Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs.
A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.
Laboratory of Structure-Function Biochemistry, Department of Molecular Chemistry, Graduate School of Science, Kyushu University, Fukuoka, 812-8581, Japan.
N
N
Shirasu
T
T
Kuromizu
H
H
Nakao
Y
Y
Chuman
T
T
Nose
T
T
Costa
Y
Y
Shimohigashi
D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000166:cytology; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry; D004399:Dynorphins / Q000737:chemistry* / Q000378:metabolism*; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D051381:Rats; D011957:Receptors, Opioid / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000345:Affinity Labels; D004220:Disulfides; D011957:Receptors, Opioid; D013438:Sulfhydryl Compounds; D004399:Dynorphins; D003545:Cysteine