Agonist-induced, G protein-dependent and -independent down-regulation of the mu opioid receptor. The receptor is a direct substrate for protein-tyrosine kinase.
The mu opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr(394) in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr(394) was also responsible for mu opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr(394). The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr(394) has no regulatory role but phosphorylation of tyrosine residues appears essential.
Pak
Y
Y
Department of Pharmacology, University of Toronto, Ontario M5S 1A8, Canada.
O'Dowd
B F
BF
Wang
J B
JB
George
S R
SR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015536:Down-Regulation; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009270:Naloxone / Q000378:metabolism; D037342:Pertussis Toxin; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017450:Receptors, Opioid, mu / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004791:Enzyme Inhibitors; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D013912:Threonine; D009270:Naloxone; D037342:Pertussis Toxin; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins