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

doi:10.1074/jbc.270.28.16714

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

STUDIES IN ADRENAL CHROMAFFIN CELLS (*)

From the (1) Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632 and the
(2)Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Osaka, Japan

^§ To whom correspondence should be addressed:
Dept. of Pharmacology, 1301 MSRB III, University of Michigan Medical School, Ann Arbor, MI 48109-0632
.

Abstract

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-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.

Footnotes

↵* This work was supported by National Institutes of Health Grant RO1 DK27959 (to R. W. H.) and a postdoctoral fellowship from the American Heart Association of Michigan (to S.-H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SNARE

receptor for soluble NSF attachment protein

SNAP25

synaptosome-associated protein of 25 kDa

b-Rabphilin3a

brain Rabphilin3a

c-Rabphilin3a

chromaffin cell Rabphilin3a

CMV

human cytomegalovirus

GH

growth hormone

GAP

GTPase activating protein

Rp

Rabphilin3a

kb

kilobase(s)

bp

base pair(s)

PCR

polymerase chain reaction

DMPP

1,1-dimethyl-4-phenylpiperazinium.
↵² D. L. Scheuner and R. W. Holz, unpublished observations.