The GHS-R sequence (Fig. 4) provides a context for determining the amino acid residues central to ligand binding and receptor activation. Based on current knowledge of the GHS-R and the predicted structures of the GHSs, several amino acid residues in TM 3, 5, and 6 are likely to be involved in ligand binding, mediating the agonist activity of GHRP-6 and the nonpeptide GHSs. An important feature of GHS agonists is the presence of a basic N+ (22-25). Based on conservation between swine and human GHS-R and the growing body of evidence implicating the transmembrane helices and extracellular loops 3 and 4 in ligand binding in other GPC-Rs, nine acidic sites in the GHS-R stand out as potential candidates for stabilizing the positive charge (26-28). A preliminary 3D model of the swine GHS-R suggests that E124 in H3 is disposed similarly to D113 of the p adrenergic receptor and an acidic amino acid residue in the equivalent position in the somatostatin type 2 (sst2) receptor. Other candidate sites are arrayed in the extracellular loops (D196,201,204; E194,207,212,291,299). Site-directed mutagenesis experiments will discriminate which negatively charged amino acid residues are important for GHS agonism.
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