FUNCTIONAL DIVERSITY OF C2 DOMAINS OF SYNAPTOTAGMIN FAMILY - MUTATIONAL ANALYSIS OF INOSITOL HIGH POLYPHOSPHATE BINDING DOMAIN

Synaptotagmins I and II are inositol high polyphosphate series (inosit ol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphospha te, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca2+-regulated exocytosis of neurosec retory vesicles. In this study, we analyzed the inositol high polyphos phate series binding site in the C2B domain by site-directed mutagenes is and compared the IP4 binding properties of the C2B domains of multi ple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine resid ues, at positions 327, 328, and 332 in the middle of the C2B domain, w hich is not conserved in the C2A domain, were found to be essential fo r IF, binding in synaptotagmin II, When these lysine residues were alt ered to glutamine, the IP4 binding ability was completely abolished. T he primary structures of the IP4 binding sites are highly conserved am ong synaptotagmins I through IV. However, synaptotagmin III did not sh ow significant binding ability, which may be due to steric hindrance b y 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.