Clapp Peter, Capper Austin B, Taussig Ronald
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Methods Enzymol. 2002;345:241-51. doi: 10.1016/s0076-6879(02)45020-3.
Initial steps in the identification of the Gs alpha-binding site present in mammalian adenylyl cyclases can be achieved with the use of the yeast genetic system described. It must be stressed that this system serves as a means to identify mutants that are candidates; biochemical analysis of these mutants is a next and necessary step in the confirmation of these phenotypes. The system described can be readily adapted for the isolation of additional classes of mammalian adenylyl cyclase mutants including mutants with altered regulation toward forskolin, catalytic abnormalities, or enhanced sensitivities toward activators. In addition, this system can be employed for the isolation of constitutively active adenylyl cyclase mutants, or by coexpressing other adenylyl cyclase isoforms and their known regulators, mutations in the binding sites for these molecules can be elucidated.
利用所描述的酵母遗传系统，可以完成鉴定哺乳动物腺苷酸环化酶中存在的Gsα结合位点的初步步骤。必须强调的是，该系统是一种鉴定候选突变体的手段；对这些突变体进行生化分析是确认这些表型的下一步且必要的步骤。所描述的系统可以很容易地用于分离其他类型的哺乳动物腺苷酸环化酶突变体，包括对福斯高林调节改变的突变体、催化异常的突变体或对激活剂敏感性增强的突变体。此外，该系统可用于分离组成型活性腺苷酸环化酶突变体，或者通过共表达其他腺苷酸环化酶同工型及其已知调节剂，可以阐明这些分子结合位点的突变。
