Distinct differences between morphine- and [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin-mu-opioid receptor complexes demonstrated by cyclic AMP-dependent protein kinase phosphorylation.
The present study demonstrates a conditional, agonist-dependent phosphorylation of the mu-opioid receptor (MOR-1) by cyclic AMP-dependent protein kinase (PKA) in membrane preparations of MOR-1-transfected neuroblastoma Neuro2A cells. Opioid agonist-dependent phosphorylation occurs in a time- and concentration-dependent manner (EC50 approximately 40 nM) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 microM morphine and 6 nM PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and beta-endorphin) stimulated phosphorylation of MOR-1 by PKA, the potent mu-opioid-selective peptide [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) or other enkephalin analogues such as [D-Ala2]-Met5-enkephalinamide (DALA), [D-Ala2,D-Leu5]-enkephalin (DADLE), and Met5-enkephalin had no effect. The lack of the effect of DAMGO on MOR-1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist-dependent conformations of MOR-1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist-receptor conformations.
Chakrabarti
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Department of Pharmacology, University of Minnesota Medical School, Minneapolis, USA.
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D000701:Analgesics, Opioid / Q000494:pharmacology*; D000918:Antibody Specificity; D002227:Carbazoles; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives / Q000494:pharmacology; D004745:Enkephalins / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D009020:Morphine / Q000494:pharmacology*; D009447:Neuroblastoma; D009474:Neurons / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D010766:Phosphorylation; D011233:Precipitin Tests; D011758:Pyrroles / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology
D000701:Analgesics, Opioid; D002227:Carbazoles; D004745:Enkephalins; D004791:Enzyme Inhibitors; D007211:Indoles; D011758:Pyrroles; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004744:Enkephalin, Methionine; C057416:KT 5720; C022118:enkephalinamide-Met, Ala(2)-; D016308:Enkephalin, Leucine-2-Alanine; D009020:Morphine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases