Subcellular localization and kinetic characterization of guanine nucleotide binding proteins in normal rat and human pancreatic islets and transformed β cells

Abstract

The subcellular localization and the kinetics of the GTPase activities of monomeric and heterotrimeric GTP-binding proteins were investigated in normal rat and human pancreatic islets and were compared to those obtained using a transformed hamster β cell line (HIT cells). The [α- 32P]GTP overlay technique revealed the presence of at least four low-molecular-mass proteins (approx. 20–27 kDa) in normal rat islets, which were enriched in the secretory granule fraction compared to the membrane fraction (with little abundance of these proteins in the cytosolic fraction). In contrast, in HIT cells, these proteins (at least six) were predominantly cytosolic. Three of these proteins were immunologically 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 o) in the membrane and secretory granule fractions of normal rat and human islets, whereas at least three such Ptx substrates (36–39 kDa) were found in HIT cell membranes. Kinetic analyses of the intrinsic specific GTPase activities revealed the presence of at least three such activities ( K m for GTP of 372 nM, 2.2 μM, and 724 μM) in islet homogenates which were differentially distributed in various subcellular fractions; similar activities were also demonstrable in HIT cell homogenates. Thus, these studies demonstrate the presence of both monomeric as well as trimeric G-proteins intrinsic to the secretory granules of normal rat islets which can be ascribed to beta cells; since these G-proteins are regulated by insulinotropic lipids (as described in the accompanying article), such proteins may couple the activation of phospholipases (endogenous to islets) to the exocytotic secretion of insulin. These findings also suggest that caution is necessary in extrapolating data concerning G-proteins from cultured, transformed β cell lines to the physiology of normal islets, in view of both qualitative and quantitative differences between the two preparations.