Boekhoff I, Touhara K, Danner S, Inglese J, Lohse M J, Breer H, Lefkowitz R J
University Stuttgart-Hohenheim, Institute of Zoophysiology, 70599 Stuttgart, Federal Republic of Germany.
J Biol Chem. 1997 Feb 14;272(7):4606-12. doi: 10.1074/jbc.272.7.4606.
Phosducin, which tightly binds betagamma-subunits of heterotrimeric G-proteins, has been conjectured to play a role in regulating second messenger signaling cascades, but to date its specific function has not been elucidated. Here we demonstrate a potential role for phosducin in regulating olfactory signal transduction. In isolated olfactory cilia certain odorants elicit a rapid and transient cAMP response, terminated by a concerted process which requires the action of two protein kinases, protein kinase A (PKA) and a receptor-specific kinase (GRK3) (Schleicher, S., Boekhoff, I. Arriza, J., Lefkowitz, R. J., and Breer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1420-1424). The mechanism of action of GRK3 involves a Gbetagamma-mediated translocation of the kinase to the plasma membrane bound receptors (Pitcher, J. A., Inglese, J., Higgins, J. B. , Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M. , Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267). A protein with a molecular mass of 33 kDa that comigrates on SDS gels with recombinant phosducin and which is immunoreactive with phosducin antibodies is present in olfactory cilia. Recombinant phosducin added to permeabilized olfactory cilia preparations strongly inhibits termination of odorant-induced cAMP response and odorant-induced membrane translocation of GRK3. In addition, the cAMP analogue dibutyryl cAMP stimulates membrane targeting of the receptor kinase. This effect is presumably due to PKA-mediated phosphorylation of phosducin, which diminishes its affinity for binding to the Gbetagamma-subunit, thereby making Gbetagamma available to function as a membrane anchor for GRK3. A specific PKA inhibitor blocks the odorant-induced translocation of the receptor kinase. Consistent with this formulation, a non-phosphorylatable mutant of phosducin (phosducin Ser-73 --> Ala) is an even more effective inhibitor of desensitization and membrane targeting of GRK3 than the wild-type protein. A phosducin mutant that mimics phosphorylated phosducin (phosducin Ser-73 --> Asp) lacks this property and in fact recruits GRK3 to the membrane and potentiates desensitization. These results suggest that phosducin may act as a phosphorylation-dependent switch in second messenger signaling cascades, regulating the kinetics of desensitization processes by controlling the activity of Gbetagamma-dependent GRKs.
磷光蛋白能紧密结合异源三聚体G蛋白的βγ亚基，据推测它在调节第二信使信号级联反应中发挥作用，但迄今为止其具体功能尚未阐明。在此，我们证明了磷光蛋白在调节嗅觉信号转导中的潜在作用。在分离出的嗅觉纤毛中，某些气味剂会引发快速且短暂的cAMP反应，该反应通过一个协同过程终止，这一过程需要两种蛋白激酶，即蛋白激酶A（PKA）和受体特异性激酶（GRK3）的作用（施莱歇尔，S.，伯克霍夫，I.，阿里扎，J.，莱夫科维茨，R. J.，和布雷尔，H.（1993年）《美国国家科学院院刊》90，1420 - 1424）。GRK3的作用机制涉及Gβγ介导的激酶向质膜结合受体的转位（皮彻，J. A.，英格利斯，J.，希金斯，J. B.，阿里扎，J. L.，凯西，P. J.，金，C.，贝诺维奇，J. L.，夸特拉，M. M.，卡龙，M. G.，和莱夫科维茨，R. J.（1992年）《科学》257，1264 - 1267）。一种分子量为33 kDa的蛋白质存在于嗅觉纤毛中，它在SDS凝胶上与重组磷光蛋白迁移率相同，并且与磷光蛋白抗体发生免疫反应。添加到透化的嗅觉纤毛制剂中的重组磷光蛋白强烈抑制气味剂诱导的cAMP反应的终止以及气味剂诱导的GRK3向膜的转位。此外，cAMP类似物二丁酰cAMP刺激受体激酶的膜靶向作用。这种效应可能是由于PKA介导的磷光蛋白磷酸化，这降低了其与Gβγ亚基结合的亲和力，从而使Gβγ能够作为GRK3的膜锚发挥作用。一种特异性PKA抑制剂可阻断气味剂诱导的受体激酶转位。与此说法一致的是，磷光蛋白的非磷酸化突变体（磷光蛋白丝氨酸73→丙氨酸）比野生型蛋白更能有效抑制GRK3的脱敏和膜靶向作用。一种模拟磷酸化磷光蛋白的磷光蛋白突变体（磷光蛋白丝氨酸73→天冬氨酸）缺乏这种特性，实际上它会将GRK3招募到膜上并增强脱敏作用。这些结果表明，磷光蛋白可能在第二信使信号级联反应中作为一种磷酸化依赖性开关，通过控制Gβγ依赖性GRK的活性来调节脱敏过程的动力学。
