Characterization of a third ghrelin receptor, GHS-R3a, in channel catfish reveals novel expression patterns and a high affinity for homologous ligand

A novel third channel catfish growth hormone secretagogue (ghrelin) receptor, GHS-R3a, gene was characterized. Identification and analysis of the genomic organization of channel catfish GHS-R3a revealed differences in exon/intron structure relative to the previously published GHS-R1a and GHS-R2a sequences. Amino acid sequence alignment of catfish GHS-R3a with -R1a and -R2a revealed 48 and 52% sequence identity, respectively. Phylogenetic analysis predicted a new clade of GHS-R3a receptors found only in fish, with representation in the teleost infradivisions Osteoglossomorpha, Clupeomorpha, and Euteleostei. In functional analyses, homologous catfish ghrelin increased intracellular Ca2+ concentration in human embryonic kidney (HEK) 293 cells stably expressing catfish GHS-R3a. On the contrary, intracellular Ca2+ concentration was unaffected by treatment with the synthetic growth hormone secretagogues GHRP-6 and hexarelin. Realtime PCR results indicated high expression of GHS-R3a in the brain and gonads, demonstrating tissue specificity among the catfish GHS-Rs. The effects of fasting and refeeding on all three ghrelin receptors were evaluated in catfish brain, pituitary, stomach, and Brockmann bodies. Most notably, GHS-R3a was the only receptor observed to significantly increase (2.9-6.3-fold) in brain, pituitary, and stomach within 4 days of fasting (P