Abstract
This study was to identify specific regions in kappa opioid receptors that accounted for binding selectivity of kappa ligands. Six chimeric mu/kappa receptors were constructed from cloned rat kappa and mu opioid receptors and transiently expressed in COS-1 cells. All six chimeric mu/kappa receptors bound [3H] diprenorphine with high affinities, indicating that these chimeras retain opioid receptor conformation. Binding affinities of three peptide ligands (dynorphin A, alpha-neo-endorphin, and dynorphin B) and three nonpeptide ligands (norbinaltorphimine, U50,488H, and U69,593) for chimeras were determined and compared to those for mu and kappa opioid receptors. The second extracellular loop and the adjoining C-terminal portion of the fourth transmembrane helix were essential for the high affinity binding of dynorphin A, alpha-neo-endorphin, and dynorphin B to the kappa receptor. The third extracellular loop and the sixth and seventh transmembrane helices played an important role in determining the selectivity of nor-binaltorphimine for the kappa over the mu receptor. U50,488H and U69,593 appeared to require the whole kappa receptor except the second extracellular loop to attain high affinity binding. Thus, the kappa opioid receptor has differential binding domains for peptide and non-peptide ligands.
Highlights
This study watso identify specific regions Kionpioid re- putative transmembrane helices (TMHs)’ separated by intraceptors that accounted for binding selecotfivKitlyigands. and extracellular loops, characteristics of G protein-coupled
For chimera IV, HindIIUAflIII frag- quence of the fragment produced by Polymerase chain reaction (PCR) and those in the ment ofRMOR in pRdCMV and AflIIIIApaI fragment of RKOR in pcDNA3 were ligated into HindIII and ApaI sites of pcDNA3
Since sequences within the TMH 5 and the N-terminal half of the i3 loop are highly homologous between p and K receptors(Fig. 11, the exchange essentially involves the e2 loop and the C-terminal portion of HindIII fragment ofRMOR in pRdCMV were ligated the TMH 4
Summary
Dept. of Pharmacology, Temple University Schoool f Medicine, 3420 N. Oid agonists [17, 18].nor-BNI is a highly selective K antagonist ’ The abbreviations used are: TMH, transmembranehelix; aa, amino acid(s); e2 loop, the second extracellular loop; e3 loop, the third extracellular loop; i3 loop, the third intracellular loop; nor-BNI, nor-binaltorphimine; nt, nucleotide(s1;PCR, polymerase chain reaction; RMOR, the rat p opioid receptor; RKOR, the rat K opioid receptor. PeptidaenNdonpeptidBeindinDgomains of K OpioRideceptors receptor would facilitate computerized modeling of the receptor structure
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