N-terminal-specific anti-B-50 (GAP-43) antibodies inhibit Ca(2+)-induced noradrenaline release, B-50 phosphorylation and dephosphorylation, and calmodulin binding.
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 Ser41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca2+. 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.
Hens
J J
JJ
Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, Utrecht University, The Netherlands.
De Wit
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Boomsma
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Mercken
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Oestreicher
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D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D019922:GAP-43 Protein; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism*; D019319:Okadaic Acid; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D013329:Structure-Activity Relationship; D013574:Synaptosomes; D013696:Temperature
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D004988:Ethers, Cyclic; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D019319:Okadaic Acid; D004533:Egtazic Acid; D002118:Calcium; D009638:Norepinephrine