MOLECULAR MECHANISMS OF GUANYLATE CYCLASE ACTIVATION

描述：（改编自申请人的摘要）这是一个修订的 一项研究鸟苷酸蛋白参与的提案 环化酶调节蛋白（GCRP）在调节ANF刺激的 鸟苷酸环化酶活性。 这种蛋白质最近被克隆出来， 在申请人的实验室中表达的蛋白质已经显示出放大 共转染实验中的ANF依赖性环化酶活性。 申请人 假设;（1）鸟苷酸环化酶存在于活性和非活性 形式，ANF和ATP的存在促进了 前者;（2）非活性形式被自身抑制位点抑制 存在于环化酶的激酶样结构域中，和（3）在存在 ANF和ATP、GCRP和/或其剪接变体GCRP-2，与ANF和ATP、GCRP和/或其剪接变体GCRP-2相关。 环化酶并诱导构象变化， 对活性部位的限制。 为了评估这一假设， 申请人提出：（1）在鸟苷酸上定位自抑制位点 环化酶分子，并确定是否 ANF和ATP通过释放抑制性约束激活环化酶，（2） 研究ANF是否诱导GCRP和GCRP-2与 鸟苷酸环化酶和（3）检查是否有限的蛋白水解或结合在 自抑制位点诱导活性位点的构象变化 的环化酶。

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.