Evidence That the Rab3a-binding Protein, Rabphilin3a, Enhances Regulated Secretion.

Rabphilin3a had been identified in brain as a Rab3a-binding protein and may serve as an effector for Rab3a function. We have cloned a splice variant of brain-Rabphilin3a from a bovine adrenal chromaffin cell cDNA library and investigated the function of the protein in regulated exocytosis in bovine chromaffin cells. The predicted amino acid sequence of chromaffin cell (c-) Rabphilin3a was identical with that of brain (b-) Rabphilin3a except for a 6-amino-acid insert VFSLSA in the amino-terminal half of the protein. An antibody directed against a carboxyl-terminal peptide recognized an 85-kDa protein in COS7 cells transfected with the cDNA in a mammalian expression vector. A band of similar mobility was enriched in a fraction of highly purified chromaffin granule membranes, consistent with the Rabphilin3a being associated with chromaffin granule membranes. Overexpression of either chromaffin cell or brain Rabphilin3a by transfection with the corresponding cDNAs in mammalian expression vectors enhanced DMPP-induced secretion of co-expressed human growth hormone (GH) approximately 30%. Chromaffin cells transfected with a plasmid with the entire coding sequence of c-Rabphilin3a inserted in the antisense orientation inhibited secretion of co-expressed GH by approximately 30%. Rabphilin3a mutants lacking one or both of the carboxyl-terminal C2 domains strongly inhibited DMPP-stimulated exocytosis. The single C2 domain deletion also strongly inhibited Ca(2+)-dependent secretion from digitonin-permeabilized cells. These data indicate that Rabphilin3a is a positive regulator of exocytosis. Because the C2 deletion mutants contain the amino-terminal Rab3a-GTP binding domain, they may inhibit secretion by competing with endogenous Rabphilin3a for interaction with Rab3a-GTP without being able to mimic the functional effects of full-length Rabphilin3a.