Binding domain characterization of growth hormone secretagogue receptor

[摘要] Background and Objectives: Activation of ghrelin receptor growth hormone secretagoguereceptor (GHS-R) by endogenous or synthetic ligands amplifies pulsatile release of growthhormone (GH) and enhances food intake, very relevant to development and growth. GHS-Ris a G-protein coupled receptor that has great druggable potential. Understanding the preciseligand and receptor interactions is crucial to advance the application of GHS-R. Materialsand Methods: We used radiolabeled ligand-binding assay and growth hormone releaseassay to assess the binding and functional characteristics of GHS-R to synthetic agonistsMK-0677 and GHS-25, as well as to endogenous peptide ligand ghrelin. We analyzed theligand-dependent activity of GHS-R by measuring aequorin-based [Ca++]i responses. Todefine a ligand-binding pocket of GHS-R, we generated a series of human/puffer fish GHS-Rchimeras 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 invitro competitive binding assay. Remarkably, the in vivo GH secretagogue activity is higherwith the synthetic agonists MK-0677 and GHS-25 than that of ghrelin. Importantly, the activitywas completely abolished in GHS-R knockout mice. In GHS-R chimera analysis, we identifiedthe C-terminal region, particularly the transmembrane domain 6 (TM6), to be critical for theligand-dependent activity. Our site-directed mutagenesis study further revealed that aminoacid residues D99 and W276 in GHS-R are essential for ligand binding. Interestingly, criticalresidues distinctively interact with different ligands, MK-0677 activation depends on E124,while ghrelin and GHS-25 preferentially interact with F279. Conclusion: The ligand-bindingpocket of human GHS-R is mainly defined by interactive residues in TM6 and the adjacentregion 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.

[关键词] ghrelin;growth hormone secretagogue;growth hormone secretagogue receptor;receptor binding