Actions of Rab3 effector domain peptides in chief cells from guinea pig stomach

Rab3 proteins are low molecular weight guanine nucleotide-binding proteins that belong to the Ras superfamily and are believed to play a role in the final steps of exocytosis. To examine potential interactions of these proteins with signaling pathways that mediate pepsinogen secretion from gastric chief cells, we synthesized peptides corresponding to the effector domain of Rab3. In the absence of added calcium [calcium concentration ([Ca²⁺ ]) < 1 nM], a maximal concentration (15 μM) of the Rab3 effector domain peptide or Rab3AL peptide, containing alanine and leucine substitutions, stimulated the release of 62 and 66%, respectively, of total pepsinogen from streptolysin O-permeabilized chief cells. A Rab2AL peptide, corresponding to the Rab2 effector domain, and modified (scrambled and truncated) Rab3AL peptides did not alter secretion from permeabilized cells. An additive secretory response was observed when 5 μM Rab3AL peptide was combined with increasing calcium ([Ca²⁺] < 1 nM to 3 μM). In contrast, adding up to 3 mM adenosine 3',5'-cyclic monophosphate (cAMP) had no effect on Rab3AL peptide-induced secretion, and Rab3AL peptide did not alter endogenous cAMP production. The addition of a nonhydrolyzable GTP analogue [0.01 to 100 μM guanosine 5'-O-(3-thiotriphosphate)] potentiated the secretory response to Rab3AL peptide. This potentiated response indicates that other GTP-binding proteins are involved in calcium-independent secretion. Preincubation of cells with streptolysin O (10-30 min), to allow egress of cytosolic constituents, enhanced the response to Rab3AL peptide, suggesting that the target(s) for this peptide is (are) anchored to chief cell membranes. In summary, Rab3 effector domain peptides stimulate exocytosis from gastric chief cells in a specific, dose-dependent manner that is independent of changes in cellular mediators, such as calcium and cAMP. Moreover, these results suggest that, in gastric chief cells, the effector domain regions of Rab3 proteins may regulate pepsinogen secretion by interacting with membrane-associated target molecules.