PT  - JOURNAL ARTICLE
AU  - Lamian, Vahideh
AU  - Rich, Adam
AU  - Ma, Zhengping
AU  - Li, James
AU  - Seethala, Ramakrishna
AU  - Gordon, David
AU  - Dubaquie, Yves
TI  - Characterization of Agonist-Induced Motilin Receptor Trafficking and Its Implications for Tachyphylaxis
AID  - 10.1124/mol.105.017111
DP  - 2006 Jan 01
TA  - Molecular Pharmacology
PG  - 109--118
VI  - 69
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/69/1/109.short
4100  - http://molpharm.aspetjournals.org/content/69/1/109.full
SO  - Mol Pharmacol2006 Jan 01; 69
AB  - 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.