Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand receptor interaction.

In this study, two novel GHRHR receptor splice variants, named chicken GHRHR-v1 (cGHRHR-v1) and cGHRHR-v2 respectively, were identified from chicken pituitary using RT-PCR assay. cGHRHR-v1 is characterized by an N-terminal deletion of 36 amino acid residues, including an aspartate at position 56 (Asp(56)) conserved in G protein-coupled receptor B-I subfamily. cGHRHR-v2 is a carboxyl-terminal truncated receptor variant with four putative transmembrane domains, which arose from alternative use of a splice acceptor site on intron 8. Using the pGL3-CRE-luciferase reporter system, the functionality of the two variants was examined in Chinese hamster ovary cells. cGHRHR-v1 was shown to be capable of transmitting signal upon agonist stimulation, but cGHRHR-v2 could not. Both GHRH and pituitary adenylate cyclase-activating peptide (PACAP) could activate cGHRHR-v1 at high dosages (GHRH >/=10(-8) M; PACAP >/=10(-6) M) and GHRH was much more potent than PACAP, suggesting that cGHRHR-v1 is a functional membrane-spanning receptor with an impairment in high-affinity ligand binding, rather than in receptor activation and ligand-binding specificity. This finding also points out the possibility that Asp(56) is not a critical determinant for receptor activation and direct ligand-receptor interaction. To substantiate this hypothesis, using site-directed mutagenesis, two receptor mutants with replacement of Asp(56) by Ala or Gly were generated. Expectedly, chicken or human GHRH could still activate both receptor mutants with reduced potencies (about 2- to 14-fold less potent). Taken together, our findings not only suggest that cGHRHR variants may play a role in controlling normal pituitary functions, but also support that Asp(56) is nonessential for receptor activation and direct ligand-receptor interaction.

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