GLUCOSE-DEPENDENT AND GTP-DEPENDENT STIMULATION OF THE CARBOXYL METHYLATION OF CDC42 IN RODENT AND HUMAN PANCREATIC-ISLETS AND PURE BETA-CELLS - EVIDENCE FOR AN ESSENTIAL ROLE OF GTP-BINDING PROTEINS IN NUTRIENT-INDUCED INSULIN-SECRETION

Several GTP-binding proteins (G-proteins) undergo posttranslational mo difications (isoprenylation and carboxyl methylation) in pancreatic be ta cells. Herein, two of these were identified as CDC42 and rap 1, usi ng Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but n ot in acinar cells of the pancreas. CDC42 undergoes a guanine nucleoti de-specific membrane association and carboxyl methylation in normal ra t islets, human islets, and pure beta (HIT or INS-1) cells. GTP gamma S-dependent carboxyl methylation of a 23-kD protein was also demonstra ble in secretory granule fractions from normal islets or beta cells. A FC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferas es) blocked the carboxyl methylation of CDC42 in five types of insulin -secreting cells, without blocking GTP gamma S-induced translocation, implying that methylation is a consequence (not a cause) of transfer t o membrane sites. High glucose (but not a depolarizing concentration o f K+) induced the carboxyl methylation of CDC42 in intact cells, as as sessed after specific immunoprecipitation. This effect was abrogated b y GTP depletion using mycophenolic acid and was restored upon GTP repl etion by coprovision of guanosine. In contrast, although rap 1 was als o carboxyl methylated, it was not translocated to the particulate frac tion by GTP gamma S; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimu lation). AFC also impeded nutrient-induced (but not K+-induced) insuli n secretion from islets and beta cells under static or perifusion cond itions, whereas an inactive structural analogue of AFC failed to inhib it insulin release. These effects were reproduced not only by S-adenos ylhomocysteine (another methylation inhibitor), but also by GTP deplet ion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-pr oteins such as CDC42 is an obligate step in the stimulus-secretion cou pling of nutrient-induced insulin secretion, but not in the exocytotic event itself. Furthermore, AFC blocked glucose-activated phosphoinosi tide turnover, which may provide a partial biochemical explanation for its effect on secretion, and implies that certain G-proteins must be carboxyl methylated for their interaction with signaling effector mole cules, a step which can be regulated by intracellular availability of GTP.