Ž . Molecular Brain Research 52 1997 1–16 Research report Anti-synapsin monoclonal antibodies: epitope mapping and inhibitory effects on phosphorylation and Grb2 binding Paola Vaccaro a , Luciana Dente a , Franco Onofri a , Adriana Zucconi a , Sabrina Martinelli a , Flavia Valtorta b , Paul Greengard c , Gianni Cesareni a , Fabio Benfenati a, ) a Department of Experimental Medicine and Biochemical Sciences and Department of Biology, UniÕersity of Rome ‘Tor Vergata’, Õia di Tor Vergata 135, 00133 Roma, Italy b DIBIT, S. Raffaele Sci. Institute ‘B. Ceccarelli’ and CNR Cellular and Molecular Pharmacology Centers, Department of Medical Pharmacology, UniÕersity of Milan, Milan, Italy c Laboratory of Molecular and Cellular Neuroscience, Rockefeller UniÕersity, New York, NY, USA Accepted 17 June 1997 Abstract The synapsins are a family of major neuron-specific synaptic vesicle-associated phosphoproteins which play important roles in synaptic function. In an effort to identify molecular tools which can be used to perturb the activity of the synapsins in in vitro as well as Ž . in vivo experiments, we have localized the epitopes of a panel of monoclonal antibodies mAbs raised against synapsins I and II and Ž . have characterized their ability to interfere with the interactions of the synapsins with protein kinases, actin and Src homology-3 SH3 domains. The epitopes of the six mAbs were found to be concentrated in the N-terminal region within domains A and B for the synapsin Ž II-reactive mAbs 19.4, 19.11, 19.51 and 19.21, and in two C-terminal clusters in the proline-rich domains D for synapsin I mAbs 10.22, . Ž . 19.51, 19.11 and 19.8 and G for synapsin II mAb 19.8 . The synapsin II-specific mAbs 19.4 and 19.21, whose overlapping epitopes are adjacent to phosphorylation site 1, specifically inhibited synapsin II phosphorylation by endogenous or exogenous cAMP-dependent protein kinase. While all the anti-synapsin I mAbs were unable to affect the interactions of synapsin I both with Ca 2q rcalmodulin-depen- dent protein kinase II and with actin monomers and filaments, mAbs 19.8 and 19.51 were found to inhibit the binding of Grb2 SH3 domains to the proline-rich C-terminal region of synapsin I. q 1997 Elsevier Science B.V. Keywords: Synapsin; Synaptic vesicle; Protein kinase A; CaM kinase II; SH3 domain; Actin; Monoclonal antibody; Epitope mapping; Peptide library 1. Introduction Ž . The synapsins synapsin Ia, Ib, IIa and IIb comprise a family of major neuron-specific synaptic vesicle phospho- proteins which play important roles in the development and function of presynaptic terminals. Synapsins I and II Abbreviations: BSA, bovine serum albumin; CaM, Ca 2q rcalmodulin; CaMPK, Ca 2q rcalmodulin-dependent protein kinase; DTT, dithio- threitol; FCS, fetal calf serum; HRP, horseradish peroxidase; mAb, monoclonal antibody; NTCB, 2-nitro-3-thiocyanobenzoic acid; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PKA, cAMP-dependent protein knase; PEG, polyethylene glycol; SDS, sodium dodecyl sulfate; SH3, Src homology-3; TBS, Tris-buffered saline; TBS T, Tris-buffered saliner0.5% Tween-20; TU, ampicillin transducing units. ) Corresponding author. Department of Biomedical Sciences, Section of Physiology, University of Modena, via Campi 287, I-41100 Modena, Ž . Italy. Fax: q39 59 42-8236; E-mail: benfenat@c220.unimo.it are encoded by distinct genes, while the a and b isoforms result from differential splicing of the primary transcripts. Each synapsin isoform is composed of a mosaic of individ- ual and shared domains. All four synapsins share a large Ž . N-terminal region domains A–C which comprises more than half of the molecule and exhibit a combination of distinct C-terminal domains which are either shared by two Ž isoforms domain D for synapsins IarIb, domain G for . synapsins IIarIIb, or domain E for synapsins IarIIa or Ž are isoform-specific domains F, H and I for synapsins Ib, . w x IIa and IIb, respectively 11,37 . The synapsins are targets of multiple signal transduction pathways in the brain. All four synapsins are phosphory- Ž . lated by cAMP-dependent protein kinase PKA and 2q Ž . Ca rcalmodulin-dependent protein kinase I CaMPKI Ž . on a serine residue site 1 located within domain A. In addition, synapsin I, but not synapsin II, is phosphorylated by Ca 2q rcalmodulin-dependent protein kinase II 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.