Differences in motilin receptor desensitization after stimulation with motilin or motilides are due to alternative receptor trafficking

Backgrounds & aimsThe motilin receptor (MTLR) is an important therapeutic target for treatment of hypomotility disorders. The negative outcome in clinical trials with the motilin agonist, ABT-229, indicated that desensitization may limit the therapeutic usefulness of motilides. We therefore compared the mechanisms involved in the intracellular trafficking of the MTLR after stimulation with motilin, erythromycin-A (EM-A) or ABT-229.MethodsDesensitization was studied by measuring changes in Ca2+ rises and by receptor binding studies in CHO cells co-expressing the Ca2+ indicator apoaequorin and the MTLR, C-terminally tagged with EGFP. Receptor phosphorylation was studied by immunoprecipitation. MTLR-EGFP trafficking to organelles and translocation of β-arrestins were visualized by fluorescence microscopy.ResultsAgonist-induced desensitization of the MTLR was due to receptor internalization with potencies (p-int50) in the order of: ABT-229 (8.3) > motilin (7.86) > EM-A (4.77) but with no differences in the internalization kinetics (t1/2: ∼25 min). The percentage cell surface receptor loss was more profound after exposure to ABT-229 (88 ± 1%) than to motilin (63 ± 10%) or EM-A (34 ± 2%). For motilin and EM-A MTLR phosphorylation probably occurs via G protein-coupled receptor kinases while for ABT-229 phosphorylation was also protein kinase C dependent. All agonists translocated cytosolic β-arrestin-2 with greater affinity to the plasma membrane than β-arrestin-1. After internalization the MTLR co-localized with transferrin but not with cathepsin D. After stimulation with motilin and EM-A the t1/2 for MTLR resensitization was 3 h and 1 h, respectively but amounted 26 h for ABT-229.ConclusionOur results suggest that the resensitization kinetics determine the desensitization properties of the motilin agonists.