Evidence that 2 Nonoverlapping High-Affinity Calmodulin-Binding Sites are Present in the Head of Synapsin-1

Abstract

Calmodulin is an important element in the regulation of nerve terminal exocytosis by Ca2+. Calmodulin has been shown to interact with the synaptic vesicle phosphoproteins synapsins Ia and Ib [Okabe, T. and Sobue, K. (1987) FEBS Lett. 213, 184-188; Hayes, N. V. L., Bennett, A. E and Baines, A. J. (1991) Biochem. J. 275, 93-97]. These proteins are thought to provide regulated linkages between synaptic vesicles and cytoskeletal elements. It is well established that calmodulin modulates synapsin I activities via calmodulin-dependent protein-kinase-LT-catalysed phosphorylation. The direct binding of calmodulin to synapsin I suggests a second mode of regulation in addition to phosphorylation. In this study, we present evidence indicating that two sites for calmodulin 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-naphthyl)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 stoichiometry 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 calmodulin and synapsin I were generated in vitro, corresponding to one and two calmodulin molecules bound/synapsin I. Defined fragments of synapsin I were generated with the reagent 2-nitro-5-thiocyanobenzoic acid, which cleaves at cysteine residues. Cysteine-specific cleavage of whole synapsin I after cross-linking to biotinylated calmodulin generated a pair of polypeptide complexes (approximately 46 kDa and 38 kDa), the masses of which indicated cross-linking of calmodulin to the N-terminal and middle regions of synapsin I. Purified N-terminal and middle fragments each showed a Ca2+-dependent interaction with calmodulin affinity columns. Two calmodulin-binding fragments (7.4 kDa and 6.5 kDa) were generated using Staphylococcus aureus V8 protease digestion of synapsin I. These fragments were isolated by calmodulin affinity chromatography and reverse-phase HPLC. N-terminal sequence analysis indicated that the fragments originated from two non-overlapping areas of the synapsin I head region, and that each was contained within one of the 2-nitro-5-thiocyanobenzoic-acid-derived calmodulin-binding fragments. The 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 synapsin I to F-actin and bundling of F-actin by synapsin I. Direct binding of calmodulin to synapsin I may represent a non-covalent. mode of regulation of synapsin I activity in addition to covalent regulation by phosphorylation