Received for publication December 2, 2004.
Revised January 17, 2005.
Accepted for publication January 25, 2005.

Demonstration of a specific site of covalent labeling of the human motilin receptor using a biologically active photolabile motilin analogue

Abstract

The motilin receptor belongs to a group of Class I G protein-coupled receptors that also includes growth hormone secretagogue and ghrelin receptors. These represent clinically useful targets for pharmacotherapy. Their potentially unique structures and the molecular basis of their binding are not yet clear. We previously reported the initial affinity labeling of a region within this receptor (cyanogen bromide fragment extending from the first to the second extracellular loop) using a position one photolabile motilin analogue. To extend our understanding of the molecular basis of motilin binding, we have developed an additional radioiodinatable motilin analogue probe having site of covalent attachment in position five. This was a full agonist that bound to the motilin receptor specifically and with high affinity, and that efficiently established a single covalent bond to its receptor. Sequential chemical and enzymatic cleavage of labeled wild type and mutant motilin receptor constructs established that the region of labeling was within the third extracellular loop. This was further localized to Phe³³² using radiochemical Edman degradation sequencing. These data provide the first spatial approximation constraint that can be utilized in the docking of this peptide ligand to its receptor. We hope that a series of such constraints can be determined to provide adequate structural information to begin to elucidate the conformation of this agonist-bound receptor, and to ultimately be useful in the rational design of drugs acting at this important target.

Key words: G protein-coupled receptor, ligand binding, motilin, motlin receptor, photoaffinity labeling, receptor conformation

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