Real-time analysis of molecular interaction of retinoid receptors and receptor-interacting protein 140 (RIP140).
Receptor interacting protein 140 (RIP140) is a coregulator for a large number of transcription factors. RIP140 interacts with retinoic acid receptor (RAR) and retinoid X receptor (RXR) with or without ligands. The C-terminal domain of RIP140 (RIP-C') contains a novel sequence (1063-1076, LTKTNPILYYMLQK) and has been shown to interact with RAR and RXR ligand dependently in two-hybrid interaction and pull-down assays. To examine the kinetic characteristics of molecular interaction of RIP-C' with RAR and RXR, a surface plasmon resonance technology (BIAcore) was applied for real-time analyses of this molecular interaction with highly purified proteins. A modified pull-down assay using purified proteins was also conducted to obtain supporting data. The effect of retinoid ligands on this type of interaction was addressed. By using receptor mutants, it was demonstrated that the activation function-2 domain and the ability to form dimers of the receptors are required for an efficient interaction of receptor with RIP140. Finally, with a mutagenesis approach, we determined the effects of specific point mutations on the kinetics of RIP-C' interaction with RAR/RXR.
Chen
Yixin
Y
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Kerimo
Ann
A
Khan
Shaukat
S
Wei
Li-Na
LN
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D007700:Kinetics; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000078723:Nuclear Receptor Interacting Protein 1; D010446:Peptide Fragments / Q000737:chemistry; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D047488:Retinoid X Receptors; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D009687:Nuclear Proteins; D000078723:Nuclear Receptor Interacting Protein 1; D010446:Peptide Fragments; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors