Hsu Y N, Edwards S C, Wecker L
Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, Tampa 33612-4799, U.S.A.
J Neurochem. 1997 Dec;69(6):2427-31. doi: 10.1046/j.1471-4159.1997.69062427.x.
Studies determined whether alpha4beta2 or alpha3beta2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for alpha4beta2 and 500 nM for alpha3beta2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing alpha4beta2 receptors were incubated with [gamma-32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the alpha4 subunit was present. Phosphorylation of alpha4 subunits of alpha4beta2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing alpha3beta2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the alpha3 subunit. Results suggest that the PKA-mediated phosphorylation of alpha4 and not alpha3 subunits may explain the differential inactivation by nicotine of these receptor subtypes expressed in oocytes.
研究确定了非洲爪蟾卵母细胞中表达的α4β2或α3β2神经元烟碱样受体是否为环磷酸腺苷依赖性蛋白激酶（PKA）的作用底物，以及尼古丁是否会影响受体磷酸化。将亚基的cRNA共注射到卵母细胞中，并在不存在或存在尼古丁（α4β2受体为50 nM，α3β2受体为500 nM）的情况下将细胞孵育24小时。尼古丁不干扰受体的分离。当将从表达α4β2受体的卵母细胞中分离出的受体与[γ-32P]ATP和PKA的催化亚基一起孵育，通过电泳分离并用放射自显影法可视化时，出现了一种标记的磷蛋白，其预测的α4亚基分子大小与之一致。α4β2受体的α4亚基在与尼古丁孵育的前5分钟内磷酸化增加，并持续24小时。相比之下，从表达α3β2受体的卵母细胞中分离出的受体未显示出与α3亚基大小相对应的标记磷蛋白。结果表明，PKA介导α4而非α3亚基的磷酸化可能解释了尼古丁对卵母细胞中表达的这些受体亚型的不同失活作用。
