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 the growth hormone secretagogue receptors. Together, these represent a new Class 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 photoaffinity labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional motilin receptor (CHO-MtlR; 175,000 sites per cell, with K_i = 2.3 ± 0.4 nM motilin and EC₅₀ = 0.3 ± 0.1 nM motilin) and a radioiodinatable peptide analogue of human motilin that incorporated a photolabile p-benzoyl-L-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, [Bpa¹,Ile ¹³]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentration-dependent manner (EC ₅₀ 1.5 ± 0.4 nM). This photolabile ligand bound specifically and with high affinity to the motilin receptor (K_i = 12.4 ± 1.0 nM), and covalently labeled that molecule within its Mr = 45,000 deglycosylated 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 representing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains support their functional importance as well.