Wittpoth C, Scholich K, Yigzaw Y, Stringfield T M, Patel T B
Department of Pharmacology, University of Tennessee, The Health Science Center, 874 Union Avenue, Memphis, TN 38163, USA.
Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9551-6. doi: 10.1073/pnas.96.17.9551.
The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with beta gamma subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G(salpha) (G(salpha)) or Ca(2+)/calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by beta gamma subunits, the presence of both of these regions formed another beta gamma interaction site that was sufficient to observe G(salpha)- or Ca(2+)/CaM-stimulated activity. Inhibition of AC activity attributable to interaction of beta gamma subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of beta gamma on various effectors. Moreover, the C1 region of ACI was sufficient to observe G(ialpha1)-elicited inhibition of Ca(2+)/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G(ialpha1), the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of G(ialpha1) are mediated on the C1 regions of both ACI and ACV. The effects of beta gamma on ACI can be mediated by interactions with the C1a region and a beta gamma interacting site formed by the C1b and C2 domains of this enzyme.
腺苷酸环化酶（AC）的两个大的胞质结构域（C1和C2），当单独表达并混合在一起时，可重构能够被各种调节剂调节的酶活性。因此，我们使用了来自I型AC（ACI）和V型AC（ACV）的C1或其C1a亚结构域以及C2区域，来确定ACI上与异源三聚体G蛋白的βγ亚基相互作用的区域。此外，我们还使用了一种嵌合C1结构域（VC1aIC1b），其中C1a区域源自ACV，C1b区域源自ACI。通过将C1或C1a或VC1aIC1b结构域与ACI或ACV的C2区域混合，我们发现ACI的C1a区域（氨基酸236 - 471）足以观察到βγ介导的酶活性抑制，该活性可被组成型激活的G（sα）（G（sα）*）或Ca（2+）/钙调蛋白（CaM）刺激。尽管ACI的C1b区域和C2结构域本身不足以被βγ亚基抑制活性，但这两个区域的存在形成了另一个βγ相互作用位点，足以观察到G（sα）*或Ca（2+）/CaM刺激的活性。由βγ亚基在两个位点中任何一个位点相互作用导致的AC活性抑制，被一种肽（QEHA）阻断，该肽先前已被证明可抑制βγ对各种效应器的作用。此外，ACI的C1区域足以观察到G（iα1）引发的对Ca（2+）/CaM刺激活性的抑制。尽管ACV的C1a区域足以被G（iα1）抑制活性，但来自ACI或ACV的C1b区域的存在增加了对抑制性G蛋白抑制的敏感性。因此，G（iα1）的抑制作用是通过与ACI和ACV的C1区域相互作用介导的。βγ对ACI的作用可通过与C1a区域以及由该酶的C1b和C2结构域形成的βγ相互作用位点相互作用来介导。
