Bicarbonate regulated adenylyl cyclase

描述(申请人提供)：多个生理过程(即呼吸、血流、脑脊液和房水形成以及精子激活)受二氧化碳和/或重碳酸盐的调节。已知的唯一受碳酸氢盐或二氧化碳调节的哺乳动物信号蛋白是碳酸氢盐响应的‘可溶性’腺苷环化酶(SAC)。哺乳动物SAC酶在分子、生化和结构上不同于G蛋白调节的跨膜腺苷酸环化酶，后者介导细胞对激素的反应。在这项资助之前的支持下，我们已经证实SAC是精子中细胞外碳酸氢盐的细胞传感器，也是鳞片中细胞外pH的细胞传感器(通过细胞内碳酸氢盐的波动)。我们现在建议通过测试SAC在其他生理过程中是否作为碳酸氢盐和/或二氧化碳传感器来继续这一项目。在这一应用的初步数据中，我们表明，体细胞表达不同于先前在雄性生殖细胞中发现的那些碳酸氢盐响应性SAC的亚型。我们计划：(1)从碳酸氢盐反应的组织和细胞系中提纯SAC的体细胞形式，并测定其多肽氨基酸序列。(2)克隆这些新的SAC亚型。(3)利用原生酶和异源表达的重组SAC亚型鉴定其生化性质。(4)确定在碳酸氢盐/二氧化碳调节的各种生理系统中是否表达SAC亚型以及表达哪些SAC亚型。

DESCRIPTION (provided by applicant): Multiple physiological processes (i.e., breathing, blood flow, cerebrospinal fluid and aqueous humor formation, and sperm activation) are modulated by carbon dioxide and/or bicarbonate. The only known mammalian signaling protein modulated by bicarbonate or carbon dioxide is the bicarbonate responsive 'soluble' adenylyl cyclase (sAC). Mammalian sAC enzymes are molecularly, biochemically, and structurally distinct from the G protein regulated transmembrane adenylyl cyclases which mediate cellular responses to hormones. With previous support from this grant, we have confirmed that sAC serves as a cellular sensor for extracellular bicarbonate in sperm and as a cellular sensor for extracellular pH (via fluctuations of intracellular bicarbonate) in the epidydymis. We now propose to continue this project by testing whether sAC serves as the bicarbonate and/or carbon dioxide sensor in other physiological processes. In preliminary data for this application we show that somatic cells express isoforms of bicarbonate responsive sAC distinct from those previously identified in male germ cells. We plan to: (1) purify the somatic forms of sAC from bicarbonate responsive tissues and cell lines and determine their peptide amino acid sequences. (2) Clone these novel sAC isoforms. (3) Characterize their biochemical properties using both the native enzymes and heterologously expressed, recombinant sAC isoforms. (4) Ascertain whether and which sAC isoforms are expressed in various bicarbonate/carbon dioxide regulated physiological systems.