title: Characterization of agonist-induced motilin receptor trafficking and its implications for tachyphylaxis.
abstract: The motilin receptor (MR) is a member of the seven-transmembrane receptor family and is expressed throughout the gastrointestinal tract of humans and other species. Motilin, the natural MR peptide ligand, has profound stimulatory effects on gastrointestinal contractility, indicating a therapeutic potential for MR modulators. However, long-term clinical use of certain MR agonists is limited by tachyphylaxis, a reduced responsiveness to repeated compound exposure. This study was meant to characterize the ligand-induced endocytosis of MR and to test whether receptor trafficking contributes to tachyphylaxis. A cell-based assay was developed by fusing a green fluorescent protein (GFP) moiety to the motilin receptor, and high-content biology instrumentation was used to quantify time and dose dependence of MR-GFP endocytosis. Maximal internalization of MR-GFP was induced after 45 min of constant exposure to 80 nM motilin. This process was disrupted by nocodazole, suggesting an essential role for microtubules. Internalized MR-GFP vesicles disappeared within 15 to 45 min of motilin withdrawal but did not overlap with the lysosomal compartment, indicating that MR-GFP escaped degradation and was recycled back to the plasma membrane. It is noteworthy that the kinetics of MR-GFP redistribution varied substantially when stimulated with motilin, erythromycin, 6,9-hemiacetal 8,9-anhydro-4''-deoxy-3'-N-desmethyl-3'-N-ethylerythromycin B (ABT-229), or N-[(1S)-1-[[[(1S)-1-(aminocarbonyl)-3-phenylpropyl]amino]carbonyl]-3-phenylpropyl]-2'-(1,3-benzodioxol-5-ylmethyl)tetrahydro-1',3'-dioxo-spiro[piperidine-4,5'(6'H)-[1H][1,2,4]triazolo[1,2-a]pyridazine]-8'-carboxamide (BMS-591348) at equipotent doses for Ca(2+)-mobilization. Retardation of the intracellular MR-GFP sorting cycle seemed to correlate with the tachyphylaxis-inducing properties of each compound, but not its EC(50). These results indicate that MR internalization, desensitization, and resensitization are ligand-dependent and that appropriate screening strategies may enable the development of small molecule agonists with ideal combinations of these distinct properties.
authors.affiliation: Clinical Discovery Technologies, Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Rd, North Billerica, MA 01862, USA.
authors.forename: Vahideh
authors.initials: V
authors.lastname: Lamian
authors.forename: Adam
authors.initials: A
authors.lastname: Rich
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Ma
authors.forename: James
authors.initials: J
authors.lastname: Li
authors.forename: Ramakrishna
authors.initials: R
authors.lastname: Seethala
authors.forename: David
authors.initials: D
authors.lastname: Gordon
authors.forename: Yves
authors.initials: Y
authors.lastname: Dubaquie
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D011964:Receptors, Gastrointestinal Hormone / Q000819:agonists* / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000819:agonists* / Q000378:metabolism*; D013618:Tachyphylaxis
chemical_list: D017931:DNA Primers; D011964:Receptors, Gastrointestinal Hormone; D018013:Receptors, Neuropeptide; C050802:motilin receptor; D049452:Green Fluorescent Proteins