Mutation of tyrosine 318 (Y318F) in the delta-opioid receptor attenuates tyrosine phosphorylation, agonist-dependent receptor internalization, and mitogen-activated protein kinase activation.
Opioid receptors are known for their ability to activate diverse second messenger systems. Previously, we showed that selective delta-opioid agonists were able to induce the rapid tyrosine phosphorylation of delta-opioid receptors (delta-ORs) through Src. Src-dependent tyrosine phosphorylation of delta-ORs appears to be important for activation of the mitogen-activated protein kinase cascade and for receptor sequestration into clathrin-coated endosomes, as the Src antagonist, PP1, inhibited both. In an attempt to clarify the role of tyrosine phosphorylation in delta-OR signalling and regulation, we constructed a mutant receptor in which the tyrosine located in the conserved NPXXY motif of the C-terminus was replaced by a phenylalanine (Y318F-delta-OR). Mutation of Y318 resulted in a receptor that was comparable to the wild type in its expression level in HEK-293 cells and in its affinity for opioid ligands. Both receptors showed effective coupling to G proteins and were capable of inhibiting forskolin-stimulated cAMP accumulation with similar potencies. However, the mutant receptor was able to stimulate (35)S-GTPgammaS binding with a lower EC(50) than the wild type receptor. The stimulation of tyrosine phosphorylation in delta-ORs by [D-Thr(2)]-Leu-enkephalin-Thr (DTLET) was significantly less in cells expressing the Y318F-delta-OR than in cells expressing the wild type. In addition, both rapid receptor internalization and down-regulation were markedly attenuated in the mutant. Finally, the mutant receptor was unable to induce a robust activation of the MAPK pathway, suggesting that tyrosine phosphorylation of the delta-OR protein is important for this signalling pathway. These findings implicate tyrosine phosphorylation of Y318 in receptor signalling and agonist-mediated regulation.
Department of Psychiatry, New York University School of Medicine, Millhauser Laboratories-Room HN-610, New York, NY 10016, USA. ken.kramer@nyu.edu
H K
HK
Kramer
M L
ML
Andria
S A
SA
Kushner
D H
DH
Esposito
J M
JM
Hiller
E J
EJ
Simon
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000700:Analgesics / Q000493:pharmacokinetics; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D010766:Phosphorylation; D011869:Radioligand Assay; D017465:Receptors, Opioid, delta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014316:Tritium; D014443:Tyrosine
D000700:Analgesics; D017465:Receptors, Opioid, delta; D011994:Recombinant Proteins; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine; D010649:Phenylalanine; D004743:Enkephalin, Leucine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases