Brass L F, Woolkalis M J, Manning D R
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-4283.
J Biol Chem. 1988 Apr 15;263(11):5348-55.
Platelet responses to agonists are believed to be mediated by at least two pertussis toxin-sensitive guanine nucleotide-binding (G) proteins: Gi which inhibits adenylyl cyclase and Gp, which stimulates phospholipase C. The present studies compare the properties of Gi and Gp and examine their interactions with the receptors for various platelet agonists. In permeabilized platelets and platelet membranes, pertussis toxin [32P]ADP-ribosylated a protein(s) (alpha 41) which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionally below rabbit and bovine alpha i (Mr = 41,000). Prior exposure of the platelets to an agonist inhibited the [32P]ADP-ribosylation of alpha 41 to an extent which correlated with the pattern of responses to that agonist. Thrombin, which elicited responses that were mediated by both Gi and Gp, decreased radiolabeling by greater than 90%. Epinephrine, which was functionally coupled only to Gi, decreased radiolabeling by 50%, as did vasopressin and platelet-activating factor (PAF), which were coupled only to Gp. U46619, a thromboxane analog which neither inhibited cAMP formation nor caused pertussis toxin-sensitive phosphoinositide hydrolysis, had no effect on 32P-ADP-ribosylation. These results suggest that either G alpha 41 regulates more than one enzyme or that alpha subunits from more than one G protein comigrate within alpha 41. Two-dimensional electrophoresis was used to test the latter possibility. Upon isoelectric focusing, alpha 41 resolved into two distinct subspecies. However, these appear to be minor variants rather than functionally distinct alpha subunits since: 1) both proteins produced the same proteolytic fragments after digestion with chymotrypsin or Staphylococcus aureus V8 protease and 2) preincubation of the platelets with agonists, including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited the [32P]ADP-ribosylation of both proteins to the same extent. The pattern of functional responses produced by some of the agonists was found to depend upon the conditions used for the assay. Although unable to inhibit cAMP formation in intact platelets, both PAF and vasopressin caused pertussis toxin-sensitive inhibition of adenylyl cyclase in isolated membranes. Collectively, these observations suggest that 1) in platelets a single pertussis toxin-sensitive, alpha 41-containing G protein may be involved in the regulation of both adenylyl cyclase and phospholipase C and 2) additional constraints which are altered during membrane isolation may help to determine which enzyme is coupled to which agonist.
血小板对激动剂的反应被认为至少由两种对百日咳毒素敏感的鸟嘌呤核苷酸结合（G）蛋白介导：抑制腺苷酸环化酶的Gi和刺激磷脂酶C的Gp。本研究比较了Gi和Gp的特性，并研究了它们与各种血小板激动剂受体的相互作用。在透化血小板和血小板膜中，百日咳毒素[32P]ADP-核糖基化了一种蛋白质（α41），该蛋白质在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的迁移位置略低于兔和牛的αi（Mr = 41,000）。血小板预先暴露于激动剂会抑制α41的[32P]ADP-核糖基化，其抑制程度与对该激动剂的反应模式相关。凝血酶引发的反应由Gi和Gp共同介导，使放射性标记减少超过90%。仅与Gi功能偶联的肾上腺素使放射性标记减少50%，仅与Gp偶联的血管加压素和血小板活化因子（PAF）也是如此。U46619是一种血栓素类似物，既不抑制cAMP形成也不引起百日咳毒素敏感的磷酸肌醇水解，对32P-ADP-核糖基化没有影响。这些结果表明，要么Gα41调节多种酶，要么来自多种G蛋白的α亚基在α41内共同迁移。二维电泳用于测试后一种可能性。在等电聚焦时，α41分解为两个不同的亚类。然而，这些似乎是微小的变体而非功能不同的α亚基，因为：1）两种蛋白质在用胰凝乳蛋白酶或金黄色葡萄球菌V8蛋白酶消化后产生相同的蛋白水解片段；2）血小板与激动剂预孵育，包括那些在完整血小板中似乎仅与Gp（PAF和血管加压素）或仅与Gi（肾上腺素）相互作用的激动剂，会同等程度地抑制两种蛋白质的[32P]ADP-核糖基化。发现一些激动剂产生的功能反应模式取决于测定所用的条件。虽然PAF和血管加压素在完整血小板中不能抑制cAMP形成，但它们在分离膜中引起百日咳毒素敏感的腺苷酸环化酶抑制。总体而言，这些观察结果表明：1）在血小板中，一种单一的对百日咳毒素敏感的含α41的G蛋白可能参与腺苷酸环化酶和磷脂酶C的调节；2）在膜分离过程中改变的其他限制因素可能有助于确定哪种酶与哪种激动剂偶联。
