Identification of Binding Domains of the Growth Hormone-Releasing Hormone Receptor by Analysis of Mutant and Chimeric Receptor Proteins

The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pi-tuitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for inter-action with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH re-ceptors. Two truncated receptors, GHRHDN, in which part of the N-terminal domain between the putative signal sequence and the first transmem-brane domain was deleted, and GHRHDC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were de-ficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associ-ated extracellular loops (C terminus) are alone suf-ficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between recep-tors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumu-lation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH. (Mo-lecular Endocrinology 12: 750–765, 1998