Receptor for Activated C-Kinase (RACK-1), a WD Motif-Containing Protein, Specifically Associates with the Human Type I IFN Receptor

Abstract

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-αRβL) 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-αRβL 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-αRβL, the minimum site for RACK-1 binding was mapped to aa 300–346. RACK-1 binding to IFN-αRβL 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-αRβL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-αRα), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-αRβL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-β1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-β1b. However, stimulation of cells with either IFN-β1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.