Binding Domain Characterization of Growth Hormone Secretagogue Receptor

Yuxiang Sun; Xiangcang Ye; Hilda Kennedy; Alexander G A Smith; Roy G Smith

doi:10.2478/jtim-2022-0033

Binding Domain Characterization of Growth Hormone Secretagogue Receptor

J Transl Int Med. 2022 Jul 2;10(2):146-155. doi: 10.2478/jtim-2022-0033. eCollection 2022 Jun.

Authors

Yuxiang Sun^{1

2}, Xiangcang Ye¹, Hilda Kennedy², Alexander G A Smith², Roy G Smith^{2

3}

Affiliations

¹ Department of Nutrition, Texas A&M University, College Station 77843, TX, USA.
² Huffington Center on Aging, Baylor College of Medicine, Houston 77030, TX, USA.
³ Department of Molecular Medicine, The Scripps Research Institute, La Jolla 920337, CA, USA.

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.

Keywords: ghrelin; growth hormone secretagogue; growth hormone secretagogue receptor; receptor binding.