Localization of the Rab3 Small G Protein Regulators in Nerve Terminals and Their Involvement in Ca2+-dependent Exocytosis

doi:10.1074/jbc.273.51.34580

Localization of the Rab3 Small G Protein Regulators in Nerve Terminals and Their Involvement in Ca²⁺-dependent Exocytosis*

From the ^‡Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565-0871, Japan and ^¶Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan

Abstract

The Rab3 small G protein subfamily (Rab3) consists of four members, Rab3A, -B, -C, and -D. We have recently isolated and characterized the Rab3 regulators, GDP/GTP exchange protein (GEP) and GTPase activating protein (GAP), both of which are specific for the Rab3 subfamily. Rab3 GEP stimulates the conversion of the GDP-bound inactive form to the GTP-bound active form, whereas Rab3 GAP stimulates the reverse reaction. Of the four members of the Rab3 subfamily, evidence is accumulating that Rab3A is involved in Ca²⁺-dependent exocytosis, particularly in neurotransmitter release. We first analyzed the subcellular localization of Rab3 GEP and GAP in rat brain. Subcellular fractionation analysis showed that both Rab3 GEP and GAP were enriched in the synaptic soluble fraction. Immunocytochemical analysis in primary cultured rat hippocampal neurons showed that both Rab3 GEP and GAP were concentrated at the presynaptic nerve terminals. We then examined whether Rab3 GEP and GAP were involved in Ca²⁺-dependent exocytosis by use of human growth hormone (GH) co-expression assay system of cultured PC12 cells. Overexpression of the deletion mutant of Rab3 GEP possessing the catalytic activity reduced the high K⁺-induced GH release without affecting the basal GH release, whereas that of the deletion mutant lacking the catalytic activity showed no effect on the high K⁺-induced GH release. In contrast, overexpression of Rab3 GAP or its deletion mutant possessing the catalytic activity did not affect the high K⁺-induced GH release or the basal GH release. These results indicate that Rab3 GEP and GAP are colocalized with Rab3A at the synaptic release sites and suggest that they regulate the activity of Rab3A and are involved in Ca²⁺-dependent exocytosis.

Footnotes

↵* The investigation at Osaka University Medical School was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, and Culture, Japan (1997, 1998), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan (1997, 1998), and by a grant from the Human Frontier Science Program (1997).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Present address: The Third Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466-8550, Japan.
↵‖ To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, 2-2 Yamada-oka, Suita 565-0871, Japan. Tel.: +81-6-879-3410; Fax: +81-6-879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp.
↵2 Y. Takai, manuscript in preparation.
Abbreviations:

GDI

GDP dissociation inhibitor

GEP

GDP/GTP exchange protein

GAP

GTPase activating protein

GH

growth hormone

FBS

fetal bovine serum

NMDA

N-methyl-d-aspartate

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis.
- Received August 27, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.