Differential contributions of motilin receptor extracellular domains for peptide and non-peptidyl agonist binding and activity

Bunzo Matsuura, Maoqing Dong, Shruthi Naik, Laurence J Miller, Morikazu Onji

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The family of G protein-coupled receptors that includes receptors for motilin, ghrelin, and growth hormone secretagogue has substantial potential importance as drug targets. Understanding of the molecular basis of hormone binding and receptor activation should provide insights that are helpful in the development of such drugs. We previously examined the unique second extracellular loop domain of the motilin receptor, identifying key epitopes in perimembranous locations at each end of this long loop (Matsuura, B., Dong, M., and Miller, L. J. (2002) J. Biol. Chem. 277, 9834-9839). Here, we have extended that work, examining the other predicted extracellular domains of the motilin receptor by using sequential deletions of segments ranging from one to six amino acid residues and site-directed alanine replacement mutagenesis approaches. Each construct was transiently expressed in COS cells, and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and motilin radioligand binding. Only those receptor segments that included key Cys residues in positions 25, 30, and 111 or perimembranous regions at the ends of the amino terminus and the first and third extracellular loops disrupted motilin biological activity. Each of these Cys deletions also disrupted action of erythromycin. Alanine replacements for each of the potentially important amino acid residues in the perimembranous segments revealed that residues Gly 36, Pro103, Leu109, and Phe332 were responsible for the selective negative impact on motilin biological activity, while responding normally to erythromycin. These results support the presence of functionally important disulfide bonds in the motilin receptor ectodomain and demonstrate that the structural determinants for binding and biological activity of peptide and non-peptidyl agonist ligands are distinct, with a broad extracellular perimembranous base contributing to normal motilin binding.

Original languageEnglish (US)
Pages (from-to)12390-12396
Number of pages7
JournalJournal of Biological Chemistry
Volume281
Issue number18
DOIs
StatePublished - May 5 2006

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Motilin
Erythromycin
Peptides
Bioactivity
Alanine
Ghrelin Receptor
Amino Acids
Mutagenesis
Ghrelin
COS Cells
G-Protein-Coupled Receptors
Disulfides
Pharmaceutical Preparations
Growth Hormone
Epitopes
Chemical activation
motilin receptor
Protein Domains
Hormones
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Differential contributions of motilin receptor extracellular domains for peptide and non-peptidyl agonist binding and activity. / Matsuura, Bunzo; Dong, Maoqing; Naik, Shruthi; Miller, Laurence J; Onji, Morikazu.

In: Journal of Biological Chemistry, Vol. 281, No. 18, 05.05.2006, p. 12390-12396.

Research output: Contribution to journalArticle

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