Pobiner B F, Northup J K, Bauer P H, Fraser E D, Garrison J C
Department of Pharmacology, University of Virginia, Charlottesville 22908.
Mol Pharmacol. 1991 Aug;40(2):156-67.
Angiotensin II can inhibit hormone-stimulated adenylyl cyclase in intact hepatocytes or in hepatic membrane preparations. Because the response can be blocked by pertussis toxin, the object of the present study was to determine which of the known variants of Gi can couple angiotensin II receptors to inhibition of adenylyl cyclase. The potential candidates were identified by probing RNA isolated from rat hepatocytes with cDNAs specific for the alpha subunits of known toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Hepatocytes contained no detectable RNA for the Go or Gi1 alpha subunits and similar levels of RNA coding for the Gi2 and Gi3 alpha subunits. To determine whether Gi3 could couple angiotensin receptors to inhibition of cyclase, membranes were prepared from hepatocytes whose G proteins were fully ADP-ribosylated with pertussis toxin, and the Gi3 holoprotein purified from rabbit liver was reconstituted into the membranes. The nature of the Gi3 reconstituted into the membrane was assessed by immunoblotting with antibodies specific for the Gi alpha subunits. Reconstitution of 6-10 pmol of Gi3/mg of membrane protein into the toxin-treated membranes restored the ability of 10 nM angiotensin II to inhibit adenylyl cyclase. Because pertussis toxin has nonspecific effects, an assay was developed to measure the interaction of the angiotensin receptor with reconstituted G proteins in normal membranes. In the presence of Mg2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a reduction of the affinity of the angiotensin II receptor for 125I-angiotensin II that was stable to washing and the detergents used to reconstitute G proteins into the membranes. Using this protocol to activate G proteins and "uncouple" receptors, the ability of the GDP-liganded form of Gi to restore high affinity binding was examined. Reconstitution of about 10-15 pmol of oligomeric Gi3/mg of membrane protein restored both the high affinity state of the angiotensin II receptor and the ability of GTP gamma S to shift the affinity to a lower state. The same shift in receptor affinity could be accomplished by reconstituting the Gi3 alpha subunit, resolved free of beta gamma subunits, into the membranes. Reconstitution of up to 50 pmol of Gs/mg of membrane protein had no effect on angiotensin II receptor affinity. The results suggest that a major form of Gi in hepatocytes is Gi3 and that it can couple angiotensin receptors to inhibition of adenylyl cyclase.
血管紧张素II可抑制完整肝细胞或肝细胞膜制剂中激素刺激的腺苷酸环化酶。由于该反应可被百日咳毒素阻断，本研究的目的是确定已知的Gi变体中哪一种能将血管紧张素II受体与腺苷酸环化酶的抑制作用偶联起来。通过用已知毒素敏感鸟嘌呤核苷酸结合调节蛋白（G蛋白）α亚基的cDNA探测从大鼠肝细胞分离的RNA来鉴定潜在的候选者。肝细胞中未检测到Go或Gi1α亚基的RNA，编码Gi2和Gi3α亚基的RNA水平相似。为了确定Gi3是否能将血管紧张素受体与环化酶的抑制作用偶联起来，从G蛋白被百日咳毒素完全ADP-核糖基化的肝细胞制备膜，并将从兔肝纯化的Gi3全蛋白重构到膜中。通过用针对Giα亚基的特异性抗体进行免疫印迹来评估重构到膜中的Gi3的性质。将6 - 10 pmol的Gi3/mg膜蛋白重构到经毒素处理的膜中，可恢复10 nM血管紧张素II抑制腺苷酸环化酶的能力。由于百日咳毒素有非特异性作用，开发了一种测定法来测量正常膜中血管紧张素受体与重构G蛋白的相互作用。在Mg2+存在下，鸟苷5'-O-(3-硫代三磷酸)(GTPγS)导致血管紧张素II受体对125I-血管紧张素II的亲和力降低，这种降低对洗涤以及用于将G蛋白重构到膜中的去污剂稳定。使用该方案激活G蛋白并“解偶联”受体，检查了GDP结合形式的Gi恢复高亲和力结合的能力。将约10 - 15 pmol的寡聚Gi3/mg膜蛋白重构可恢复血管紧张素II受体的高亲和力状态以及GTPγS将亲和力转变为较低状态的能力。通过将不含βγ亚基的Gi3α亚基重构到膜中，可实现受体亲和力的相同转变。将高达50 pmol的Gs/mg膜蛋白重构对血管紧张素II受体亲和力没有影响。结果表明，肝细胞中Gi的主要形式是Gi3，并且它可以将血管紧张素受体与腺苷酸环化酶的抑制作用偶联起来。
