Premium
N‐Terminal‐Specific Anti‐B‐50 (GAP‐43) Antibodies Inhibit Ca 2+ ‐Induced Noradrenaline Release, B‐50 Phosphorylation and Dephosphorylation, and Calmodulin Binding
Author(s) -
Hens Jacques J. H.,
De Wit Marina,
Boomsma Frans,
Mercken Marc,
Oestreicher A. Beate,
Gispen Willem Hendrik,
De Graan Pierre N. E.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64031127.x
Subject(s) - dephosphorylation , calmodulin , phosphorylation , protein kinase c , biochemistry , synaptosome , phosphatase , biology , neurotransmitter , kinase , microbiology and biotechnology , endogeny , chemistry , receptor , in vitro , enzyme
B‐50 (GAP‐43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin‐binding domain of B‐50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B‐50 into streptolysin O‐permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N‐terminal residues 39–43 of rat B‐50 dose‐dependently inhibited Ca 2+ ‐induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C‐terminal‐directed (residues 132–213) anti‐B‐50 antibodies were without effect in the same dose range. NM2 inhibited PKC‐mediated B‐50 phosphorylation at Ser 41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32 P‐B‐50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B‐50 in the absence of Ca 2+ . Similar concentrations of NM6 did not affect B‐50 phosphorylation or dephosphorylation or B‐50/calmodulin binding. We conclude that the N‐terminal residues 39–43 of the rat B‐50 protein play an important role in the process of Ca 2+ ‐induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca 2+ ‐ and phosphorylation‐dependent fashion.