Skiba N P, Artemyev N O, Hamm H E
Department of Physiology and Biophysics, University of Illinois, College of Medicine, Chicago 60680, USA.
J Biol Chem. 1995 Jun 2;270(22):13210-5. doi: 10.1074/jbc.270.22.13210.
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 Kd of 2.5 microM, whereas the binding of P gamma-68-87 to G alpha tGTP gamma S was approximately 15-fold less (Kd = 40 microM) 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 -Ile86-Ile87 together with 9 adjacent residues provides inhibitory interaction of P gamma with P alpha beta. The carboxyl-terminal G alpha tGTP gamma S binding site of P gamma is different from but adjacent to its PDE inhibitory site. During the visual transduction process, G alpha tGTP 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.
转导素α亚基（Gαt）的GTP结合形式与环鸟苷酸磷酸二酯酶（PDE）的γ亚基（Pγ）之间的相互作用是光信号转导过程中效应器激活的关键事件。Pγ的羧基末端一半参与与Gαt的相互作用以及对PDE活性的抑制。在此，我们结合使用合成肽和诱变方法来定位Pγ羧基末端区域与Gαt和Pαβ相互作用的特定区域，并确定了参与抑制PDE活性的残基。我们发现，对应于Pγ第68 - 87位残基的合成肽完全抑制胰蛋白酶激活的PDE。肽Pγ - 63 - 87以2.5 μM的解离常数（Kd）与Gαt GTPγS结合，而Pγ - 68 - 87与Gαt GTPγS的结合力约低15倍（Kd = 40 μM），这表明羧基末端Pγ区域68 - 87包含一个与Pαβ相互作用的位点，也是αt结合位点的一部分。为了更精确地在羧基末端区域内定位Gαt和Pαβ位点，通过定点诱变产生了一组羧基末端突变体。缺失第63 - 69位和70 - 76位残基会减少突变体与αt的结合，而与缺失多达11个氨基酸残基的羧基末端截短突变体的结合不变。相反，Pγ从δ1到δ11的羧基末端截短导致其抑制活性逐渐降低。因此，极端羧基末端的疏水序列 - Ile86 - Ile87以及9个相邻残基提供了Pγ与Pαβ的抑制性相互作用。Pγ的羧基末端Gαt GTPγS结合位点与其PDE抑制位点不同但相邻。在视觉转导过程中，Gαt GTP可能结合到Pγ的该区域，导致极端羧基末端从Pαβ上的抑制位点移位，从而导致PDE激活。
