Molecular and Functional Studies of Inhibitory G Protein in RINm5F Cells

Abstract

Inhibitory G proteins (G1) play an important role in cell proliferation. In order to characterize G1 proteins in RINm5F (RIN) cells, we first established RIN cells in cell culture. Immunoblot analysis was performed on extracted G proteins using Western blot techniques and a G1-specific antibody. We identified three prominent bands consistent with three distinct inhibitory α subunits of membrane-bound G protein (G1) in RIN cells. In contrast, we identified only one prominent distinct inhibitory α subunit of G protein in an equal quantity of membrane-protein in our control (normal rat pancreas). In several cell types, G1 is known to mediate the inhibitory action of somatostatin on intracellular cyclic AMP (cAMP) accumulation. Therefore, we studied the action of the long-acting analogue of somatostatin, octreotide (SMS), on basal and 3-isobutyl-1-methylxanthine-stimulated cAMP accumulation in RIN cells. SMS did not inhibit cAMP accumulation or tritiated thymidine incorporation into DNA (TTID) in RIN cells. However, when treatment with SMS is supplemented with the nonhydrolyzable analogue of guanine nucleotide, Gpp(NH)p (Gpp), which is known to dissociate G proteins into its constitutive subunits, then SMS + Gpp induced an inhibitory action and significantly reduced cAMP accumulation and TTID. These data are consistent with the concept of qualitatively and functionally altered inhibitory G protein expression in the insulinproducing, islet cell (RINm5F) rat insulinoma tumor cell line. Further study of human tumors will lead to new insights into the clinical implications of G proteinmediated signal transduction in insulinoma.