title: Differences in motilin receptor desensitization after stimulation with motilin or motilides are due to alternative receptor trafficking.
abstract: OBJECTIVE
  The 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.
  
  
  METHODS
  Desensitization 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 beta-arrestins were visualized by fluorescence microscopy.
  
  
  RESULTS
  Agonist-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 (t(1/2): approximately 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 beta-arrestin-2 with greater affinity to the plasma membrane than beta-arrestin-1. After internalization the MTLR co-localized with transferrin but not with cathepsin D. After stimulation with motilin and EM-A the t(1/2) for MTLR resensitization was 3h and 1h, respectively but amounted 26h for ABT-229.
  
  
  CONCLUSIONS
  Our results suggest that the resensitization kinetics determine the desensitization properties of the motilin agonists.
authors: Centre for Gastroenterological Research, Catholic University of Leuven, Gasthuisberg O&N 1, bus 701, B-3000 Leuven, Belgium.
authors: Anna
authors: A
authors: Mitselos
authors: Pieter
authors: P
authors: Vanden Berghe
authors: Theo L
authors: TL
authors: Peeters
authors: Inge
authors: I
authors: Depoortere
mesh_terms: D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D009037:Motilin / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D014162:Transfection; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D011964:Receptors, Gastrointestinal Hormone; D018013:Receptors, Neuropeptide; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C050802:motilin receptor; D009037:Motilin; C114264:Alemcinal; D004917:Erythromycin