Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits
Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits
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DOI：
10.1073/pnas.93.11.5407
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发表时间：
1996-05-28
影响因子：
11.1
通讯作者：
Hamm, HE
中科院分区：
文献类型：
--
作者：
Artemyev, NO;Natochin, M;Hamm, HE
cGMP磷酸二酯酶（PDE）是脊椎动物感光细胞的关键效应酶，其调节第二信使cGMP的水平。PDE由催化性α和β亚基（P α和P β）和两个抑制性γ亚基（P γ）组成，所述抑制性γ亚基在黑暗中阻断PDE活性。主要的抑制区域已经定位于P γ的C末端。最后一个C-末端残基-IleIle形成对抑制PDE活性至关重要的重要疏水结构域。在这项研究中，P γ的突变体被设计用于交联实验，以鉴定P α和P β亚基上与P γ C末端结合的区域。在其中一个突变体中，68位的半胱氨酸被丝氨酸取代，P γ的最后四个C-末端残基被单个半胱氨酸取代。用光探针4-（N-马来酰亚胺基）二苯甲酮（MBP）在半胱氨酸残基处标记该突变体P γ 83 Cys。标记的P γ 83 CysMBP突变体是比未标记的突变体更有效的PDE活性抑制剂，表明疏水MBP探针模拟P γ疏水C末端。一个特定的，高产量的交联高达70%之间实现的P γ 83 CysMBP和PDE催化亚基。P α和N-末端截短的P β（缺少147个氨基酸残基）以相同的效率交联到P γ 83 CysMBP上。使用来自交联PDE的胰蛋白酶片段的质谱分析，我们鉴定了与P α的aa残基751-763交联的位点。P β的相应区域，P β-749-761，也可以结合到P γ C末端。我们的数据表明，P γ通过结合PDE的催化位点阻断PDE活性，该催化位点靠近NKXD基序，NKXD基序是与cGMP的鸟嘌呤环相互作用的共有序列。
cGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic alpha and beta subunits (P alpha and P beta) and two inhibitory gamma subunits (P gamma) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of P gamma. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of P gamma were designed for cross-linking experiments to identify regions on P alpha and P beta subunits that bind to the P gamma C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of P gamma were replaced with a single cysteine. This mutant, P gamma 83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled P gamma 83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the P gamma hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the P gamma 83CysMBP and PDE catalytic subunits. P alpha and the N-terminally truncated P beta (lacking 147 aa residues) cross-linked to P gamma 83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of P alpha. The corresponding region of P beta, P beta-749-761, also may bind to the P gamma C terminus. Our data suggest that P gamma blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.