Inositol phosphate stimulation by LH requires the entire α Asn 56 oligosaccharide

Abstract

Lentil lectin-bound, fucose-enriched hTSH was reported to stimulate both cAMP and inositol phosphate (IP) intracellular signalling pathways, whereas fucose-depleted hTSH stimulated only the cAMP pathway. Gonadotropins activate the cAMP pathway and in several studies higher concentrations activate the IP pathway. Since only the 10% of α subunit Asn 56 oligosaccharides (Asn 52 in humans) are fucosylated, the higher glycoprotein hormone concentrations required for IP pathway activation might be related to the abundance of competent hormone isoforms. Lentil lectin-fractionated equine (e)LHα and eFSHα preparations were combined with a truncated, des(121–149)eLHβ preparation. All four hybrid hormone preparations induced IP accumulation in porcine theca cells, suggesting that activation of the IP pathway was not dependent on fucosylation at α subunit Asn 56. However, the presence of Asn 56 carbohydrate was necessary for increased IP accumulation. Intact, rather than Asn 56-deglycosylated eLH preparations provoked a biphasic steroidogenic response by rat testis Leydig cells, suggesting that Gα i stimulation was also sensitive to loss of Asn 56 carbohydrate. While rat granulosa cells responded to human FSH preparations in a biphasic manner, a classical sigmoidal response was obtained to eFSH and Asn 56-deglycosylated eFSH, suggesting that the equine preparations did not activate Gα i. Purified oLHα Asn 56 oligosaccharides inhibited FSH-stimulated steroidogenesis in rat granulosa cell cultures indicating a direct role for carbohydrate in FSH action. The same carbohydrate preparation inhibited hCG-stimulated fluorescence energy transfer suggesting oligosaccharide involvement in activated LH receptor self-association.