Identification of peptide ligand-binding domains within the human motilin receptor using photoaffinity labeling

The cDNA encoding the human motilin receptor was recently cloned and found to represent a G protein-coupled receptor that is structurally related to t he growth hormone secretagogue receptors. Together, these represent a new C lass I receptor family. Our aim in the present work is to gain insight into the molecular basis of binding of motilin to its receptor using photoaffin ity labeling. To achieve this, we developed a Chinese hamster ovary cell li ne that overexpressed functional motilin receptor (CHO-MtIR; 175,000 sites per cell, with K-i = 2.3 +/- 0.4 nM motilin and EC50 = 0.3 +/- 0.1 nM motil in) and a radioiodinatable peptide analogue of human motilin that incorpora ted a photolabile p-benzoyl-L-phenyl-alanine (Bpa) residue into its pharmac ophoric domain. This probe, [Bpa(1),Ile(13)]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentratio n-dependent manner (EC50 = 1.5 0.4 nm). This photolabile ligand bound speci fically and with high affinity to the motilin receptor (K-i = 12.4 +/- 1.0 nM), and covalently labeled that molecule within its M-r = 45,000 deglycosy lated core. Cyanogen bromide cleavage demonstrated its covalent attachment to fragments of the receptor having apparent M-r = 6,000 and M-r = 31,000. These were demonstrated to represent fragments that included both the first and the large second extracellular loop domains, with the latter represent ing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains suppor t their functional importance as well.