PAROTID RAB3/RAB-GDI ISOFORMS--EXPRESSION/LOCALIZATION

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The long-term objective of the studies described in the present proposal is to elucidate the role of Rab proteins, as well as accessory proteins which regulate Rab protein function, in regulated exocytosis in the parotid gland. The specific aims of this proposal are to: 1) examine the expression and localization of Rab3 and rab-GDP dissociation inhibitor (rab-GDI) isoforms; and 2) examine agonist- induced subcellular redistribution of these isoforms in rat parotid gland. The salivary glands perform many important functions that affect the oral and gastrointestinal environment including protein secretion. Hence, an understanding of the basic mechanisms that regulate salivary gland secretion is essential. Although Rab proteins are involved in various stages of vesicular transport and secretion, the expression and function of these important proteins in salivary glands have not been studied. Four Rab3 isoforms (A, B, C and D) and at least two rab-GDI isoforms have been identified in mammalian cells. Rab3 isoform expression in the parotid gland will be examined by RT-PCR using Rab3- specific primers. The PcR products obtained will be cloned and sequenced to identify the Rab3 isoforms expressed in this tissue. Rab3 and rab-GDI expression will also be examined by Northern and Western blotting using isoform-specific DNA probes and antibodies, respectively. Preliminary studies from this laboratory indicate that at least one Rab3 isoform, Rab3D, and one rab-GDI isoform are expressed in rat parotid gland. The association of Rab3 and rab-GDI isoforms expressed in the parotid gland with various subcellular fractions, including secretory granules, will be examined by cellular fractionation and Western blotting. Precise subcellular localization of these proteins will also be studied by immunofluorescence and immunogold electron microscopy. The effects of agents which stimulate amylase secretion on the subcellular localization of Rab3 and rab-GDI isoforms will be examined by quantitative Western blotting. The studies outlined in this proposal should indicate a role for Rab3 and rab-GDI proteins in mediating regulated secretion in the parotid gland.