Simpson Rachel E, Ciruela Antonio, Cooper Dermot M F
The Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
The Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
J Biol Chem. 2006 Jun 23;281(25):17379-17389. doi: 10.1074/jbc.M510992200. Epub 2006 Apr 13.
Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.
钙调蛋白（CaM）介导钙离子（Ca2+）对8型腺苷酸环化酶（AC8）的刺激作用。早期研究在AC8中鉴定出两个CaM结合位点；一个位于N端，对AC8活性显然并非必需，另一个位于C端，对Ca2+刺激至关重要（顾，C.，以及库珀，D.M.F.（1999年）《生物化学杂志》274，8012 - 8021）。本研究利用突变型CaM和修饰的AC8物种进行结合和功能研究，探讨这两个CaM结合结构域的作用及其在调节AC8活性中的相互作用。我们报告称，AC8的N端CaM结合结构域在招募CaM方面发挥作用，且这种招募对于体内Ca2+刺激AC8至关重要。使用CaM的Ca2+不敏感突变体，我们发现部分结合的CaM可与AC8结合，但只有完全结合的Ca2+/CaM才能刺激AC8活性。此外，部分结合的CaM在体内抑制AC8活性。结果表明，CaM与AC8的N端预先结合，我们认为AC8的C端利用这种招募的CaM来介导Ca2+刺激。
