Zhu X, Birnbaumer L
Department of Anesthesiology, School of Medicine, University of California at Los Angeles 90095, USA.
Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2827-31. doi: 10.1073/pnas.93.7.2827.
Stimulatory guanine nucleotide binding protein (Gs)-coupled receptors activated by luteinizing hormone, vasopressin, and the catecholamine isoproterenol (luteinizing hormone receptor, type 2 vasopressin receptor, and types 1 and 2 beta-adrenergic receptors) and the Gi-coupled M2 muscarinic receptor (M2R) were expressed transiently in COS cells, alone and in combination with Gbeta gamma dimers, their corresponding Galphas (Galpha(s), or Galpha(i3)) and either Galpha(q) or Galpha(16). Phospholipase C (PLC) activity, assessed by inositol phosphate production from preincorporated myo[3H]inositol, was then determined to gain insight into differential coupling preferences among receptors and G proteins. The following were observed: (i) All receptors tested were able to stimulate PLC activity in response to agonist occupation. The effect of the M2R was pertussis toxin sensitive. (ii) While, as expected, expression of Galpha(q) facilitated an agonist-induced activation of PLC that varied widely from receptor to receptor (400% with type 2 vasopressin receptor and only 30% with M2R), expression of Galpha(16) facilitated about equally well the activation of PLC by any of the tested receptors and thus showed little if any discrimination for one receptor over another. (iii) Gbeta gamma elevated basal (agonist independent) PLC activity between 2- and 4-fold, confirming the proven ability of Gbeta gamma to stimulate PLCbeta. (iv) Activation of expressed receptors by their respective ligands in cells coexpressing excess Gbeta gamma elicited agonist stimulated PLC activities, which, in the case of the M2R, was not blocked by pertussis toxin (PTX), suggesting mediation by a PTX-insensitive PLC-stimulating Galpha subunit, presumably, but not necessarily, of the Gq family. (v) The effects of Gbeta gamma and the PTX-insensitive Galpha elicited by M2R were synergistic, suggesting the possibility that one or more forms of PLC are under conditional or dual regulation of G protein subunits such that stimulation by one sensitizes to the stimulation by the other.
促性腺激素、血管加压素和儿茶酚胺异丙肾上腺素激活的刺激性鸟嘌呤核苷酸结合蛋白（Gs）偶联受体（促性腺激素受体、2型血管加压素受体以及1型和2型β-肾上腺素能受体）和Gi偶联的M2毒蕈碱受体（M2R）在COS细胞中瞬时表达，单独表达以及与Gβγ二聚体、它们相应的Gα（Gαs或Gαi3）以及Gαq或Gα16共同表达。然后通过从预先掺入的肌醇[3H]肌醇产生肌醇磷酸来评估磷脂酶C（PLC）活性，以深入了解受体和G蛋白之间的差异偶联偏好。观察到以下情况：（i）所有测试的受体在激动剂占据时都能够刺激PLC活性。M2R的作用对百日咳毒素敏感。（ii）正如预期的那样，Gαq的表达促进了激动剂诱导的PLC激活，其在不同受体之间差异很大（2型血管加压素受体为400%，而M2R仅为30%），而Gα16的表达对任何测试受体的PLC激活促进作用大致相同，因此对一种受体与另一种受体几乎没有区别。（iii）Gβγ使基础（不依赖激动剂）PLC活性提高2至4倍，证实了Gβγ刺激PLCβ的已证实能力。（iv）在共表达过量Gβγ的细胞中，各自的配体激活表达的受体引发了激动剂刺激的PLC活性，就M2R而言，这种活性不受百日咳毒素（PTX）的阻断，这表明是由一种对PTX不敏感的PLC刺激Gα亚基介导的，大概但不一定是Gq家族的。（v）M2R引发的Gβγ和对PTX不敏感的Gα的作用是协同的，这表明一种或多种形式的PLC可能受到G蛋白亚基的条件性或双重调节，使得一种的刺激会使对另一种的刺激敏感。
