Tilapia GnRH receptors: signal transduction and internalization rate
Ayelet Avitan & Berta Levavi-Sivan
Multiple subtypes of GnRH receptor (GnRHR) are present in individual vertebrate species. We found two distinct GnRHRs in tilapia, classified as type 1 and 3 (tGnRHR1/3). Amino-acid similarity between the receptors was calculated at 59%, with the different amino acids scattered throughout the receptors. We compared the sequence analysis and signal transduction of the two tGnRHRs, using the human GnRHR type 1 as a control. Sequence analysis revealed that all three receptors exhibit recognition motifs of Galpha q/11, while only tGnRHR3 and the hGnRHR1 revealed also, one recognition motif of Galpha s. We found that both tilapia receptors and the human receptor contain one PKA phosphorylation site. However, tGNRHR3 has five PKC phosphorylation sites whereas both tGnRHR1 and hGnRHR1 have only two sites. This diversity is further supported by the differential signal-transduction pathways: all three receptors activate the PKC pathway (as reflected by measurement of IPs accumulation), but only tGnRHR3 activates the PKA pathway (as reflected by activation of the reporter construct CRE-luciferase). All three receptors were also found to activate the phosphorylation of MAP kinase (ERK-1/2).
tGnRHR3 is highly expressed in the posterior part of the pituitary which contains LH and FSH cells. Hence, we characterized tGnRHR3 in terms of both LH release rate and receptor internalization rate in response to continuous exposure to GnRH.
Constant exposure of tilapia pituitary fragments to sGnRHa resulted in an increased secretion rate for 3 h, followed by a gradual decline to the basal secretion rate which lasted for 22 h. A chimera between tGnRHR3 and green florescence protein (GFP) was prepared and used to observe the changes in receptor distribution and translocation, activated by agonist with time. The receptor is initially localized at the plasma membrane and upon activation by sGnRHa undergoes relatively rapid endocytosis.