Ligand Activation Domain of Human Orphan Growth Hormone (GH) Secretagogue Receptor (GHS-R) Conserved from Pufferfish to Humans

Abstract

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.

Pituitary Growth Hormone Secretion in the Turbot, a Phylogenetically Recent Teleost, Is Regulated by a Species-Specific Pattern of Neuropeptides

“…Geris et al [88]reported in chicken, using a perifusion system, that the GH secretagogue L-692, 249 had only 20–25% the in vitro potency of the endogenous releasing factors GHRH and TRH. Recent cloning of a gene in pufferfish (Spheroides nephelus) which shares significant identity to the human GHRP receptor [89]suggests that GHRP may also play an important role in teleosts. Shepherd et al [90]have shown that in tilapia GHRP-2 stimulated GH release in vivo with the same potency as GHRH.…”

Ghrelin: a hormone regulating food intake and energy homeostasis

“…However, although these peptides show high receptor affinity, they are unable to stimulate GH secretion from somatotroph cells (Ghigo et al 2001). This sequence of seven amino acids has been universally conserved among different species from fish and reptiles to birds and mammals (Palyha et al 2000), which suggests that ghrelin has an important physiological significance.…”

Bullfrog Ghrelin Is Modified by n-Octanoic Acid at Its Third Threonine Residue

“…In previous functional studies of the GHS-R in non-mammalian species, artificial ligands such as GHRPs and nonpeptidyl GHSs have been utilized. A GHS-R homologue identified in the pufferfish shares 58% identity to human GHS-R and is activated by GHRP-6 and nonpeptidyl GHSs (17), suggesting that GHS binding to the GHS-R is not species-specific. These GHS species, however, bind relatively weakly to pufferfish, compared with human GHS-R, possibly due to structural differences between the two receptors.…”