Binding Domain Characterization of Growth Hormone Secretagogue Receptor.
This study investigated how synthetic and endogenous ligands bind to and activate the growth hormone secretagogue receptor (GHS-R), a G-protein coupled receptor involved in growth hormone (GH) release and appetite regulation. Using radiolabeled ligand-binding assays, calcium-response (aequorin-based) assays, GH release assays in mice, receptor chimeras (human/puffer fish domain swaps), and site-directed mutagenesis, researchers characterized the structural basis of ligand-receptor interactions. The study found that synthetic agonists MK-0677 and GHS-25 displayed high binding affinity and, notably, greater in vivo GH secretagogue activity compared to the endogenous peptide ghrelin. GHS-R knockout mice showed complete abolition of activity, confirming receptor specificity. Chimera analysis identified the C-terminal region, particularly transmembrane domain 6 (TM6), as critical for ligand-dependent activation. Site-directed mutagenesis pinpointed residues D99 and W276 as essential for ligand binding, while E124 was selectively important for MK-0677, and F279 was preferentially involved in ghrelin and GHS-25 interactions. The study is primarily mechanistic and largely preclinical (in vitro and animal models), limiting direct translation to human therapeutic contexts. Its findings advance structural understanding of GHS-R and may inform future drug design efforts.
Why this grade: The study combines in vitro binding/functional assays with in vivo mouse experiments (including knockout models), but includes no human subjects, limiting its direct clinical applicability.
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.
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