Distinct Functional Properties of Rab3A and Rab3B in PC12 Neuroendocrine Cells

doi:10.1074/jbc.271.12.6963

Distinct Functional Properties of Rab3A and Rab3B in PC12 Neuroendocrine Cells (*)

From the Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294

^§ An Established Investigator of the American Heart Association during the course of this study. To whom correspondence should be addressed:
Dept. of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., Birmingham, AL 35294
. Tel.: 205-934-3122; Fax: 205-934-1445.

Abstract

Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [³H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [³H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca-evoked secretion when expressed in PC12 cells.

Footnotes

↵* This work was supported by National Institutes of Health Grant DK50830 and American Cancer Society Grant CB-141. 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:

LDCV

large dense core vesicle

MEM

minimal essential medium

GST

glutathione S-transferase

NE

norepinephrine

PBS

phosphate-buffered saline

GTPS

guanosine 5′-3-O-(thio)triphosphate.
↵² E. Weber, T. Jilling, and K. L. Kirk, unpublished observations.
- Received December 6, 1995.
- Revision received January 11, 1996.