Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label.
The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.
Zhang
Yan
Y
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA.
McCurdy
Christopher R
CR
Metzger
Thomas G
TG
Portoghese
Philip S
PS
D001120:Arginine / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005470:Fluorometry; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017465:Receptors, Opioid, delta / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics
D003432:Cross-Linking Reagents; D009281:Naphthalenes; D019920:Photoaffinity Labels; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D012694:Serine; C027717:2,3-naphthalenedicarboxaldehyde; D001120:Arginine; D008239:Lysine; D003545:Cysteine