Smith Karen E, Gu Chen, Fagan Kent A, Hu Biao, Cooper Dermot M F
Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Biol Chem. 2002 Feb 22;277(8):6025-31. doi: 10.1074/jbc.M109615200. Epub 2001 Dec 13.
Ca(2+)-sensitive adenylyl cyclases (ACs) depend on capacitative Ca(2+) entry (CCE) for their regulation. Residence of the endogenous Ca(2+)-inhibitable adenylyl cyclase of C6-2B glioma cells in cholesterol-enriched caveolae is essential for its regulation by CCE (Fagan, K. A., Smith, K. E., and Cooper, D. M. F. (2000) J. Biol. Chem. 275, 26530-26537). In the present study, we established that depletion of cellular cholesterol ablated the regulation by CCE of a Ca(2+)-stimulable adenylyl cyclase, AC8, heterologously expressed in HEK293 cells. We considered the possibility that a calmodulin-binding domain in the N terminus of AC8, which is not required for in vitro regulation by Ca(2+), might play a targeting role. Deletion and mutation of the N terminus did attenuate the enzyme's sensitivity to CCE without altering its in vitro responsiveness to Ca(2+)/calmodulin. Both N terminus-deleted AC8 and wild type AC8 were expressed at the plasma membrane, as shown by imaging analysis of green fluorescence protein-tagged constructs. However, not only wild type AC8 but also the CCE-insensitive mutants occurred in caveolar fractions of the plasma membranes, even though a Ca(2+)-insensitive adenylyl cyclase, AC7, was excluded from caveolae. Finally, the AC8 mutants were no more responsive to nonphysiological elevation of Ca(2+) than the wild type. We conclude that (i) not all adenylyl cyclases reside in caveolae, (ii) the calmodulin-binding domain in the N terminus of AC8 does not play a role in caveolar targeting, (iii) the N terminus does play a role in associating AC8 with factors that confer sensitivity to CCE, and (iv) residence of Ca(2+)-sensitive adenylyl cyclases in caveolae is essential but not sufficient for regulation by CCE.
钙敏感型腺苷酸环化酶（ACs）的调节依赖于容量性钙内流（CCE）。C6-2B胶质瘤细胞内源性钙抑制型腺苷酸环化酶定位于富含胆固醇的小窝，这对其受CCE调节至关重要（费根，K.A.，史密斯，K.E.，和库珀，D.M.F.（2000年）《生物化学杂志》275，26530 - 26537）。在本研究中，我们证实，细胞胆固醇耗竭消除了在HEK293细胞中异源表达的钙刺激型腺苷酸环化酶AC8受CCE的调节。我们考虑了一种可能性，即AC8 N端的钙调蛋白结合结构域虽在体外对钙调节并非必需，但可能起靶向作用。N端的缺失和突变确实减弱了该酶对CCE的敏感性，而未改变其体外对钙/钙调蛋白的反应性。如对绿色荧光蛋白标记构建体的成像分析所示，N端缺失的AC8和野生型AC8均在质膜表达。然而，不仅野生型AC8，而且CCE不敏感的突变体也出现在质膜的小窝部分，尽管钙不敏感型腺苷酸环化酶AC7被排除在小窝之外。最后，AC8突变体对非生理性钙升高的反应并不比野生型更强。我们得出结论：（i）并非所有腺苷酸环化酶都定位于小窝；（ii）AC8 N端的钙调蛋白结合结构域在小窝靶向中不起作用；（iii）N端确实在使AC8与赋予CCE敏感性的因子结合中起作用；（iv）钙敏感型腺苷酸环化酶定位于小窝对CCE调节是必要的，但不是充分的。
