The chicken type III GnRH receptor homologue is predominantly expressed in the pituitary, and exhibits similar ligand selectivity to the type I receptor

Two GnRH isoforms (cGnRH-I and GnRH-II) and two GnRH receptor subtypes (cGnRH-R-I and cGnRH-R-III) occur in chickens. Differential roles for these molecules in regulating gonadotrophin secretion or other functions are unclear. To investigate this we cloned cGnRH-R-III from a broiler chicken and compared its structure, expression and pharmacological properties with cGnRH-R-I. The broiler cGnRH-R-III cDNA was 100% identical to the sequence reported in the red jungle fowl and white leghorn breed. Pituitary cGnRH-R-III mRNA was ∼1400-fold more abundant than cGnRH-R-I mRNA. Northern analysis indicated a single cGnRH-R-III transcript. A pronounced sex and age difference existed, with higher pituitary transcript levels in sexually mature females versus juvenile females. In contrast, higher expression levels occurred in juvenile males versus sexually mature males. Functional studies in COS-7 cells indicated that cGnRH-R-III has a higher binding affinity for GnRH-II than cGnRH-I (Kd: 0·57 vs 19·8 nM) with more potent stimulation of inositol phosphate production (ED50: 0·8 vs 4·38 nM). Similar results were found for cGnRH-R-I, (Kd: 0·51 vs 10·8 nM) and (ED50: 0·7 vs 2·8 nM). The initial rate of internalisation was faster for cGnRH-R-III than cGnRH-R-I (26 vs 15·8%/min). Effects of GnRH antagonists were compared at the two receptors. Antagonist #27 distinguished between cGnRH-R-I and cGnRH-R-III (IC50: 2·3 vs 351 nM). These results suggest that cGnRH-R-III is probably the major mediator of pituitary gonadotroph function, that antagonist #27 may allow delineation of receptor subtype function in vitro and in vivo and that tissue-specific recruitment of cGnRH-R isoforms has occurred during evolution

Evolution of vertebrate GnRH receptors from the perspective of a basal vertebrate

This minireview provides the current status on gonadotropin-releasing hormone receptors (GnRH-R) in vertebrates, from the perspective of a basal vertebrate, the sea lamprey, and provides an evolutionary scheme based on the recent advance of whole genome sequencing. In addition, we provide a perspective on the functional divergence and evolution of the receptors. In this review we use the phylogenetic classification of vertebrate GnRH receptors that groups them into three clusters: type I (mammalian and non-mammalian), type II, and type III GnRH receptors. New findings show that the sea lamprey has two type III-like GnRH receptors and an ancestral type GnRH receptor that is more closely related to the type II-like receptors. These two novel GnRH receptors along with lGnRH-R-1 share similar structural features and amino acid motifs common to other known gnathostome type II/III receptors. Recent data analyses of the lamprey genome provide strong evidence that 2 whole rounds of genome duplication (2R) occurred prior to the gnathostome-agnathan split. Based on our current knowledge, it is proposed that lGnRH-R-1 evolved from an ancestor of the type II receptor following a vertebrate-shared genome duplication and that the two type III receptors resulted from a duplication within lamprey of a gene derived from a lineage shared by many vertebrates

Molecular Cloning and Pharmacological Characterization of Two Novel GnRH Receptors in the Lamprey (Petromyzon marinus)

This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (IGnRH-R-2 and IGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands IGnRH-II and -III induced an inositol phosphate (IP) response at IGnRH-R-2 and IGnRH-R-3, whereas the ligand IGnRH-I did not stimulate an IP response. IGnRH-II was a more potent activator of lGnRH-R-3 than IGnRH-II. Stimulation of IGnRH-IIR-2 and IGnRH-R-3 testing all three IGnRH ligands did not elicit a cAMP response. IGnRH-R-2 has a higher binding affinity in response to IGnRH-R-2 than IGnRH-R-2, whereas IGnRH-R-3 has a higher binding affinity in response to IGnRH-R-2 than IGnRH-III. IGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that IGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and IGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate. (Endocrinology 153: 3345-3356, 2012

Virus Survival in Water and Wastewater Systems

Center for Water and the Environmen