The Kiss/Gpr54 system is considered to be an essential regulator of the reproductive axis in most vertebrate species. The existence of paralogous kisspeptin genes as well as kisspeptin receptor genes has quite recently been reported in several fish and amphibian species. Thus, the fishes may provide excellent animal models for the study of general principles underlying the kisspeptin and gpr54 receptor systems of vertebrates. The pharmacological profile of sea bass gpr54-1b and gpr54-2b was examined to characterize the ligand-receptor interactions and the signaling pathways of two gpr54 in sea bass (Dicentrarchus labrax). For this aim, the open reading frames of sea bass gpr54-1b and gpr54-2b were subcloned in the expression vector pcDNA3 and these plasmids were transiently transfected into Chinese hamster ovary cells in combination with pCRE-luc or pSRE-luc reporter vectors. In these reporter vectors the luciferase gene (luc) is transcriptionally regulated by a cyclic AMP response element (CRE) or by a serum response element (SRE), allowing to differentiate between protein kinase C and protein kinase A signal transduction pathways. We tested graded concentrations (10-5 to 10-10M) of sea bass kiss1 and kiss2 decapeptides as well as longer kisspeptins (kiss1-15 and kiss2-12) on the double transfected cells. The recorded luciferase activities showed that sea bass gpr54-1b exhibits high sensitivity toward kiss1-derived peptides, with a preferential binding to the kiss1-15. Activation of this receptor generated both SRE and CRE specific signalling, although the stimulation of the CRE pathway was more effective. On the other hand, a different ligand-receptor interaction is observed for the sea bass gpr54-2b that clearly appears to discriminate between long and short kisspeptin forms, showing higher potency and affinity for longer ones. It can be concluded that this receptor is activated by the two ligands, however it exhibits preferential binding for the kiss2-12. In this case, activation of SRE driven luciferase was slightly higher than CRE-luc. All together these data show distinct binding preferences of these receptors for sea bass kisspeptins and different activation levels of the studied signaling pathways.

Supported by GV (PROMETEO/2010/003 to S.Z.), EU (LIFECYCLE, FP7222719 to S.Z.». F.E. was supported by a JAE Predoctoral fellowship (CSIC).

Poster presentado en el 8º Congreso de la Asociación Ibérica de Endocrinología Comparada celebrado en Madrid del 5 al 7 septiembre 2011

Peer Reviewed