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This Article Full Text Full Text (PDF) All Versions of this Article: 294/2/G460    most recent 00244.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Tokunaga, H. Articles by Onji, M. PubMed PubMed Citation Articles by Tokunaga, H. Articles by Onji, M.

HORMONES AND SIGNALING

Mutational analysis of predicted intracellular loop domains of human motilin receptor

¹Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Tohon, Ehime, Japan; and ²Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona

Submitted 1 June 2007 ; accepted in final form 21 November 2007

Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin- and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63–66, 135–137, and 296–301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.

erythromycin; G protein-coupled receptor; mutagenesis