Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.
Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.
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NCB-20 细胞中 Ca(2) 抑制性腺苷酸环化酶的克隆和表达。
DOI：
10.1073/pnas.89.15.6716
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发表时间：
1992
影响因子：
11.1
通讯作者：
Cooper,DM
中科院分区：
文献类型：
--
作者：
Yoshimura,M;Cooper,DM
已从NCB-20细胞中克隆了编码腺苷酸环化酶[ATP焦磷酸裂解酶（环化），EC 4.6.1.1]的cDNA，其中腺苷酸环化酶活性在生理浓度下被Ca 2+抑制。通过聚合酶链反应（PCR）分离cDNA克隆（5.8个腺苷酸酶），使用简并引物通过比较三种腺苷酸环化酶序列（I型、II型和III型）和随后的文库筛选而设计。北方分析显示，在心脏组织中表达对应于该cDNA的mRNA（6.1个腺苷酸酶），这是Ca（2+）-可降解腺苷酸环化酶的主要来源。该克隆编码的蛋白质的1165个氨基酸，其亲水性的配置文件是非常相似的其他哺乳动物腺苷酸环化酶，最近被克隆。这种蛋白质和其他腺苷酸环化酶之间的一个显着差异是一个漫长的氨基末端区域之前的第一次跨膜跨度。在人胚肾细胞系293中瞬时表达该cDNA揭示了与对照转染细胞相比，响应于毛喉素的cAMP产生增加了3倍。在纯化的质膜从转染细胞，腺苷酸环化酶的活性增加也被检测到，这是容易抑制亚微摩尔Ca 2+。因此，这种腺苷酸环化酶似乎代表了在NCB-20细胞、心脏组织和其他地方遇到的Ca（2+）-可降解形式。它的鉴定应允许确定的结构特征，确定腺苷酸环化酶的调节模式由Ca 2+。
A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+.