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  • Ghrelin contains an n octanoylation


    Ghrelin contains an n-octanoylation at its Ser3 residue (Kojima et al., 1999). While des-acyl ghrelin is found in high concentrations in the blood, only the lipidated form is active at GHSR (Bednarek et al., 2000). We previously showed that ghrelin interacts with the membrane via its lipid modification (Vortmeier et al., 2015). Further, membrane-bound ghrelin was found to possess a small α-helical core and disordered termini in Neurotensin to free ghrelin, which displays no helical character. This demonstrated that the environment was crucial to the structure of ghrelin and suggested that the receptor-bound state may yet differ further. Peptide-binding GPCRs comprise a large subset of the GPCR family and structural knowledge is growing (Wu et al., 2017). However, only a few of these structures were determined with their respective peptide bound. These include the receptors for neurotensin (White et al., 2012), endothelin (Shihoya et al., 2016), opioid peptides (Fenalti et al., 2015), C5a (Liu et al., 2018), apelin (Ma et al., 2017), and chemokines (Burg et al., 2015, Qin et al., 2015). Nuclear magnetic resonance (NMR) has added structural and dynamic studies of peptides binding their receptors including bradykinin (Joedicke et al., 2018, Lopez et al., 2008), NPY (Kaiser et al., 2015, Yang et al., 2018), and dynorphin (O'Connor et al., 2015). An emerging theme in these studies is the large binding surface areas of peptide ligands compared with small-molecule ligands over two sites. One site correlates with the canonical orthosteric site of rhodopsin-like GPCRs, while an additional site is located distally in the N terminus, as in chemokine receptors (Burg et al., 2015, Qin et al., 2015), or extracellular loop 2 (ECL2), as in NPY type 2 receptor (Kaiser et al., 2015). Initial studies of ghrelin found that the N-terminus of ghrelin including the lipid-modified Ser3 residue was critical for receptor binding, and the first five residues were suggested to represent the minimal binding motif (Bednarek et al., 2000). However, this short peptide possessed a binding affinity two orders of magnitude lower than full-length ghrelin and could not activate GHSR in isolated membranes (Torsello et al., 2002). This suggested a role for residues outside of the first five amino acids. We report here an extension of this binding motif to at least nine residues at a site in GHSR distinct from the orthosteric binding pocket. We measured peptide secondary structure and receptor-proximal residues using isotopically labeled ghrelin in complex with GHSR. We incorporated this data into a modeling method that built the ligand-receptor complex while accounting for high flexibility. Resulting models were filtered against the experimental NMR data and were compared against existing and novel mutational analysis. The final ensemble of models identifies an extended binding pocket from the central transmembrane (TM) bundle out to GHSR's extracellular loops.
    Introduction Prior to the discovery of its endogenous ligand, ghrelin receptor was first identified as growth hormone secretagogue-receptor (GHS-R), the endogenous receptor for the artificial GH-releasing peptide (GHRP), in pigs and humans (Howard et al., 1996). In fact, two GHS-R molecules of varying length were identified, GHS-R1a and GHS-R1b. GHS-R1a is a functional G-protein coupled receptor (GPCR) having seven transmembrane domains (TMDs) and induced by agonist-dependent intracellular Ca2+. GHS-R1b is an alternatively spliced variant, consisting of TMDs 1–5, identical to its GHS-R1a, and terminates in a portion of the connected intron. As such, GHS-R1b does not induce Ca2+ signaling (Howard et al., 1996; Kojima et al., 1999). According to fundamental structural features, the GHS-Rs belong to a family of paralogous receptors including motilin, neuromedin U, neurotensin, and GPR39 (Kaiya et al., 2014), each having roles in gastrointestinal physiology (Kojima and Kangawa, 2005).