Cloning and Characterization of a Functional Type II Gonadotropin-Releasing Hormone Receptor with a Lengthy Carboxy-Terminal Tail from an Ancestral Vertebrate, the Sea Lamprey

Abstract

A full-length transcript encoding a functional type II GnRH receptor was cloned from the pituitary of the sea lamprey, Petromyzon marinus. The current study is the first to identify a pituitary GnRH receptor transcript in an agnathan, which is the oldest vertebrate lineage. The cloned receptor retains the conserved structural features and amino acid motifs of other known GnRH receptors and notably includes a C-terminal intracellular tail of approximately 120 amino acids, the longest C-terminal tail of any vertebrate GnRH receptor identified to date. The lamprey GnRH receptor was shown to activate the inositol phosphate (IP) signaling system; stimulation with either lamprey GnRH-I or lamprey GnRH-III led to dose-dependent responses in transiently transfected COS7 cells. Furthermore, analyses of serially truncated lamprey GnRH receptor mutants indicate perturbations of the C-terminal tail disrupts IP accumulation, however, the tailless lamprey GnRH receptor was not only functional but was also capable of stimulating IP levels equal to wild type. Expression of the receptor transcript was demonstrated in the pituitary and testes using RT-PCR, whereas in situ hybridization showed expression and localization of the transcript in the proximal pars distalis of the pituitary. The phylogenetic placement and structural and functional features of this GnRH receptor suggest that it is representative of an ancestral GnRH receptor. In addition to having an important role in lamprey reproductive processes, the extensive C-terminal tail of this lamprey GnRH receptor may have great significance for understanding the evolutionary change of this vital structural feature within the GnRH receptor family.

Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus

“…A comparative analysis of GnRH receptors across the vertebrate lineage can provide significant insight into the molecular evolution of this receptor family. In this light, the GnRH receptor from the sea lamprey, Petromyzon marinus (Silver et al 2005), provides an ideal model to analyze basal, or ancestral-like functions and functional elements that are involved in ligand binding, signaling and internalization.…”

“…The GnRH receptor is unique among all GPCRs in that the type I mammalian GnRH receptors lack the highly conserved intracellular carboxy-terminal (C-terminal) tail, which has been shown to be a vital structural element required for several key functions, such as G-protein coupling and second messenger activation, ligand binding, cell surface expression and ligand-dependant internalization (Koenig & Edwardson 1997, Heding et al 1998, Blomenrohr et al 1999, Bockaert et al 2003, Ronacher et al 2004. Based simply on the presence or absence of a C-terminal tail, the GnRH receptors can be divided into two groups; the type I tail-less GnRH receptors, which have only been identified in mammals and the C-terminal tailcontaining type II GnRH receptors, which have been identified across the vertebrate lineage (Okubo et al 2001, Ikemoto et al 2004, Silver et al 2005.…”

“…The lamprey expresses two forms of GnRH, lamprey GnRH-I and lamprey GnRH-III, both of which are produced in the hypothalamus and have been shown to regulate the reproductive axis (Sherwood et al 1986, Sower et al 1993, Deragon & Sower 1994, Suzuki et al 2000, Silver et al 2004. Recently, a GnRH receptor cDNA was cloned from the sea lamprey, Petromyzon marinus; this contained a C-terminal tail of 120 amino acids (aa), the longest of any previously described GnRH receptor to date (Silver et al 2005). To better describe the lamprey GnRH receptor, a series of functional and pharmacological assays was performed in the current study, including examination of cAMP responses, analysis of receptor binding kinetics, whole cell competitive binding assays and internalization studies using a series of C-terminal tail truncations to better our understanding of the significance of certain regions of the lengthy C-terminal tail, as well as functional aspects of the receptor.…”

Contribution of Chicken GnRH-II and Lamprey GnRH-III on Gonadotropin Secretion

“…Although lGnRH-III is not a natural ligand of the type I or type II GnRHR, the lGnRH-III receptor shares different characteristics of both type I and type II GnRHRs (Silver et al, 2005;Silver and Sower, 2006). The presence of lGnRH-III (or a related analog) have been reported in brain extracts from humans, sheep, cows and rats (Dees et al, 1999;Hiney et al, 2002;Yahalom et al, 1999;Yu et al, 2000), suggesting a biological activity of this GnRH isoform in several species.…”

7TM Receptors

“…Receptors for all three ligands exist in amphibians but only GnRH I and GnRH II (and their cognate receptors) have been found in mammals (Sealfon et al, 1997; Millar, 2002). Type I and Type II GnRH receptors (provisonal nomenclature) have been cloned from numerous species (most of which express two or three types of GnRHR) and grouped phylogenetically (Silver et al, 2005). Type I GnRHRs are expressed primarily by pituitary gonadotrophs in mammals and mediate central control of reproduction.…”