Purification of two types of gonadotropin receptors from carp ovarian follicles: overlapping recognition by two different ligands

Abstract

Teleostean gonadotropin receptors were solubilized from the plasma membrane preparation of carp ovarian follicles by lithium diiodosalicylate and Triton X-100. Solubilization resulted fourfold increase in GTH binding activity as compared to the crude plasma membrane preparation. An addition of 25% glycerol and protease inhibitors to the solubilized receptor retained more than 90% original activity at -20 οC for 30 days. Gel filtration through Sephadex G 100 significantly increased the specific binding capacity from 70 fmol/mg protein (soluble receptor) to 250 fmol/mg protein. Peak I of gel filtration showing the receptor activity was further purified by affinity chromatography on purified salmon GTH II - Sepharose with a remarkable increase in specific binding capacity from 250 fmol/mg protein (gel filtration peak) to 2300 pmol/mg protein for salmon GTH I ligand and 2800 pmol/mg protein for GTH II ligand. In SDS-PAGE affinity eluate the active peak showed two distinct bands corresponding to 66 and 62 kDa molecular masses. These two proteins were clearly separated in FPLC Mono S chromatography, 62 kDa as GTHR I and 66 kDa as GTHR II. The former preferably binds to GTH I ligand, while the latter to GTH II, although both demonstrated overlapping recognition to both the ligands. GTH receptor protein I (GTHR I) and II (GTHR II) were purified 42,000- and 54,000-fold, respectively. Competitive binding inhibition studies indicate GTH I and ovine FSH a better ligand for GTHR I, while GTH II and ovine LH were preferable ligands for GTHR II. Biological relevance of these two receptor proteins was ascertained by monitoring the specific binding capacity of GTHR I and II at different stages of the annual reproductive cycle. GTHR I-GTH I was a prevailing complex during preparatory and pre-spawning stages, while GTHR II-GTH II was the dominant one at the maturational and final maturational stages. It may be concluded there are two GTH receptor proteins, each having a preferred ligand. The overlapping ligand-binding activity and profile of each receptor ligand complex suggest their link to seasonal development and maturation of carp ovarian follicle.