Abstract
BACKGROUND AND OBJECTIVES: Activation of ghrelin receptor growth hormone secretagogue receptor (GHS-R) by endogenous or synthetic ligands amplifies pulsatile release of growth hormone (GH) and enhances food intake, very relevant to development and growth. GHS-R is a G-protein coupled receptor that has great druggable potential. Understanding the precise ligand and receptor interactions is crucial to advance the application of GHS-R. MATERIALS AND METHODS: We used radiolabeled ligand-binding assay and growth hormone release assay to assess the binding and functional characteristics of GHS-R to synthetic agonists MK-0677 and GHS-25, as well as to endogenous peptide ligand ghrelin. We analyzed the ligand-dependent activity of GHS-R by measuring aequorin-based [Ca(++)](i) responses. To define a ligand-binding pocket of GHS-R, we generated a series of human/puffer fish GHS-R chimeras by domain swapping, as well as a series of mutants by site-directed mutagenesis. RESULTS: We found that the synthetic ligands have high binding affinity to GHS-R in the in vitro competitive binding assay. Remarkably, the in vivo GH secretagogue activity is higher with the synthetic agonists MK-0677 and GHS-25 than that of ghrelin. Importantly, the activity was completely abolished in GHS-R knockout mice. In GHS-R chimera analysis, we identified the C-terminal region, particularly the transmembrane domain 6 (TM6), to be critical for the ligand-dependent activity. Our site-directed mutagenesis study further revealed that amino acid residues D99 and W276 in GHS-R are essential for ligand binding. Interestingly, critical residues distinctively interact with different ligands, MK-0677 activation depends on E124, while ghrelin and GHS-25 preferentially interact with F279. CONCLUSION: The ligand-binding pocket of human GHS-R is mainly defined by interactive residues in TM6 and the adjacent region of the receptor. This novel finding in GHS-R binding domains advances the structural/ functional understanding of GHS-R, which will help to select/design better GHS-R agonists/ antagonists for future therapeutic applications.