The catalytic and GAF domains of the rod cGMP phosphodiesterase (PDE6) heterodimer are regulated by distinct regions of its inhibitory γ subunit
The catalytic and GAF domains of the rod cGMP phosphodiesterase (PDE6) heterodimer are regulated by distinct regions of its inhibitory γ subunit
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DOI：
10.1074/jbc.m103316200
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发表时间：
2001-07-20
影响因子：
4.8
通讯作者：
Cote, RH
中科院分区：
文献类型：
--
作者：
Mou, HM;Cote, RH
视网膜视杆细胞光感受器中视觉转导的中枢效应物cGMP磷酸二酯酶（PDE 6）是一种催化异源二聚体（α β），低分子量抑制性γ亚基与其结合形成非活化PDE全酶（α β γ（2））。尽管已知γ与α β紧密结合，但每个γ亚基与α β的结合亲和力、γ上与α β相互作用的结构域以及γ与α β上的调节和催化区域之间的变构相互作用还没有很好地理解。我们在这里显示，当牛视杆PDE 6的调节性GAF结构域被cGMP占据时，γ亚基结合到催化性α β二聚体上的两个不同位点（K-D1 <1 pM，K-D2 = 3 pM）。当cAMP占据非催化位点时，与α β的结合异质性不存在。γ上的两个主要结构域可以独立地与α β相互作用，其中γ的N-末端一半结合的亲和力比其C-末端抑制区域大50倍。γ的N-末端一半负责α β上γ和cGMP结合位点之间的正协同性，但对催化活性没有影响。使用合成肽，我们确定了γ的氨基酸序列的区域，结合α β，恢复高亲和力cGMP结合低亲和力非催化位点，并延迟cGMP交换与两个非催化位点。亚基异质性、γ与α β相互作用的多个位点以及γ与GAF结构域的正协同性都可能有助于在视杆细胞光感受器的视觉转导期间精确控制PDE 6的激活和失活动力学。
The central effector of visual transduction in retinal rod photoreceptors, cGMP phosphodiesterase (PDE6), is a catalytic heterodimer (alpha beta) to which low molecular weight inhibitory gamma subunits bind to form the nonactivated PDE holoenzyme (alpha beta gamma (2)). Although it is known that gamma binds tightly to alpha beta, the binding affinity for each gamma subunit to alpha beta, the domains on gamma that interact with alpha beta, and the allosteric interactions between gamma and the regulatory and catalytic regions on alpha beta are not well understood. We show here that the gamma subunit binds to two distinct sites on the catalytic alpha beta dimer (K-D1 < 1 pM, K-D2 = 3 pM) when the regulatory GAF domains of bovine rod PDE6 are occupied by cGMP. Binding heterogeneity of to alpha beta is absent when cAMP occupies the noncatalytic sites. Two major domains on gamma can interact independently with alpha beta with the N-terminal half of gamma binding with 50-fold greater affinity than its C-terminal, inhibitory region. The N-terminal half of gamma is responsible for the positive cooperativity between gamma and cGMP binding sites on alpha beta but has no effect on catalytic activity. Using synthetic peptides, we identified regions of the amino acid sequence of gamma that bind to alpha beta, restore high affinity cGMP binding to low affinity noncatalytic sites, and retard cGMP exchange with both noncatalytic sites. Subunit heterogeneity, multiple sites of gamma interaction with alpha beta, and positive cooperativity of gamma with the GAF domains are all likely to contribute to precisely controlling the activation and inactivation kinetics of PDE6 during visual transduction in rod photoreceptors.