Modification of the calcium and calmodulin sensitivity of the type I adenylyl cyclase by mutagenesis of its calmodulin binding domain.
Modification of the calcium and calmodulin sensitivity of the type I adenylyl cyclase by mutagenesis of its calmodulin binding domain.
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发表时间：
1993-11
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通讯作者：
Z. Wu;S. Wong;D. Storms
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作者：
Z. Wu;S. Wong;D. Storms
I型腺酰环化酶在体外受钙离子和钙调蛋白的直接刺激，在体内也受细胞内钙离子升高的刺激。体内Ca~(2+)对酶的刺激作用可能是由于酶与Ca~(2+)和钙调蛋白的直接相互作用，也可能是由于Ca~(2+)激活的蛋白激酶对酶的间接刺激机制。在这项研究中，我们在钙调蛋白结合域中进行了几个点突变，以确定该酶对钙的敏感性是否可以通过突变来改变。突变体酶的催化活性与野生型I型腺酰环化酶相当。用Ser-507取代Cys-507对该酶的钙调素或钙敏感性没有显著影响。然而，用天冬氨酸取代Lys-504可使细胞对钙离子的敏感性降低4倍。Arg-503取代Phe-503后，钙离子和钙调蛋白的刺激作用被取消。随着Arg-503突变体的出现，细胞内钙离子对I型腺苷环化酶活性的刺激作用也大大减弱，表明细胞内钙离子对该酶的刺激主要是由于与钙调素和钙离子的直接相互作用。这些数据表明，该酶的钙敏感性可以通过钙调素结合结构域的点突变来调节，并表明该酶在体内可以直接受到钙离子和钙调蛋白的调节。
The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vitro, and the enzyme is also stimulated by increases in intracellular Ca2+ in vivo. Ca2+ stimulation of the enzyme in vivo may be due to direct interactions of the enzyme with Ca2+ and calmodulin or to an indirect mechanism involving stimulation of the enzyme by Ca(2+)-activated protein kinases. In this study, we have made several point mutations within the calmodulin binding domain to determine if the Ca2+ sensitivity of the enzyme can be modified by mutagenesis. The catalytic activities of the mutant enzymes were comparable to wild type type I adenylyl cyclase. Substitution of Cys-507 with Ser-507 did not have significant effects on the calmodulin or Ca2+ sensitivity of the enzyme. However, replacement of Lys-504 with Asp caused a 4-fold decrease in sensitivity to Ca2+. Ca2+ and calmodulin stimulation were abolished by substitution of Phe-503 with Arg-503. Stimulation of type I adenylyl cyclase activity in vivo by intracellular Ca2+ was also greatly diminished with the Arg-503 mutant indicating that Ca2+ stimulation of the enzyme in vivo is due primarily to direct interactions with calmodulin and Ca2+. These data demonstrate that the Ca2+ sensitivity of this enzyme can be modulated by point mutagenesis within the putative calmodulin binding domain and indicate that the enzyme can be directly regulated by Ca2+ and calmodulin in vivo.