ABSTRACT
Agonist activation of the delta-opioid receptor leads to internalization via G betagamma recruitment of G protein coupled receptor kinase-2, which phosphorylates the receptor at several sites, including Ser363, allowing beta-arrestin binding and localization to clathrin coated pits. Using human embryonic kidney cells expressing a delta-opioid receptor we tested the hypothesis that prevention of receptor coupling to G protein by treatment with pertussis toxin (PTX) will block these processes. PTX treatment did not reduce phosphorylation of delta-opioid receptor Ser363 in response to the agonist [D-Pen2, D-Pen5]enkephalin, or recruitment of beta-arrestin 2-green fluorescent protein to the membrane and only slowed, but did not prevent, [D-Pen2, D-Pen5]enkephalin-induced internalization. Similarly, PTX treatment only partially prevented the ability of the delta-opioid peptide agonists deltorphin II and [Met5]enkephalin and the non-peptide agonist BW373U86 to induce receptor internalization. No internalization was seen with morphine, oxymorphindole or the putative delta(1)-opioid agonist TAN-67 in the presence or absence of PTX, even though TAN-67 showed a strong activation of G protein, as measured by guanosine-5'-O-(3-[(35)S]thio)triphosphate binding. The ability of an agonist to stimulate phosphorylation at Ser363 was predictive of its capacity to induce internalization. The results suggest a role for G protein in delta-opioid receptor internalization, but show that alternative G protein independent pathways exist.
Subject(s)
GTP-Binding Proteins/physiology , Receptors, Opioid, delta/metabolism , Analgesics, Opioid/pharmacology , Arrestins/genetics , Arrestins/metabolism , Benzamides/pharmacology , Cell Line, Transformed , Dose-Response Relationship, Drug , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Enkephalin, Methionine/pharmacology , Green Fluorescent Proteins/genetics , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Morphine/pharmacology , Oligopeptides/pharmacology , Pertussis Toxin/pharmacology , Phosphorylation/drug effects , Phosphorylation/physiology , Piperazines/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Protein Transport/drug effects , Protein Transport/physiology , Quinolines/pharmacology , Receptors, Opioid, delta/agonists , Serine/metabolism , Sulfur Isotopes/metabolism , Transfection/methods , beta-Arrestin 2 , beta-ArrestinsABSTRACT
Ligands from the naltrexamine series have consistently demonstrated agonist activity at kappa opioid receptors (KOR), with varying activity at the mu opioid receptor (MOR). Various 6 beta-cinnamoylamino derivatives were made with the aim of generating ligands with a KOR agonist/MOR partial agonist profile, as ligands with this activity may be of interest as treatment agents for cocaine abuse. The ligands all displayed the desired high affinity, nonselective binding in vitro and in the functional assays were high efficacy KOR agonists with some partial agonist activity at MOR. Two of the new ligands (12a, 12b) have been evaluated in vivo, with 12a acting as a KOR agonist and therefore somewhat similar to the previously evaluated analogues 3-6, while 12b displayed predominant MOR agonist activity.
