Mutational analysis of predicted intracellular loop domains of human motilin receptor

Hitoo Tokunaga, Bunzo Matsuura, Maoqing Dong, Laurence J Miller, Teruhisa Ueda, Shinya Furukawa, Yoichi Hiasa, Morikazu Onji

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume294
Issue number2
DOIs
StatePublished - Feb 2008

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Motilin
Erythromycin
GTP-Binding Proteins
Calcium
Pharmacologic Actions
COS Cells
G-Protein-Coupled Receptors
Phenylalanine
Histidine
Mutagenesis
Alanine
motilin receptor
Mutation

Keywords

  • Erythromycin
  • G protein-coupled receptor
  • Mutagenesis

ASJC Scopus subject areas

  • Gastroenterology
  • Physiology

Cite this

Mutational analysis of predicted intracellular loop domains of human motilin receptor. / Tokunaga, Hitoo; Matsuura, Bunzo; Dong, Maoqing; Miller, Laurence J; Ueda, Teruhisa; Furukawa, Shinya; Hiasa, Yoichi; Onji, Morikazu.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 294, No. 2, 02.2008.

Research output: Contribution to journalArticle

Tokunaga, Hitoo ; Matsuura, Bunzo ; Dong, Maoqing ; Miller, Laurence J ; Ueda, Teruhisa ; Furukawa, Shinya ; Hiasa, Yoichi ; Onji, Morikazu. / Mutational analysis of predicted intracellular loop domains of human motilin receptor. In: American Journal of Physiology - Gastrointestinal and Liver Physiology. 2008 ; Vol. 294, No. 2.
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