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Characterization of Agonist-Induced Motilin Receptor Trafficking and Its Implications for Tachyphylaxis

Clinical Discovery Technologies (V.L., Y.D.), Metabolic Disease Research (Z.M., R.S., D.G.), Discovery Chemistry (J.L.), Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey; and Biological Sciences, State University of New York—Brockport, Brockport, New York (A.R.)

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 Ca²⁺-mobilization. Retardation of the intracellular MR-GFP sorting cycle seemed to correlate with the tachyphylaxis-inducing properties of each compound, but not its EC₅₀. 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.

Received for publication July 21, 2005.

Accepted for publication October 11, 2005.

Address correspondence to: Dr. Yves Dubaquie, Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Rd, North Billerica, MA 01862. E-mail: yves.dubaquie{at}bms.com