Molecular basis underlying the poly C binding protein 1 as a regulator of the proximal promoter of mouse mu-opioid receptor gene.
Previous studies showed poly C binding protein 1 (PCBP) participating in the mu-opioid receptor (MOR) gene regulation via binding to a single-stranded (ss) DNA element. In this report, we therefore investigate the molecular basis of PCBP regulating the MOR gene expression. Various truncated PCBPs, including one domain (KH1, KH2, variable or KH3), two- (K12, K2v or Kv3) or three-sequential domains (K12v or K2v3), were constructed. The MOR ssDNA binding abilities of these truncated PCBPs were examined using electrophoretic mobility shift assay (EMSA). KH1 domain possessed a strong MOR ssDNA binding activity. Variable domain displayed no binding, and KH2 or KH3 domain possessed a weak MOR ssDNA binding activity. Binding of two-domain PCBPs indicated an additive effect of two-domain combinations. Interestingly, K2v3, a three-domain PCBP, displayed as strong ssDNA binding as that of K12v, suggesting synergism of KH2, KH3 and variable domains for the binding activity. Functional analysis demonstrated one-domain PCBPs exhibiting no transactivation on the MOR proximal promoter. Two-domain PCBPs displayed approximately 20% activity, while three-domain PCBPs displayed 70%-85% of full-length PCBP activity. Taken together, these results suggested that no single domain possessed sufficient functional activity to serve as an independent transactivation domain, and the combination of three sequential domains was necessary for its optimal activity to activate the MOR proximal promoter. In summary, our data suggested that cooperativity of three sequential domains is essential for PCBP functioning as a MOR gene regulator. Various ways in which this cooperativity could occur are discussed.
Malik
Adnan K
AK
Department of Biology, Seton Hall University, 208 McNulty Hall, 400 South Orange Ave. South Orange, NJ 07079, USA.
Flock
Kelly E
KE
Godavarthi
Chaitanya L
CL
Loh
Horace H
HH
Ko
Jane L
JL
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000502:physiology*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma; D010759:Phosphorus Isotopes / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C404943:Oprm protein, mouse; C121375:Pcbp1 protein, mouse; D010759:Phosphorus Isotopes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D017450:Receptors, Opioid, mu; D008715:Methionine