SUBCELLULAR-LOCALIZATION AND KINETIC CHARACTERIZATION OF GUANINE-NUCLEOTIDE-BINDING PROTEINS IN NORMAL RAT AND HUMAN PANCREATIC-ISLETS AND TRANSFORMED BETA-CELLS

The subcellular localization and the kinetics of the GTPase activities of monomeric and heterotrimeric GTP-binding proteins were investigate d in normal rat and human pancreatic islets and were compared to those obtained using a transformed hamster beta cell line (HIT cells). The [alpha-P-32]GTP overlay technique revealed the presence of at least fo ur low-molecular-mass proteins (approx. 20-27 kDa) in normal rat islet s, which were enriched in the secretory granule fraction compared to t he membrane fraction (with little abundance of these proteins in the c ytosolic fraction). In contrast, in HIT cells, these proteins (at leas t six) were predominantly cytosolic. Three of these proteins were immu nologically identified as rab3A, rac2 and CDC42Hs in islets as well as in HIT cells. In addition, pertussis toxin augmented the ribosylation of at least one heterotrimeric G-protein of about 39 kDa (probably G( i) and/or G(0)) in the membrane and secretory granule fractions of nor mal rat and human islets, whereas at least three such Ptx substrates ( 36-39 kDa) were found in HIT cell membranes. Kinetic analyses of the i ntrinsic specific GTPase activities revealed the presence of at least three such activities (K-m for GTP of 372 nM, 2.2 mu M, and 724 mu M) in islet homogenates which were differentially distributed in various subcellular fractions; similar activities were also demonstrable in HI T cell homogenates. Thus, these studies demonstrate the presence of bo th monomeric as well as trimeric G-proteins intrinsic to the secretory granules of normal rat islets which can be ascribed to beta cells; si nce these G-proteins are regulated by insulinotropic lipids (as descri bed in the accompanying article), such proteins may couple the activat ion of phospholipases (endogenous to islets) to the exocytotic secreti on of insulin. These findings also suggest that caution is necessary i n extrapolating data concerning G-proteins from cultured, transformed beta cell lines to the physiology of normal islets, in view of both qu alitative and quantitative differences between the two preparations.