ACTIONS OF RAB3 EFFECTOR DOMAIN PEPTIDES IN CHIEF CELLS FROM GUINEA-PIG STOMACH

Rab3 proteins are low molecular weight guanine nucleotide-binding prot eins that belong to the Ras superfamily and are believed to play a rol e in the final steps of exocytosis. To examine potential interactions of these proteins with signaling pathways that mediate pepsinogen secr etion from gastric chief cells, we synthesized peptides corresponding to the effector domain of Rab3. In the absence of added calcium [calci um concentration ([Ca2+]) < 1 nM], a maximal concentration (15 mu M) o f the Rab3 effector domain peptide or Rab3AL peptide, containing alani ne and leucine substitutions, stimulated the release of 62 and 66%, re spectively, of total pepsinogen from streptolysin O-permeabilized chie f cells. A Rab2AL peptide, corresponding to the Rab2 effector domain, and modified (scrambled and truncated) Rab3AL peptides did not alter s ecretion from permeabilized cells. An additive secretory response was observed when 5 mu M Rab3AL peptide was combined with increasing calci um ([Ca2+] < 1 nM to 3 mu M. In contrast, adding up to 3 mM adenosine 3',5'-cyclic monophosphate (cAMP) had no effect on Rab3AL peptide-indu ced secretion, and Rab3AL peptide did not alter endogenous cAMP produc tion. The addition of a nonhydrolyzable GTP analogue [0.01 to 100 mu M guanosine 5'-O-(3-thiotriphosphate)] potentiated the secretory respon se to Rab3AL peptide. This potentiated response indicates that other G TP-binding proteins are involved in calcium-independent secretion. Pre incubation of cells with streptolysin O (10-30 min), to allow egress o f cytosolic constituents, enhanced the response to Rab3AL peptide, sug gesting that the target(s) for this peptide is (are) anchored to chief cell membranes. In summary, Rab3 effector domain peptides stimulate e xocytosis from gastric chief cells in a specific, dose-dependent manne r that is independent of changes in cellular mediators, such as calciu m and cAMP. Moreover, these results suggest that, in gastric chief cel ls, the effector domain regions of Rab3 proteins may regulate pepsinog en secretion by interacting with membrane-associated target molecules.