DAMGO recognizes four residues in the third extracellular loop to discriminate between mu- and kappa-opioid receptors.
Previously, we reported that replacement of the region from the fifth transmembrane domain to the C-terminus of kappa-opioid receptor with the corresponding region of mu-opioid receptor gives high affinity for [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), a mu-opioid receptor-selective ligand, to the resultant chimeric receptor, suggesting that the difference in the amino acid sequence within this region is critical for the discrimination between mu- and kappa-opioid receptors by DAMGO. In the present study, we constructed further six mu/kappa-chimeric receptors and revealed that at least two separate regions around the third extracellular loop are critical for the discrimination between mu- and kappa-opioid receptors by DAMGO. Furthermore, we constructed several mutant receptors by a site-directed mutagenesis technique and found that the difference between Glu297 of kappa-opioid receptor and Lys303 of mu-opioid receptor in one region, and the difference between Ser310, Tyr312 and Tyr313 of kappa-opioid receptor and Val316, Trp318 and His319 of mu-opioid receptor in the other region, are critical for the discrimination between these receptors by DAMGO. The mutant receptor, kappa (E297K + Y313H + Y312W + S310V), in which the Glu297, Ser310, Tyr312 and Tyr313 of kappa-opioid receptor were changed to Lys, Val, Trp and His, respectively, bound to DAMGO with high affinity (Kd = 8.7 +/- 1.2 nM) and efficiently mediated the inhibitory effect of DAMGO on intracellular cAMP accumulation. The present results showed that these four amino acid residues act as determinants for the discrimination between mu- and kappa-opioid receptors by DAMGO.
Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
T
T
Seki
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Minami
T
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Nakagawa
Y
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Ienaga
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Morisada
M
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Satoh
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology*; D005110:Extracellular Space / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D004745:Enkephalins; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D000262:Adenylyl Cyclases