Identification of two C-terminal amino acids, Ser(355) and Thr(357), required for short-term homologous desensitization of mu-opioid receptors.
Our recent study suggests that a cluster of Ser/Thr residues (T(354)S(355)S(356)T(357)) at the intracellular carboxyl tail of rat mu-opioid receptor (MOR1) is required for the development of short-term homologous desensitization. To investigate the functional role played by individual serine or threonine residue of this (TSST) cluster in the agonist-induced mu-opioid receptor desensitization, point mutant (T354A), (S355A), (S356A) and (T357A) mu-opioid receptors were prepared and stably expressed in human embryonic kidney 293 cells (HEK 293 cells). Similar to wild-type mu-opioid receptors, mutant (T354A) and (S356A) mu-opioid receptors stably expressed in HEK 293 cells developed homologous desensitization after 30 min pretreatment of DAMGO ([D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin), a specific mu-opioid receptor agonist. Substituting Ser(355)or Thr(357) with alanine resulted in a significant attenuation of agonist-induced mu-opioid receptor desensitization. In HEK 293 cells stably expressing double mutant (S355A/T357A) mu-opioid receptors, DAMGO pretreatment failed to significantly affect the efficacy and potency by which DAMGO inhibits forskolin-stimulated adenylyl cyclase activity. Consistent with the general belief that agonist-induced phosphorylation of guanine nucleotide binding protein (G protein)-coupled receptors is involved in homologous desensitization. Treating HEK 293 cells expressing wild-type mu-opioid receptors with 5 microM DAMGO for 30 min induced the receptor phosphorylation. Mutation of Ser(355) and Thr(357) also greatly impaired DAMGO-induced mu-opioid receptor phosphorylation. These results suggest that two C-terminal amino acids, Ser(355) and Thr(357), are required for short-term homologous desensitization and agonist-induced phosphorylation of mu-opioid receptors expressed in HEK 293 cells.
Department of Physiology, Chang Gung University School of Medicine, Tao-Yuan, Taiwan, ROC. hlwns@mail.cgu.edu.tw
Hung-Li
HL
Wang
Wen-Teng
WT
Chang
Chia-Yu
CY
Hsu
Pei-Chen
PC
Huang
Yu-Wen
YW
Chow
Allen H
AH
Li
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D002478:Cells, Cultured; D006801:Humans; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017450:Receptors, Opioid, mu / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D017450:Receptors, Opioid, mu; D013912:Threonine; D012694:Serine; D000409:Alanine