Dohlman H G, Song J, Ma D, Courchesne W E, Thorner J
Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA.
Mol Cell Biol. 1996 Sep;16(9):5194-209. doi: 10.1128/MCB.16.9.5194.
Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.
Sst2是新发现的RGS（G蛋白信号调节因子）家族的原型。缺乏信息素诱导型SST2基因产物的细胞在接触信息素后无法恢复生长。相反，在长期晕圈生物测定中，Sst2的过量产生显著提高了从信息素诱导的停滞中恢复的速率，并且在信息素反应的短期测定（Ste4，Gβ亚基的磷酸化）中可检测到信号传导减弱。当GPA1基因产物（Gα亚基）缺失时，信息素反应途径持续活跃，因此生长停止。尽管Sst2持续诱导（用特异性抗Sst2抗体观察到），gpa1δ突变体仍处于生长停滞状态，这表明Sst2的作用需要Gpa1的存在。Sst2（698个氨基酸残基）的N端结构域（第3至307位氨基酸残基）与牛GTP酶激活蛋白和人神经纤维瘤病肿瘤抑制蛋白的催化区域具有序列相似性；Sst2的C端结构域中的片段（第417至685位氨基酸残基之间）与其他RGS蛋白同源。N端和C端结构域对于Sst2在体内的功能都是必需的。通过蔗糖密度梯度分级分离判断，与Sst2在结合并影响Gpa1活性方面的作用一致，大多数Sst2与膜相关，并与Gpa1在质膜上共定位。此外，从细胞提取物中，可以分离出与Gpa1形成复合物的Sst2（以谷胱甘肽S-转移酶融合蛋白形式表达）；这种结合在从细胞膜中提取这些蛋白质所需的去污剂和盐条件下仍然存在。同样，表达GTP酶缺陷型GPA1突变体的SST2+细胞对信息素的敏感性增加，而sst2细胞则没有。这些结果表明Sst2和Gpa1发生物理相互作用，并提示Sst2是Gpa1的直接负调节因子。
