MOLECULAR MECHANISMS OF GUANYLATE CYCLASE ACTIVATION

描述：(改编自申请人的摘要)这是一份修订后的 关于调查一种名为鸟苷的蛋白质参与的建议 环化调节蛋白(GCRP)对心钠素刺激的调节作用 鸟苷环化酶活力。这种最近被克隆的蛋白质 在申请人的实验室中的表达已经被证明放大了 共转染实验中ANF依赖的环化酶活性。申请人 假设：(1)鸟苷环化酶以活性和非活性形式存在 形式，ANF和ATP的存在促进了 前者；(2)非活性形式被自身抑制部位抑制 存在于环化酶的激酶样结构域中，以及(3)在存在 ANF和ATP、GCRP和/或其剪接变异体GCRP-2与 环化酶并诱导构象变化，从而释放抑制作用 对活动站点施加的限制。为了评估这一假设， 申请人建议：(1)在鸟粪上定位自身抑制部位 使用定点突变环化酶分子并确定是否 心钠素和三磷酸腺苷通过释放抑制约束激活环化酶，(2) 研究ANF是否诱导GCRP和GCRP-2与 鸟苷环化酶和(3)检测有限的蛋白分解或结合在 自抑制部位在活性部位引起构象变化 是循环酶的。

DESCRIPTION: (Adapted from the Applicant's Abstract) This is a revised proposal to investigate the involvement of a protein called the guanylate cyclase regulatory protein (GCRP) in the regulation of ANF-stimulated guanylate cyclase activity. This protein which was recently cloned and expressed in the applicant's laboratory has been shown to amplify ANF-dependent cyclase activity in cotransfection experiments. The applicant hypothesizes that; (1) guanylate cyclase exists in an active and inactive form and that the presence of ANF and ATP promotes the development of the former; (2) the inactive form is suppressed by an autoinhibitory site present in the kinase-like domain of the cyclase and (3) in the presence of ANF and ATP, GCRP, and/or its splice variant GCRP-2, associates with the cyclase and induces a conformational change which releases the inhibitory constraint imposed on the active site. To evaluate this hypothesis the applicant proposes to: (1) locate the autoinhibitory site on the guanylate cyclase molecule using site-directed mutagenesis and to determine whether ANF and ATP activate the cyclase by releasing the inhibitory constraint, (2) investigate whether ANF induces the association of GCRP and GCRP-2 with guanylate cyclase and (3) examine whether limited proteolysis or binding at the autoinhibitory site induces a conformational change at the active site of the cyclase.