Anti-synapsin monoclonal antibodies: epitope mapping and inhibitory effects on phosphorylation and Grb2 binding.
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 in vivo experiments, we have localized the epitopes of a panel of monoclonal antibodies (mAbs) raised against synapsins I and II and 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 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, 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 Ca2+/calmodulin-dependent 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.
Vaccaro
P
P
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Roma, Italy.
Dente
L
L
Onofri
F
F
Zucconi
A
A
Martinelli
S
S
Valtorta
F
F
Greengard
P
P
Cesareni
G
G
Benfenati
F
F
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D018604:Epitope Mapping; D051380:GRB2 Adaptor Protein; D010446:Peptide Fragments / Q000276:immunology; D019151:Peptide Library; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D016704:Synapsins / Q000276:immunology*
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D010446:Peptide Fragments; D019151:Peptide Library; D011506:Proteins; D011994:Recombinant Proteins; D016704:Synapsins; D003545:Cysteine