the hypervariable region of the C-terminus, and the geranylgeranyl-acceptor cysteines have roles in this interaction. The GDP-bound form of Rab3a is the preferred substrate of geranylgeranyl transferase II. [ABSTRACT FROM AUTHOR]

A novel ras-related gene (rab28) was identified by a PCR-based cloning approach and subsequent screening of rat fat cell and brain cDNA libraries. The deduced amino acid sequence of the cDNA is distantly related with members of the Rab family (31-33% sequence identity, mainly restricted to the six GTP-binding motifs). Cloning of the human homologue of Rab28 by a PCR-based approach revealed the existence of two isoforms (hRab28s, hRab28L) which differ only by a 95-bp insertion within the coding region. This insertion generates an alternative sequence of the 30 C-terminal amino acids of the protein. Both C-termini of the human homologues comprise farnesylation motifs, but differ strikingly in a stretch of 13 amino acids. By PCr, mRNA of hRab28S was detected in most tissues investigated (cortex, liver, kidney, skeletal muscle, adipose tissue, tests and urothelium), whereas hRab28L was predominant in testis. Recombinant Rab28 proteins showed specific binding of radiolabeled guanosine 5'-O-[γ-thio]triphosphate and rapidly hydrolysed [a-32P]GTP: there was no difference in the GTP binding characteristics of the two isoforms hRab28S and hRab28L. It is suggested that the isoforms are derived from the same gene by alternative mRNA splicing, and that their functions differ in a parameter unrelated to its basic role as a GTPase. [ABSTRACT FROM AUTHOR]

We have demonstrated previously that the GTP-binding protein γ12 subunit is a selective substrate for phosphorylation by protein kinase C among various γ subunits in vitro, and that a serine residue in the N-terminal region is involved. In the present study, we first determined that the site of phosphorylation was Ser1 with antibodies developed against two N-terminal peptides containing phosphorylated Ser1 and Ser2, respectively. Using an antibody recognizing phosphorylated γ12 and Swiss 3T3 cells rich in this protein, γ12 was found to be phosphorylated by stimulation of quiescent cells with various reagents, such as phorbol 12-myristate 13-acetate (PMA), NaF, fetal calf serum, lysophosphatidic acid, endothelin, and growth factors. Pertussis toxin completely and partially prevented phosphorylation of γ12 induced by lysophosphatidic acid and fetal calf serum and by endothelin, respectively, suggesting a contribution of Gi/o. Phosphorylation of γ12 was limited when cells were stimulated by a single reagent, even with PMA, a strong activator of protein kinase C, whereas simultaneous stimulation with lysophosphatidic acid and either PMA or platelet-derived growth factor induced a synergistic increase of phosphorylation, suggesting physiological roles for GTP-binding proteins and protein kinase C in combination. Phosphorylated γ12 was also detected in various tissues of untreated rats. Its decrease by pertussis toxin treatment also suggested the involvement of Gi/o in vivo. [ABSTRACT FROM AUTHOR]

Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a novel approach to study catalysis by G-proteins. in these studies, we have used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]Gsa mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild-type enzyme at the same rate. On the other hand, modification of Gsa by cholera toxin, which catalyses ADP-ribosylation of Arg201 of Gsa, decreased the rates of hydrolysis of both GTP and its analog by 95 %. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]Gsa mutant and to the essential role of Gin227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the tale-limiting step of the GTPase reaction. [ABSTRACT FROM AUTHOR]

The ORrab2 gene encodes a GTP-binding. protein of 23.169 kDa. The deduced amino acid sequence shows that ORrab2 has the motifs conserved among small GTP-binding proteins in plants and that it shares sequence identity with Atrab2 (93.0%), Hrab2 (85.2%}. Hrab4 (5i.9%}, Hrab1 (46.2%). YPT (40.7ck), Hrab3B (40.0%). Hrab3A (38.1%). SEC4 (38.1%), Hrab5 (34.3%) and Hrab6 (32.4%). To analyze the biochemical properties of this protein, an ORrab2 cDNA was overexpressed in Escherichia coli and the protein purified by Ni2+-nitrilotriacetic acid agarose and hydroxyapatite column chromatogra- phy. The molecular mass of the protein bearing a His-tag is approximately 28.2 kDa. The guanine-nucleo- tide binding and hydrolyzing activity of ORrab2 increased with non-ionic C12E10 (polyoxyethylene 10-lauryl ether) and ionic Chaps detergent treatment. ORrab2 bound maximally 1.03 mol of [γ-35 S]GTP[S]/ tool of protein with a Ka value of 56.83 nM. The ratios koff GDP /koff GTP of ORrab2 were 3.63 for the control, 3.7 in the presence of C12E10, and 3.83 with Chaps, indicating that ORrab2 has a higher affinity for GTP than GDP. The rate (kcat) of P1 release against [γ-32P]GTP bound ORrab2 in a steady state and the rate of hydrolysis of [γ-32P]GTP (kGTPase) were calculated to be 432×10-4 ± 8×10-4 min-1 and 172×10-4 ±2 × 10-4 min-1, respectively, in the presence of 0.1% C12E10 and 1 mM MgSO4. [ABSTRACT FROM AUTHOR]