Evidence that Two Non‐Overlapping High‐Affinity Calmodulin‐Binding Sites are Present in the Head Region of Synapsin I

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. & Sobue, K. (1987) FEBS Lett. 213, 184–188; Hayes, N. V. L., Bennett, A. F. & 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‐II‐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 Kd value of = 36±14 nM for binding to calmodulin labelled with acetyl‐N′‐(5‐sulpho‐1‐naphthyl)ethylene diamine. The Kd values for synapsin I phosphorylated at various sites were as follows: site I 18% 11 nM; sites II and III 35±14nM; sites I–III 16±9nM. 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. Copyright © 1994, Wiley Blackwell. All rights reserved