Ligand-regulated internalization of the opioid receptor-like 1: a confocal study.
To better understand opioid receptor-like 1 (ORL1) internalization, we fused the C terminus of ORL1, the nociceptin (noc) receptor, to the N terminus of a green fluorescent protein and used the fusion protein to characterize receptor endocytosis in live human embryonic kidney cells. The fusion altered neither the affinity of the receptor for noc or other ORL1 receptor ligands nor the ability of the receptor to mediate agonist-induced binding of GTPgamma(35)S, i.e. coupling with heterotrimeric G protein. Confocal microscopy showed that the fluorescent receptor was mostly associated (>75%) with the periplasmic membrane. In the presence of 0.1 microm noc, approximately 80% of receptors were internalized, and half-maximum internalization was reached in approximately 12 min at 22 C and approximately 6 min at 37 C. After washing, a normal receptor level was recovered within 70 min at 22 C. The lack of internalization in the presence of 0.45 m sucrose suggests that noc-induced receptor endocytosis mainly occurred via clathrin-coated pits. Coincubation of the recombinant cells with noc and tetramethylrhodamine-transferrin showed that ORL1 was mainly internalized through the endosome compartment. Lofentanil and Ro64-6198 ([(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one]) promoted endocytosis of the fluorescent receptor as efficiently as noc. Among the two ORL1 receptor antagonists, J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), but not III-BTD, blocked the noc-induced internalization of the fluorescent receptor. Two partial agonists were dramatically less efficient than noc to promote ORL1 internalization. They recruited very little (the pseudopeptide [Phe(1)psi(CH(2)-NH)Gly(2)]-noc-(1-13)NH(2)) or no (the hexapeptide Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH(2)) G protein receptor kinase type 2 coupled to red fluorescent protein 1 at the membrane, suggesting that subsequent receptor phosphorylation necessary for internalization via coated pits is altered. Thus, partial agonists that induce a prolonged cell response without causing substantial receptor internalization may be good tools for further clinical treatments.
Corbani
Maithé
M
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 5089, 205 route de Narbonne 31017 Toulouse cedex 4, France. maithe.corbani@ipbs.fr
Gonindard
Christophe
C
Meunier
Jean-Claude
JC
D001692:Biological Transport; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D007668:Kidney / Q000196:embryology*; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009292:Narcotic Antagonists; D018847:Opioid Peptides / Q000235:genetics; D021381:Protein Transport; D011957:Receptors, Opioid / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases; D000094942:Nociceptin Receptor; D000097573:Red Fluorescent Protein; D000097629:Nociceptin
D017868:Cyclic AMP-Dependent Protein Kinases; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins; D009292:Narcotic Antagonists; D018847:Opioid Peptides; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D051552:beta-Adrenergic Receptor Kinases; D000094942:Nociceptin Receptor; D000097573:Red Fluorescent Protein; D000097629:Nociceptin; C000722283:OPRL1 protein, human