
<ArticleTitle>Identification of peptide ligand-binding domains within the human <prot><prot>motilin</prot> receptor</prot> using photoaffinity labeling.</ArticleTitle>
<AbstractText>The cDNA encoding the human <prot><prot>motilin</prot> receptor</prot> was recently cloned and found to represent a G protein-coupled receptor that is structurally related to the <prot>growth hormone secretagogue receptor</prot>s. 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 <prot>motilin</prot> to its receptor using photoaffinity labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional <prot><prot>motilin</prot> receptor</prot> (CHO-MtlR; 175,000 sites per cell, with K(i) = 2.3 +/- 0.4 nm motilin and EC(50) = 0.3 +/- 0.1 nm motilin) and a radioiodinatable peptide analogue of human <prot>motilin</prot> that incorporated a photolabile p-benzoyl-l-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, <prot>[Bpa(1),Ile(13)]motilin</prot>, 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 <prot><prot>motilin</prot> receptor</prot> (K(i) = 12.4 +/- 1.0 nm), and covalently labeled that molecule within its M(r) = 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 <prot>motilin</prot> with these receptor domains support their functional importance as well.</AbstractText>
