Endocrine Abstracts

Polycystic ovary syndrome (PCOS) is a multifactorial, complex endocrine disorder affecting a significant proportion of the global population. Aberrant secretion and/or action of gonadotropins is implicated in PCOS, but, to date, we have only limited knowledge about the precise mechanisms involved. We therefore studied LHR expression, signalling and trafficking in GLCs from women with and without PCOS. GLCs from women with PCOS exhibited enhanced Gαs-cAMP signalling response to LH, without changes in receptor gene expression. Dose response studies revealed increased potency and efficacy of LH-induced cAMP in PCOS GLCs, indicating increased LHR sensitivity. We have previously demonstrated that LHR activation of cAMP signalling in HEK 293 cells requires receptor internalization to very early endosomes (VEEs), where Gs-cAMP signalling and recycling are tightly regulated by the adaptor protein APPL1. We have now shown that LH-mediated cAMP signalling in control GLC cultures is regulated by APPL1 in a similar manner and induces an LH-dependent increase in CYP19 expression. However, in PCOS GLCs, inhibition of APPL1 dramatically reduces, rather than enhancing, LH-mediated cAMP levels, suggesting that APPL1 is driving this enhanced LHR activity, via altered phosphorylation of APPL1. Furthermore, pretreatment with insulin of GLCs from non-PCOS women mimics the ‘switch’ in APPL1-dependent regulation of LH-mediated cAMP signalling, while in PCOS samples, GLCs are resistant to insulin stimulation. We propose that insulin receptor (InsR), known to also be regulated by APPL1, underlies the enhanced APPL1-dependent cAMP signaling. To investigate this further, APPL1 was immunoprecipitated from control GLCs, indicating LH-mediated increase in PKA-dependent phosphorylation of APPL1 at serine 410. In summary, we have shown that LHR activity is tightly controlled at a spatial level, and we suggest that alterations in serine/threonine and tyrosine phosphorylation of APPL1 accounts for the aberrant cAMP response to LH in PCOS GLCs.