Hens J J, De Wit M, Boomsma F, Mercken M, Oestreicher A B, Gispen W H, De Graan P N
Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, Utrecht University, The Netherlands.
J Neurochem. 1995 Mar;64(3):1127-36. doi: 10.1046/j.1471-4159.1995.64031127.x.
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.
B - 50（生长相关蛋白43，GAP - 43）是一种突触前蛋白激酶C（PKC）底物，与去甲肾上腺素释放的分子机制有关。为了评估B - 50的PKC磷酸化位点和钙调蛋白结合结构域在神经递质释放调节中的重要性，我们将两种抗B - 50单克隆抗体引入从大鼠大脑皮质分离的经链球菌溶血素O通透的突触体中。针对大鼠B - 50 N端39 - 43位残基的NM2抗体剂量依赖性地抑制了Ca(2 +)诱导的放射性标记和内源性去甲肾上腺素从通透突触体中的释放。C端导向（132 - 213位残基）的抗B - 50抗体NM6在相同剂量范围内无作用。NM2抑制突触体细胞膜和通透突触体中PKC介导的B - 50在Ser41位点的磷酸化，抑制内源性突触体磷酸酶对32P - B - 50的去磷酸化，并在无Ca2 +的情况下抑制钙调蛋白与突触体B - 50的结合。相似浓度的NM6不影响B - 50的磷酸化、去磷酸化或B - 50/钙调蛋白结合。我们得出结论，大鼠B - 50蛋白的N端39 - 43位残基在Ca(2 +)诱导的去甲肾上腺素释放过程中起重要作用，可能是作为一个局部钙调蛋白库，以Ca(2 +)和磷酸化依赖的方式受到调节。
