Fujihara H, Walker L A, Gong M C, Lemichez E, Boquet P, Somlyo A V, Somlyo A P
Departments of Molecular Physiology and Biological Physics, Pathology and Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011, USA.
Mol Biol Cell. 1997 Dec;8(12):2437-47. doi: 10.1091/mbc.8.12.2437.
Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10(-6) M, 48 h; ; ) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)gammaS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPgammaS-induced translocation of cytosolic RhoA () to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPgammaS, which prevents immunoprecipitation of non-ADP-ribosylated RhoA. Dissociation of cytosolic RhoA-rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.
用嵌合毒素DC3B（10⁻⁶ M，48小时）预处理完整的兔门静脉平滑肌，ADP-核糖基化内源性RhoA，包括与rhoGDI复合的胞质RhoA，并抑制去氧肾上腺素诱导收缩的强直期以及去氧肾上腺素、内皮素和三磷酸鸟苷（GTP）γS引起的力的Ca²⁺敏感性，但不抑制佛波酯引起的Ca²⁺敏感性。DC3B还抑制GTPγS诱导的胞质RhoA向膜部分的转位。在DC3B处理的肌肉中，即使暴露于GTPγS后，一小部分膜相关的RhoA也可被免疫沉淀，而GTPγS可阻止非ADP-核糖基化RhoA的免疫沉淀。用SDS使胞质RhoA-rhoGDI复合物解离可恢复RhoA的免疫沉淀性和ADP核糖基化能力，这表明ADP-核糖基化位点（Asn 41）和RhoA插入环（Wei等人，1997）均被rhoGDI掩盖，并且两种蛋白质的长轴在异源二聚体中平行。我们得出结论，RhoA在G蛋白介导而非蛋白激酶C介导的Ca²⁺敏感性中起重要作用，并且ADP核糖基化通过干扰其与膜相关效应器的结合在体内抑制RhoA的Ca²⁺敏感作用。
