EVIDENCE THAT 2 NONOVERLAPPING HIGH-AFFINITY CALMODULIN-BINDING SITESARE PRESENT IN THE HEAD REGION OF SYNAPSIN-I

Calmodulin is an important element in the regulation of nerve terminal exocytosis by Ca2+. Calmodulin has been shown to interact with the sy naptic vesicle phosphoproteins synapsins Ia and Ib [Okabe, T. and Sobu e, K. (1987) FEBS Lett. 213, 184-188; Hayes, N. V. L., Bennett, A. E a nd Baines, A. J. (1991) Biochem. J. 275, 93-97]. These proteins are th ought to provide regulated linkages between synaptic vesicles and cyto skeletal elements. It is well established that calmodulin modulates sy napsin I activities via calmodulin-dependent protein-kinase-LT-catalys ed phosphorylation. The direct binding of calmodulin to synapsin I sug gests a second mode of regulation in addition to phosphorylation. In t his study, we present evidence indicating that two sites for calmoduli n binding exist in the N-terminal head region of synapsins Ia and Ib. In unphosphorylated synapsin I, these sites had a K-d value of = 36+/- 14 nM for binding to calmodulin labelled with acetyl-N'-(5-sulpho-1-na phthyl)ethylene diamine. The K-d values for synapsin I phosphorylated at various sites were as follows: site I 18+/-11 nM; sites II and III 35+/-14 nM; sites I-III 16+/-9 nM. The fluorescence data indicated a s toichiometry of not less than 2 mol calmodulin bound to 1 mol synapsin I at saturation in each case. Consistent with this stoichiometry, two chemically cross-linked species (96 kDa and 116 kDa) containing calmo dulin and synapsin I were generated in vitro, corresponding to one and two calmodulin molecules bound/synapsin I. Defined fragments of synap sin I were generated with the reagent 2-nitro-5-thiocyanobenzoic acid, which cleaves at cysteine residues. Cysteine-specific cleavage of who le synapsin I after cross-linking to biotinylated calmodulin generated a pair of polypeptide complexes (approximately 46 kDa and 38 kDa), th e masses of which indicated cross-linking of calmodulin to the N-termi nal and middle regions of synapsin I. Purified N-terminal and middle f ragments each showed a Ca2+-dependent interaction with calmodulin affi nity columns. Two calmodulin-binding fragments (7.4 kDa and 6.5 kDa) w ere generated using Staphylococcus aureus V8 protease digestion of syn apsin I. These fragments were isolated by calmodulin affinity chromato graphy and reverse-phase HPLC. N-terminal sequence analysis indicated that the fragments originated from two non-overlapping areas of the sy napsin I head region, and that each was contained within one of the 2- nitro-5-thiocyanobenzoic-acid-derived calmodulin-binding fragments. Th e origins of these fragments are close to the putative sites of actin and tubulin interaction. Addition of calmodulin in the presence of Ca2 + to mixtures of F-actin and synapsin I reduced both the binding of sy napsin I to F-actin and bundling of F-actin by synapsin I. Direct bind ing of calmodulin to synapsin I may represent a non-covalent. mode of regulation of synapsin I activity in addition to covalent regulation b y phosphorylation.