Evolution of Constrained Gonadotropin-releasing Hormone Ligand Conformation and Receptor Selectivity

Abstract

Gonadotropin-releasing hormone (GnRH) is the central regulator of reproduction in vertebrates. GnRHs have recently been identified in protochordates and retain the conserved N-and C-terminal domains involved in receptor binding and activation. GnRHs of the jawed vertebrates have a central achiral amino acid (glycine) that favors a type II ␤-turn such that the N-and C-terminal domains are closely apposed in binding the GnRH receptor. However, protochordate GnRHs have a chiral amino acid in this position, suggesting that they bind their receptors in a more extended form. We demonstrate here that a protochordate GnRH receptor does not distinguish GnRHs with achiral or chiral amino acids, whereas GnRH receptors of jawed vertebrates are highly selective for GnRHs with the central achiral glycine. The poor activity of the protochordate GnRH was increased >10-fold at vertebrate receptors by replacement of the chiral amino acid with glycine or a D-amino acid, which favor the type II ␤-turn. Structural analysis of the GnRHs using ion mobility-mass spectrometry and molecular modeling showed a greater propensity for a type II ␤-turn in GnRHs with glycine or a D-amino acid, which correlates with binding affinity at vertebrate receptors. These findings indicate that the substitution of glycine for a chiral amino acid in GnRH during evolution allows a more constrained conformation for receptor binding and that this subtle single amino acid substitution in a site remote from the ligand functional domains has marked effects on its structure and activity.In vertebrates, gonadotropin-releasing hormone (GnRH) 2 is synthesized in hypothalamic neurones and conducted a few millimeters in the hypophysial portal system to the anterior pituitary, where it binds to high affinity receptors in gonadotrophs to stimulate the release of gonadotropins (1). The gonadotropins in turn stimulate hormone and gamete production by the testes and ovaries. GnRHs have also been isolated from protochordate species (2, 3) and are thought to be secreted from neurones to directly regulate the gonads (2-4) in these representatives of vertebrate progenitors. GnRHs and GnRH receptors have also been found to directly affect vertebrate gonadal function (5), possibly reflecting the earliest role of GnRH as exemplified in protochordates (6, 7). It appears, therefore, that GnRHs have an ancient evolutionary role as regulators of reproduction, first through direct neural delivery to the gonads and later as hypothalamic neuroendocrine regulators of the gonads indirectly through gonadal stimulation by gonadotropins.To date 13 structural variants of the GnRH decapeptide have been identified in vertebrates (8), 9 from protochordates, which are vertebrate progenitors (8, 9), and a 12-amino acid homolog from an octopus species (10) (Fig. 1). All of the GnRHs are characterized by the conservation of the N-terminal residues (pGlu-His-Trp-Ser) and the C-terminal residues (Pro-Gly-NH 2 ) with the exception of two conservative substitutions (Fig. 1). In cartilaginous and bon...

Diversity of actions of GnRHs mediated by ligand-induced selective signaling

“…Structural analysis of the GnRHs using a combination of ion-mobility mass spectrometry and molecular modeling revealed a close correlation between the propensity for the formation of the βII′-type turn conformation (e.g. mammalian GnRH and a Gly 6 -substituted sea squirt GnRH) and higher binding affinity at vertebrate receptors [9] (Fig. 5).…”

“…4) [9]. In contrast, protochordate GnRH receptors from sea squirts (tunicates) did not distinguish between the GnRHs with different amino acid residues in this position suggesting that the peptide is able to interact with the receptor binding sites in a more extended form and that evolution of receptors requiring interaction with GnRH in the folded conformation only arose after the evolution of jawed vertebrates.…”

“…Thirteen structural variants of GnRH have now been identified from all the vertebrate classes, nine from protochordates and one from a species of octopus, see Refs. [2,9,95,129]. These diverse structures represent "experiments of nature" over some 600 million years and provide insight into conserved and functionally important residues.…”

“…For example, mammalian GnRH is widely conserved in amphibians and primitive bony fish, and chicken GnRH II is present in most vertebrate species, including man. An octopus GnRH and an additional Ciona GnRH comprising 12 and 16 amino acids, respectively, but retaining the conserved amino-and carboxyl-terminal domains are not shown (Reproduced from [9]). The NMR structure of GnRH I (PDB Code: 1YY1).…”

Current and future applications of GnRH, kisspeptin and neurokinin B analogues

“…Gly increases the binding affinity of these GnRHs at the mammalian receptor 36 . If Gly 6 is substituted with a D-amino acid the β-II" confirmation is enhanced and binding affinity is increased 37 .…”