Croze E, Usacheva A, Asarnow D, Minshall R D, Perez H D, Colamonici O
Department of Immunology, Berlex Biosciences, Richmond CA 94804, USA.
J Immunol. 2000 Nov 1;165(9):5127-32. doi: 10.4049/jimmunol.165.9.5127.
The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-alphaRbetaL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-alphaRbetaL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-alphaRbetaL, the minimum site for RACK-1 binding was mapped to aa 300-346. RACK-1 binding to IFN-alphaRbetaL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-alphaRbetaL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-alphaRalpha), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-alphaRbetaL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-beta1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-beta1b. However, stimulation of cells with either IFN-beta1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.
在酵母双杂交系统中，将人I型干扰素受体链2（IFNAR2c或IFN-αRβL）的胞质结构域用作诱饵，以鉴定与该受体这一区域相互作用的新蛋白。我们在此报告IFN-αRβL的胞质结构域与先前鉴定的蛋白RACK-1（活化C激酶受体）之间的特异性相互作用。使用编码IFN-αRβL胞质结构域不同区域的GST融合蛋白，将RACK-1结合的最小位点定位到第300 - 346氨基酸。RACK-1与IFN-αRβL的结合不需要RACK-1的前91个氨基酸，其中包括两个WD结构域，WD1和WD2。通过共免疫沉淀在人Daudi细胞中也检测到RACK-1与IFN-αRβL之间的相互作用，但未检测到与人I型干扰素受体链1（IFNAR1或IFN-αRα）的相互作用。结果表明，RACK-1与IFN-αRβL组成性结合，并且这种结合不受I型干扰素（IFN-β1b）刺激Daudi细胞的影响。在用IFN-β1b刺激Daudi细胞后，RACK-1本身并未发生酪氨酸磷酸化。然而，用IFN-β1b或PMA刺激细胞确实导致可检测到的免疫荧光增加以及RACK-1在细胞内的重新分布。
