van der Neut R, Pantaloni C, Nebout I, Bockaert J, Audigier Y
Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale de Pharmacologie et Endocrinologie, Montpellier, France.
J Biol Chem. 1993 Jan 5;268(1):436-41.
Proteolytic removal and genetic deletion of the amino-terminal domain of G protein alpha subunit have shown that this region is necessary for interaction with beta gamma subunits. In the alpha subunits which undergo myristoylation, myristoylation of the amino-terminal glycine modulates the affinity of alpha subunit for the beta gamma complex. To determine the role of the same glycine in nonmyristoylated alpha subunits, we substituted it for alanine in Gs alpha and characterized the properties of the mutated chain G2A Gs alpha. The mutant could still bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) as revealed by its resistance to trypsin proteolysis and was able to interact with the membrane. However, G2A Gs alpha was a poor substrate for cholera toxin-catalyzed ADP-ribosylation either in the soluble form or when membrane-associated. Addition of beta gamma subunits increased the sedimentation rate of G2A Gs alpha in sucrose gradients. Binding experiments performed on cyc- membranes reconstituted by G2A Gs alpha showed that the GTP-induced shift of isoproterenol affinity for the beta-adrenergic receptors was reduced. On the same membranes, isoproterenol, GTP gamma S and NaF were 2-fold less effective for activating adenylylcyclase when compared to cyc- membranes reconstituted by Gs alpha. This differential stimulation of adenylylcyclase was not due to an affinity change for the effector but to a decrease in the maximal activation. Thus the G2A substitution affected beta gamma-dependent properties on reconstituted membranes such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation and we propose that the decreased activation of adenylylcyclase might result from the same defect. Although not essential for association with beta gamma subunits, the amino-terminal glycine of nonmyristoylated Gs alpha might play a modulatory role in this interaction.
G蛋白α亚基氨基末端结构域的蛋白水解去除和基因缺失表明，该区域对于与βγ亚基的相互作用是必需的。在经历肉豆蔻酰化的α亚基中，氨基末端甘氨酸的肉豆蔻酰化调节了α亚基对βγ复合物的亲和力。为了确定同一甘氨酸在非肉豆蔻酰化α亚基中的作用，我们将其替换为Gsα中的丙氨酸，并对突变链G2A Gsα的特性进行了表征。突变体仍能结合鸟苷5'-(3-O-硫代)三磷酸(GTPγS)，这通过其对胰蛋白酶水解的抗性得以揭示，并且能够与膜相互作用。然而，G2A Gsα无论是以可溶形式还是与膜结合时，都是霍乱毒素催化的ADP-核糖基化的不良底物。添加βγ亚基增加了G2A Gsα在蔗糖梯度中的沉降速率。对由G2A Gsα重构的cyc-膜进行的结合实验表明，GTP诱导的异丙肾上腺素对β-肾上腺素能受体亲和力的变化减小。在同一膜上，与由Gsα重构的cyc-膜相比，异丙肾上腺素、GTPγS和NaF激活腺苷酸环化酶的效率降低了2倍。腺苷酸环化酶的这种差异刺激不是由于对效应器的亲和力变化，而是由于最大激活的降低。因此，G2A替换影响了重构膜上βγ依赖性特性，如受体偶联和霍乱毒素催化的ADP-核糖基化，我们认为腺苷酸环化酶激活的降低可能源于相同的缺陷。虽然对于与βγ亚基的结合不是必需的，但非肉豆蔻酰化Gsα的氨基末端甘氨酸可能在这种相互作用中起调节作用。
