School of Pharmacy, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Endocrinology. 2010 Mar;151(3):876-85. doi: 10.1210/en.2009-0722. Epub 2010 Jan 12.
The dimeric Gh protein is comprised of alpha (tissue transglutaminase) and beta (Calreticulin) subunits and known to be associated with FSH-, oxytocin-, or epinephrine-receptors/functions in their respective target cells. After establishing the FSH-induced activation of G alpha h/phospholipase C (PLC)-delta 1 pathway in rat Sertoli cells (SCs), we have attempted to identify a possible G alpha h-coupled novel FSH receptor (FSH-R). Remarkably, a protein with approximately 240-kDa molecular mass was coimmunoprecipitated with G alpha h in the fractionated membrane proteins of rat SCs. The protein was identified as myosin heavy polypeptide 9 (MyH9) by mass spectrometric analysis and immunoblotting. In addition, immunoprecipitation analysis reveals that MyH9 is constitutively associated with classical Gs-coupled FSH-R and inactive GDP-bound G alpha h at resting state of rat SCs, but did not interact with FSH directly as judged by Far-Western analysis. Upon the stimulation of higher levels of extracellular FSH (>1000 IU/liter), classical FSH-R induces the phosphorylation of MyH9, the dissociation of active GTP-bound G alpha h from FSH-R:MyH9 complexes, and the elicitation of G alpha h/PLC-delta 1 pathway-dependent Ca(2+)-influx in rat SCs. Furthermore, the specific inhibition of MyH9 ATPase activity with Blebbistatin dose-dependently suppressed FSH-induced G alpha h/PLC-delta 1 signaling and Ca(2+)-influx, but not intracellular cAMP accumulation in rat SCs, implying that MyH9 mediates FSH-induced activation of G alpha h/PLC-delta 1/IP(3)/Ca(2+)-influx pathway in rat SCs. This is the first to demonstrate that the filament protein MyH9 constitutively forms a ternary complex with FSH-R and inactive GDP-bound G alpha h. At higher FSH levels, this ternary complex executes an alternative signaling of classical Gs-coupled FSH-R through activating a Gs/cAMP-independent, G alpha h/PLC-delta 1 pathway in rat SCs.
二聚体 Gh 蛋白由 alpha（组织转谷氨酰胺酶）和 beta（钙网蛋白）亚基组成，已知与各自靶细胞中的 FSH、催产素或肾上腺素受体/功能相关。在确定 FSH 诱导大鼠支持细胞 (SCs) 中的 G alpha h/磷脂酶 C (PLC)-delta 1 途径激活后，我们试图鉴定一种可能的 G alpha h 偶联的新型 FSH 受体 (FSH-R)。值得注意的是，在大鼠 SC 分离的膜蛋白级分中，用 G alpha h 共沉淀的一种约 240 kDa 分子量的蛋白质。通过质谱分析和免疫印迹鉴定该蛋白为肌球蛋白重链 9 (MyH9)。此外，免疫沉淀分析表明，MyH9 在大鼠 SC 的静息状态下与经典 Gs 偶联的 FSH-R 和无活性的 GDP 结合的 G alpha h 组成性相关，但如 Far-Western 分析所示，它不直接与 FSH 相互作用。在外源 FSH 水平升高（>1000 IU/l）刺激下，经典 FSH-R 诱导 MyH9 磷酸化，活性 GTP 结合的 G alpha h 从 FSH-R:MyH9 复合物解离，并在大鼠 SC 中引发 G alpha h/PLC-delta 1 途径依赖性 Ca(2+)内流。此外，用 Blebbistatin 以剂量依赖性方式特异性抑制 MyH9 ATP 酶活性，可抑制 FSH 诱导的 G alpha h/PLC-delta 1 信号转导和 Ca(2+)内流，但不抑制大鼠 SC 中的细胞内 cAMP 积累，表明 MyH9 介导 FSH 诱导的 G alpha h/PLC-delta 1/IP(3)/Ca(2+)内流途径在大鼠 SC 中的激活。这是首次证明丝状蛋白 MyH9 与 FSH-R 和无活性的 GDP 结合的 G alpha h 组成性形成三元复合物。在更高的 FSH 水平下，该三元复合物通过激活大鼠 SC 中的 Gs/cAMP 非依赖性 G alpha h/PLC-delta 1 途径，执行经典 Gs 偶联的 FSH-R 的替代信号转导。
