A cluster of Ser/Thr residues at the C-terminus of μ-opioid receptor is required for G protein-coupled receptor kinase 2-mediated desensitization

To investigate the functional role of G protein-coupled receptor kinases (GRK) in homologous desensitization of the μ-opioid receptor, human embryonic kidney (HEK) 293 cells, which express a significant level of GRK2, were stably transfected with the cDNA encoding the rat μ-opioid receptor. Wild-type μ-opioid receptors developed homologous desensitization after 30 min pretreatment with DAMGO ([D-Ala²,N-methyl-Phe⁴,Gly-ol⁵]enkephalin), a specific μ-opioid receptor agonist. The ability of μ-opioid receptors to develop homologous desensitization was greatly impaired following the transfection of a cDNA fragment encoding the GRK2(495-689) polypeptide, which is believed to block G(βy)-mediated transduction events including the membrane translocation and activation of GRK2. The μ(CΔ45) receptor, a deletion mutant that lacks 45 C-terminal amino acids, failed to exhibit homologous desensitization after 30 min pretreatment of DAMGO. The μ(CΔ41) receptor, which differs from the μ(CΔ45) receptor by having four more Ser/Thr residues (Thr³⁵⁴Ser³⁵⁵Ser³⁵⁶Th³⁵⁷), developed GRK2-mediated desensitization. These results suggest that homologous desensitization of rat μ-opioid receptors results from the activation of GRK2 and that a cluster of Ser/Thr residues (Thr³⁵⁴Ser³⁵⁵Ser³⁵⁶Thr³⁵⁷) at the intracellular carboxyl tail plays an important role in GRK2-mediated μ-opioid receptor desensitization. Copyright (C) 2000 Elsevier Science Ltd.