Agonist-induced signaling and trafficking of the mu-opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C-terminal domain.
The human mu-opioid receptor and a mutant form, muS/ T[i3+Cter]A, in which all Ser and Thr residues from the third cytoplasmic loop and C-terminal domain were changed to Ala, were studied after expression in CHO-K1 cells. Although the mutant receptors had similar affinities for agonists and EC50 values for inhibition of adenylyl cyclase as compared to wild-type receptors, the Emax were almost 2-fold decreased, suggesting a role of the mutated residues in G-protein coupling. After chronic morphine or etorphine, the EC50 values of the agonists were about 5-fold increased at both receptors but the Emax values were not altered; upon agonist withdrawal forskolin-stimulated cAMP levels were increased to almost 200% of control levels. Sequestration and rapid down-regulation of the mu-opioid receptor were induced by DAGO and etorphine but not morphine. In contrast, the muS/T[i3+Cter]A receptor was not sequestered and was up-regulated (150-380%) after treatment with agonists. The results indicate that the Ser and Thr residues in the third cytoplasmic loop and C-terminus of the mu-opioid receptor are not involved in the limited desensitization or in the adenylyl cyclase superactivation promoted by agonists but that their integrity and/or their phosphorylation is required in the intricate and coordinately regulated pathways involved in receptor signaling and trafficking.
Capeyrou
R
R
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Unité Propre de Recherches No. 9062, Toulouse, France.
Riond
J
J
Corbani
M
M
Lepage
J F
JF
Bertin
B
B
Emorine
L J
LJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004174:Diprenorphine / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D005048:Etorphine / Q000378:metabolism / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D009020:Morphine / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry
D004745:Enkephalins; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004174:Diprenorphine; D005576:Colforsin; D013912:Threonine; D006165:Guanylyl Imidodiphosphate; D005048:Etorphine; D012694:Serine; D009020:Morphine; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases