Identification of two C-terminal amino acids, Ser³⁵⁵ and Thr³⁵⁷, required for short-term homologous desensitization of μ-opioid receptors

Our recent study suggests that a cluster of Ser/Thr residues (T³⁵⁴S³⁵⁵S³⁵⁶T³⁵⁷) at the intracellular carboxyl tail of rat μ-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 μ-opioid receptor desensitization, point mutant (T354A), (S355A), (S356A) and (T357A) μ-opioid receptors were prepared and stably expressed in human embryonic kidney 293 cells (HEK 293 cells). Similar to wild-type μ-opioid receptors, mutant (T354A) and (S356A) μ-opioid receptors stably expressed in HEK 293 cells developed homologous desensitization after 30min pretreatment of DAMGO ([D-Ala², N-methyl-Phe⁴, Gly-ol⁵]enkephalin), a specific μ-opioid receptor agonist. Substituting Ser³⁵⁵or Thr³⁵⁷ with alanine resulted in a significant attenuation of agonist-induced μ-opioid receptor desensitization. In HEK 293 cells stably expressing double mutant (S355A/T357A) μ-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 μ-opioid receptors with 5μM DAMGO for 30min induced the receptor phosphorylation. Mutation of Ser³⁵⁵ and Thr³⁵⁷ also greatly impaired DAMGO-induced μ-opioid receptor phosphorylation. These results suggest that two C-terminal amino acids, Ser³⁵⁵ and Thr³⁵⁷, are required for short-term homologous desensitization and agonist-induced phosphorylation of μ-opioid receptors expressed in HEK 293 cells.