Direct and differential interaction of beta-arrestins with the intracellular domains of different opioid receptors.
beta-arrestins have been shown to play important roles in regulation of signaling and desensitization of opioid receptors in many in vivo studies. The current study was carried out to measure the direct interaction of beta-arrestins with two functional intracellular domains, the third intracellular loop (I3L) and the carboxyl terminus (CT), of delta-, mu-, and kappa-opioid receptors (DOR, MOR, and KOR, respectively). Results from the pull-down assay using glutathione S-transferase fusion proteins demonstrated that beta-arrestins (1 and 2) were able to bind to the I3L of DOR and to the CT of DOR and KOR. Surface plasmon resonance measurement gave similar results with typical dissociation equilibrium constant (K(D)) values in the micromolar range. The site-directed mutagenesis experiment further revealed that certain specific serine/threonine residues in these receptor domains play a critical role in their interaction with beta-arrestins. Taken together, our data clearly indicated that beta-arrestins interact differentially with the functional domains of different opioid receptors; this may provide a possible molecular basis for differential regulation of opioid receptors by beta-arrestins.
Shanghai Institute of Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
B
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Cen
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Xiong
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Ma
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Pei
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics; D000071557:beta-Arrestins
D019390:Arrestins; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D013912:Threonine; D012694:Serine