Valenzuela C F, Machu T K, McKernan R M, Whiting P, VanRenterghem B B, McManaman J L, Brozowski S J, Smith G B, Olsen R W, Harris R A
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262, USA.
Brain Res Mol Brain Res. 1995 Jul;31(1-2):165-72. doi: 10.1016/0169-328x(95)00048-w.
Phosphorylation of purified bovine brain GABAA receptors by the tyrosine kinase, pp60v-src was examined. pp60v-src phosphorylated two bands of 54-62 kDa and 48-51 kDa that migrated to approximately the same position as bands recognized by antisera against the beta 2 and gamma 2 GABAA receptor subunits, respectively. Bacterially expressed proteins containing the putative large cytoplasmic loops of the beta 1 and gamma 2L subunits were phosphorylated by pp60v-src, indicating that the phosphorylation sites are located in these subunit domains. The tyrosine kinase inhibitors, genistein and the tyrphostins B-42 and B-44, inhibited muscimol-stimulated 36Cl- uptake in mouse brain membrane vesicles (microsacs). magnitude of the tyrphostin B-44-induced inhibition of muscimol-stimulated 36Cl- uptake was significantly reduced in microsacs that were lysed and resealed under conditions that inhibit phosphorylation. GABA-gated Cl- currents were also inhibited by genistein and tyrphostin B-44 in Xenopus oocytes expressing alpha 1 beta 1 and alpha 1 beta 1 gamma 2L subunits. Consequently, protein tyrosine kinase-dependent phosphorylation appears to be another mechanism of regulating the function of GABAA receptors.
研究了酪氨酸激酶pp60v-src对纯化的牛脑GABAA受体的磷酸化作用。pp60v-src使两条分别为54 - 62 kDa和48 - 51 kDa的条带发生磷酸化，它们迁移到的位置分别与抗β2和γ2 GABAA受体亚基的抗血清识别的条带大致相同。含有β1和γ2L亚基假定的大细胞质环的细菌表达蛋白被pp60v-src磷酸化，表明磷酸化位点位于这些亚基结构域中。酪氨酸激酶抑制剂金雀异黄素、酪氨酸磷酸化抑制剂B - 42和B - 44抑制了小鼠脑膜囊泡（微囊）中蝇蕈醇刺激的36Cl-摄取。在抑制磷酸化的条件下裂解并重新封闭的微囊中，酪氨酸磷酸化抑制剂B - 44对蝇蕈醇刺激的36Cl-摄取的抑制程度显著降低。在表达α1β1和α1β1γ2L亚基的非洲爪蟾卵母细胞中，金雀异黄素和酪氨酸磷酸化抑制剂B - 44也抑制了GABA门控的Cl-电流。因此，蛋白酪氨酸激酶依赖性磷酸化似乎是调节GABAA受体功能的另一种机制。
