Functional Diversity of C2 Domains of Synaptotagmin Family

doi:10.1074/jbc.270.44.26523

Functional Diversity of C2 Domains of Synaptotagmin Family

MUTATIONAL ANALYSIS OF INOSITOL HIGH POLYPHOSPHATE BINDING DOMAIN (*)

From the (1) Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan, the
(2) Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan, and the
(3) Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan

^§ To whom correspondence should be addressed. Tel.: 81-298-36-9170; Fax: 81-298-36-9040; fukuda{at}rtc.riken.go.jp.

Abstract

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP binding ability was completely abolished. The primary structures of the IP binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.

Footnotes

↵* This work was supported by grants from the Japanese Ministry of Education, Science, and Culture (to J. A. and K. M.), the Japan Society for the Promotion of Science (to M. F.), the Intractable Diseases Research Foundation (to J. A.), and the Human Frontier Science Program (to K. M.). 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:

IP

inositol 1,3,4,5-tetrakisphosphate

PCR

polymerase chain reaction

GST

glutathione S-transferase

STI-IV

synaptotagmin I-IV.
↵² M. Fukuda, unpublished data.
- Received August 30, 1995.