Hawes B E, Touhara K, Kurose H, Lefkowitz R J, Inglese J
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1994 Nov 25;269(47):29825-30.
Although a role for the beta gamma-subunits of heterotrimeric G proteins (G beta gamma) in signal transduction by several cellular systems has been established, the structural features of cellular proteins interacting with G beta gamma have yet to be fully elucidated. The G beta gamma-binding region of beta-adrenergic receptor kinase (beta ARK), a cytosolic enzyme recruited to the membrane receptor substrate by G beta gamma, has been localized to the carboxyl terminus of the enzyme. Here, we demonstrate that the amino terminus of phosducin, a 33-kDa G beta gamma-binding retinal phosphoprotein, contains sequences homologous with the G beta gamma-binding domain of beta ARK. Accordingly, a glutathione S-transferase-fusion protein containing only the amino-terminal 105 amino acids of phosducin displayed G beta gamma binding ability. This domain of phosducin contains a protein kinase A (PKA) phosphorylation site, and upon phosphorylation, the binding of full-length phosducin to G beta gamma is reduced. In addition, transient expression of phosducin in COS-7 cells significantly inhibits G beta gamma-mediated phosphoinositide hydrolysis. This inhibitory effect is completely reversed by pretreatment of cells with dibutyryl cAMP, an activator of PKA. Thus, the binding of G beta gamma to phosducin can be regulated by PKA-phosphorylation in an intact cell model system.
尽管已经确定异源三聚体G蛋白的βγ亚基（Gβγ）在多个细胞系统的信号转导中发挥作用，但与Gβγ相互作用的细胞蛋白的结构特征尚未完全阐明。β-肾上腺素能受体激酶（βARK）的Gβγ结合区域已定位到该酶的羧基末端，βARK是一种由Gβγ募集到膜受体底物的胞质酶。在这里，我们证明了视紫红质（一种33 kDa的Gβγ结合视网膜磷蛋白）的氨基末端包含与βARK的Gβγ结合域同源的序列。因此，仅包含视紫红质氨基末端105个氨基酸的谷胱甘肽S-转移酶融合蛋白具有Gβγ结合能力。视紫红质的这个结构域包含一个蛋白激酶A（PKA）磷酸化位点，磷酸化后，全长视紫红质与Gβγ的结合减少。此外，视紫红质在COS-7细胞中的瞬时表达显著抑制Gβγ介导的磷酸肌醇水解。用二丁酰cAMP（一种PKA激活剂）预处理细胞可完全逆转这种抑制作用。因此，在完整的细胞模型系统中，Gβγ与视紫红质的结合可通过PKA磷酸化来调节。
