title: RACK1 identified as the PCBP1-interacting protein with a novel functional role on the regulation of human MOR gene expression.
abstract: Poly C binding protein 1 (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 screened using the two-hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1-RACK1 interaction was confirmed via in vivo validation using the two-hybrid system, and by co-immunoprecipitation with anti-PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co-immunoprecipitation suggested that RACK1-PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over-expression 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 (3) H-diprenorphine 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 neuronal NMB cells.
authors.affiliation: Department of Biological Sciences, Seton Hall University, South Orange, NJ 07079, USA.
authors.forename: Pranjal
authors.initials: P
authors.lastname: Nahar-Gohad
authors.forename: Hamidah
authors.initials: H
authors.lastname: Sultan
authors.forename: Ysabella
authors.initials: Y
authors.lastname: Esteban
authors.forename: Alyda
authors.initials: A
authors.lastname: Stabile
authors.forename: Jane L
authors.initials: JL
authors.lastname: Ko
mesh_terms: D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D004174:Diprenorphine / Q000493:pharmacokinetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D009292:Narcotic Antagonists / Q000493:pharmacokinetics; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016601:RNA-Binding Proteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009292:Narcotic Antagonists; D009363:Neoplasm Proteins; C094604:PCBP1 protein, human; C419368:RACK1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D017450:Receptors, Opioid, mu; D014316:Tritium; D004174:Diprenorphine; D019204:GTP-Binding Proteins