THE CARBOXYL-TERMINUS OF THE GAMMA-SUBUNIT OF ROD CGMP PHOSPHODIESTERASE CONTAINS DISTINCT SITES OF INTERACTION WITH THE ENZYME CATALYTIC SUBUNITS AND THE ALPHA-SUBUNIT OF TRANSDUCIN
THE CARBOXYL-TERMINUS OF THE GAMMA-SUBUNIT OF ROD CGMP PHOSPHODIESTERASE CONTAINS DISTINCT SITES OF INTERACTION WITH THE ENZYME CATALYTIC SUBUNITS AND THE ALPHA-SUBUNIT OF TRANSDUCIN
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DOI：
10.1074/jbc.270.22.13210
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发表时间：
1995-06-02
影响因子：
4.8
通讯作者：
HAMM, HE
中科院分区：
文献类型：
--
作者：
SKIBA, NP;ARTEMYEV, NO;HAMM, HE
GTP结合形式的转导素α-亚基（G α（t））和cGMP磷酸二酯酶（PDE）的γ-亚基（P γ）之间的相互作用是光子信号转导期间效应物激活的关键事件。P γ的羧基末端的一半参与与G α（t）的相互作用，以及PDE活性的抑制。在这里，我们已经利用合成肽和诱变方法的组合来定位与G α（t）和P α β相互作用的P γ羧基末端区域的特定区域，并确定了参与抑制PDE活性的残基。我们发现对应于P γ残基68 - 87的合成肽完全抑制胰蛋白酶激活的PDE。肽P γ-63-87以2.5 μ M的K-d与G α（t）GTP γ S结合，而P γ-68-87 60 G α（t）GTP γ S的结合大约小15倍（K-d = 40 μ M），这表明羧基末端P γ区68 - 87含有与P α β相互作用的位点，也含有α（t）结合位点的一部分。为了更精确地定位羧基末端区域内的G α（t）和P α β位点，通过定点诱变产生一组羧基末端突变体。残基63 - 69和70 - 76的缺失减少了突变体与α（t）的结合，而与缺少多达11个氨基酸残基的羧基末端截短突变体的结合没有改变。与此相反，羧基末端截断的P γ从德尔塔1德尔塔11导致其抑制活性逐渐下降。因此，极端羧基末端疏水序列-Ile（86）-Ile（87）与9个相邻残基一起提供P γ与P α β的抑制性相互作用。P γ的羧基末端G α（t）GTP γ S结合位点不同于但邻近其PDE抑制位点。在视觉传导过程中，G α（t）GTP可能与P γ的该区域结合，诱导末端羧基末端从P α β上的抑制位点置换，导致PDE活化。
The interaction between the GTP-bound form of the transducin alpha-subunit (G alpha(t)) and the gamma-subunit (P gamma) of cGMP phosphodiesterase (PDE) is a key event in effector activation during photon signal transduction. The carboxyl-terminal half of P gamma is involved in interaction with G alpha(t), as well as in inhibition of PDE activity. Here we have utilized a combination of synthetic peptide and mutagenesis approaches to localize specific regions of the carboxyl-terminal region of P gamma interacting with G alpha(t) and P alpha beta and have determined residues involved in inhibition of PDE activity. We found that synthetic peptide corresponding to residues 68-87 of P gamma completely inhibit trypsin-activated PDE. The peptide P gamma-63-87 bound to G alpha(t)GTP gamma S with a K-d Of 2.5 mu m, whereas the binding of P gamma-68-87 60 G alpha(t)GTP gamma S was approximately 15-fold less (K-d = 40 mu M) suggesting that carboxyl-terminal P gamma region 68-87 contains a site for interaction with P alpha beta and also a part of the alpha(t) binding site. To map G alpha(t) and P alpha beta sites more precisely within the carboxyl-terminal region, a set of carboxyl-terminal mutants was generated by site-directed mutagenesis. Deletion of residues 63-69 and 70-76 diminished the binding of mutants to alpha(t) while binding to carboxyl-terminally truncated mutants lacking up to 11 amino acid residues was unchanged. In contrast, carboxyl-terminal truncations of P gamma from Delta 1 to Delta 11 resulted in a gradual decrease of its inhibitory activity. Thus, the extreme carboxyl-terminal hydrophobic sequence -Ile(86)-Ile(87) together with 9 adjacent residues provides inhibitory interaction of P gamma with P alpha beta. The carboxyl-terminal G alpha(t)GTP gamma S binding site of P gamma is different from but adjacent to its PDE inhibitory site. During the visual transduction process, G alpha(t)GTP Likely binds to this region of P gamma inducing a displacement of the extreme carboxyl terminus from the inhibitory site on P alpha beta, leading to PDE activation.