Dessauer C W, Tesmer J J, Sprang S R, Gilman A G
Department of Pharmacology, Dallas, Texas 75235, USA.
J Biol Chem. 1998 Oct 2;273(40):25831-9. doi: 10.1074/jbc.273.40.25831.
The stimulatory G protein alpha subunit Gsalpha binds within a cleft in adenylyl cyclase formed by the alpha1-alpha2 and alpha3-beta4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory alpha subunit Gialpha could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5'-3-O-(thio)triphosphate-Gialpha1 forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by Gialpha1. These mutations suggest binding of Gialpha within the cleft formed by the alpha2 and alpha3 helices of C1, analogous to the Gsalpha binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by Gialpha. The C1b domain of the type V enzyme contributed to affinity for Gialpha, but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein alpha subunits.
刺激性G蛋白α亚基Gsα结合在由C2结构域的α1-α2和α3-β4环形成的腺苷酸环化酶裂隙内。腺苷酸环化酶C1和C2结构域的假对称性表明同源抑制性α亚基Giα可能结合到C1内的类似裂隙中。我们证明肉豆蔻酰化的鸟苷5'-3-O-（硫代）三磷酸-Giα1与V型腺苷酸环化酶的C1（而非C2）结构域形成稳定复合物。对膜结合酶进行诱变确定了其改变会增加或大幅降低Giα1抑制的IC50的残基。这些突变表明Giα在由C1的α2和α3螺旋形成的裂隙内结合，类似于C2中的Gsα结合位点。由V型腺苷酸环化酶的C1和C2结构域混合物重构的腺苷酸环化酶活性也受到Giα的抑制。V型酶的C1b结构域有助于对Giα的亲和力，但C2的来源影响不大。在这个可溶性系统中的突变忠实地反映了在膜结合酶中观察到的表型。腺苷酸环化酶的假对称结构允许同源G蛋白α亚基对活性进行双向调节。
