Endocrine Abstracts

Androgens mediate their physiological functions via the classical nuclear receptor, AR. However, recent studies have identified a new plasma membrane (and non-genomic) androgen receptor- Zn(II) channel, termed ZIP9. Androgen activation of ZIP9 has been proposed to activate ‘non-classical’ testosterone signalling pathways, with ZIP9 coupling to G-proteins. However, how androgens and zinc interplay to modulate G protein activation remains unclear. This study therefore aimed to determine how androgens and zinc regulate ZIP9-dependent G protein coupling. HEK293 cells expressing ZIP9 were utilised to assess ZIP9-dependent cAMP production using a live kinetic cAMP reporter- GloSensor. Cells were pre-treated with 10uM forskolin for 10 minutes, then treated -/+ 100nM testosterone, 20uM zinc or testosterone/zinc co-treatment. cAMP production was monitored for up to 30 minutes. Experiments were conducted with a minimum of n = 3 in triplicate measurements. Treatment of HEK293 cells expressing ZIP9 with either testosterone or zinc alone, or in combination had no effect on cAMP production. However, pre-treatment with forskolin modulated the basal activity and Gi-coupling of ZIP9, with dose-dependent effects observed on the level of inhibitory activity dependent on ZIP9 plasmid concentration transfected. Western blot analysis showed that plasmid concentration correlated with the amount of ZIP9 expressed. At high Zip9 plasmid concentration and expression, treatment with either testosterone, zinc or a combination of both, abrogated cAMP production, suggesting ligand-gated Zip9-Gi-activation. Interestingly, at low plasmid concentration and low expression, a single treatment with zinc or testosterone did not affect basal ZIP9-Gi coupling. However, combined testosterone and zinc treatment enhanced cAMP production, suggesting a potential switch to Gs coupling. These data indicate that ZIP9 displays dual Gs/Gi coupling that is dependent on its expression level and ligand concentrations. This may have important implications in the regulation of reproductive disorders with high androgens including PCOS, with the ovarian roles important next steps to explore.