Schröder S, Lohse M J
Laboratory of Molecular Biology, University of Munich, Germany.
Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2100-4. doi: 10.1073/pnas.93.5.2100.
Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10831-10835. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits. First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin. Second, it inhibited the GTPase activity of purified G(o). The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions. Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.
磷酸视蛋白是一种主要在视网膜和松果体中表达的胞质蛋白，它能与鸟嘌呤核苷酸结合蛋白（G蛋白）的βγ亚基相互作用，从而可能调节跨膜信号传导。最近从大鼠脑中分离出了一种编码类磷酸视蛋白（PhLP）的cDNA[迈尔斯，M.F.，巴尔希特，S.，斯甘加，M. & 埃利奥特，M.（1993年）《美国国家科学院院刊》90，10831 - 10835页]。在此我们报道PhLP在大肠杆菌中的表达及其纯化。重组纯化的PhLP抑制了G蛋白βγ亚基的多种作用。首先，它抑制了百日咳毒素介导的纯化α(o)的βγ亚基依赖性ADP核糖基化。其次，它抑制了纯化的G(o)的GTP酶活性。在后者的测定中，PhLP的IC50值为89 nM，而磷酸视蛋白在17 nM时引起半数最大抑制。最后，PhLP拮抗了纯化的βγ亚基对视紫红质磷酸化的增强作用。PhLP的N末端与长得多的磷酸视蛋白N末端没有相似性，而该区域已被证明对磷酸视蛋白与βγ亚基的相互作用至关重要。因此，PhLP似乎通过一种尚不清楚的相互作用模式与G蛋白βγ亚基结合，可能代表G蛋白功能的一种内源性调节因子。
