Threonine 180 is required for G-protein-coupled receptor kinase 3- and beta-arrestin 2-mediated desensitization of the mu-opioid receptor in Xenopus oocytes

To determine the sites in the mu -opioid receptor (MOR) critical for agonis t-dependent desensitization, we constructed and coexpressed MORs lacking po tential phosphorylation sites along with G-protein activated inwardly recti fying potassium channels composed of K(ir)3.1 and K(ir)3.4 subunits in Xeno pus oocytes, Activation of MOR by the stable enkephalin analogue, [D-Ala(2) ,MePhe(4),Glyol(5)]enkephalin, led to homologous MOR desensitization in ooc ytes coexpressing both G-protein-coupled receptor kinase 3 (GRK3) and beta -arrestin 2 (arr3), Coexpression with either GRK3 or arr3 individually did not significantly enhance desensitization of responses evoked by wild type MOR activation. Mutation of serine or threonine residues to alanines in the putative third cytoplasmic loop and truncation of the C-terminal tail did not block GRK/arr3-mediated desensitization of MOR. Instead, alanine substi tution of a single threonine in the second cytoplasmic loop to produce MOR( T180A) was sufficient to block homologous desensitization. The insensitivit y of MOR(T180A) might have resulted either from a block of arrestin activat ion or arrestin binding to MOR. To distinguish between these alternatives, we expressed a dominant positive arrestin, arr2(R169E), that desensitizes G protein-coupled receptors in an agonist-dependent but phosphorylation-inde pendent manner. arr2(R169E) produced robust desensitization of MOR and MOR( T180A) in the absence of GRK3 coexpression. These results demonstrate that the T180A mutation probably blocks GRK3- and arr3-mediated desensitization of MOR by preventing a critical agonist-dependent receptor phosphorylation and suggest a novel GRK3 site of regulation not yet described for other G-p rotein-coupled receptors.