Identify PCBP1‐interacting protein and its novel functional role on human MOR gene expression

Abstract

PCBP1 is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein‐protein interaction with PCBP1. A human brain cDNA library was screed using the two‐hybrid system with PCBP1 as the bait. RACK1 protein, containing WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation, and by co‐immunoprecipitation with anti‐PCBP1 antibody using human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 overexpression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in NMB cells.*This research was supported by NIH research grant, NSF MRI grant and SHU Biological Research.