Various cellular signaling pathways induced by nociceptin activation of ORL1 (opioid receptor-like 1 receptor) develop homologous desensitization. Multiple lines of evidence suggest that agonist-induced phosphorylation of serine (Ser)/threonine (Thr) residues at intracellular carboxyl tail leads to homologous desensitization of G protein-coupled receptors. In the present study, we investigated the functional role played by C-terminal Ser/Thr residues in agonist-induced desensitization and phosphorylation of ORL1. In HEK 293 cells expressing wild-type ORL1 and ORL1(CΔ21), which lacks C-terminal 21 amino acids, nociceptin inhibition of adenylate cyclase activity exhibited homologous desensitization after 1 h pretreatment of nociceptin. In contrast, ORL1(CΔ34), which differs with ORL1(CΔ21) by lacking C-terminal Ser334, Ser335 and Ser343 residues, failed to develop agonist-induced desensitization. Point mutant (S343A) ORL1 underwent homologous desensitization after nociceptin pretreatment. Substituting Ser 334 or Ser335 with alanine greatly impaired nociceptin-induced ORL1 desensitization. In HEK 293 cells expressing double mutant (S334A/S335A) ORL1, nociceptin pretreatment failed to significantly affect the efficacy and potency by which nociceptin inhibits forskolin-stimulated cAMP formation. Mutation of Ser334 and Ser 335 also greatly reduced nociceptin-induced ORL1 phosphorylation. These results suggest that two C-terminal serine residues, Ser334 and Ser335, are required for homologous desensitization and agonist-induced phosphorylation of ORL1.
- Adenylate cyclase
- Agonist-induced phosphorylation
- HEK 293 cells
- Homologous desensitization
- Opioid receptor-like 1 receptor
ASJC Scopus subject areas
- Cell Biology