Differences in motilin receptor desensitization after stimulation with motilin or motilides are due to alternative receptor trafficking.
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.
Mitselos
Anna
A
Centre for Gastroenterological Research, Catholic University of Leuven, Gasthuisberg O&N 1, bus 701, B-3000 Leuven, Belgium.
Vanden Berghe
Pieter
P
Peeters
Theo L
TL
Depoortere
Inge
I
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
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