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    • br G protein activation through

    2024-03-30


    G protein activation through croaker ZIP9 An essential criterion for designating a steroid binding protein as a membrane steroid receptor is to demonstrate that it can transduce steroid signals in order to elicit a cellular response. Treatment with 100nMT caused G protein activation in croaker ZIP9-transfected BTB06584 australia but not in untransfected cells, as measured by increased binding of [35S]-GTPγS to plasma membrane fractions. GTPγS is a nonhydrolyzable form of GTP that replaces GDP on α subunits of G proteins when they are activated. The finding that the T-induced increase in [35S]-GTPγS binding was immunoprecipitated by a specific antibody to the stimulatory G protein α subunit (Gs), but not by an antibody directed against the inhibitory G protein α subunit (Gi), indicates that croaker ZIP9 activates a stimulatory G protein (A). A close association of ZIP9 with the Gs protein was confirmed in co-immunoprecipitation experiments. Consistent with activation of a stimulatory G protein, T treatment increased cAMP levels in croaker ZIP9-transfected cells. Ligand binding to membrane receptors is decreased when they are uncoupled from their G proteins by pharmacological agents such as GTPγS. Pretreatment with GTPγS caused a marked decrease in [3H]-T binding to croaker ZIP9-transfected cell membranes, providing further evidence that ZIP9 is directly coupled to a G protein. To date, ZIP9 is the only member of the ZIP family that has been shown to signal through G proteins.
    Regulation of intracellular free zinc levels and apoptosis through croaker ZIP9 ZIP9 would be expected to have the same zinc transport functions to increase free zinc levels in the cytoplasm as other members of the ZIP family. However, ZIP9 zinc transport activity differs from that of other ZIPs in that it is directly regulated by T in both croaker ZIP9-transfected cells and also in co-cultured G/T cells. Moreover, the zinc response to T was lost after knockdown of croaker ZIP9 expression in G/T cells, suggesting that this response solely mediated by ZIP9 (Berg et al., 2014). Often the most difficult criterion to meet in evaluating a novel protein as a hormone receptor is to demonstrate it mediates a biologically plausible function. T (20–100nM) caused a concentration-dependent increase in cell death of croaker ZIP9-transfected cells that was not mimicked by the nAR agonists mibolerone and R1881, or by estradiol-17β or cortisol (Berg et al., 2014). DNA fragmentation analysis showed the cell death was associated with apoptosis which showed the same T specificity. Interestingly, T induction of apoptosis in croaker ZIP9-transfected cells was blocked by treatment with the membrane-permeable zinc chelator, TPEN, indicating that the apoptotic response to T is mediated, at least partly, BTB06584 australia through T-induced increases in cytosolic zinc concentrations. Although the zinc signaling pathway mediating apoptosis in croaker ZIP9-transfected cells has not been identified, studies with human ZIP9 in cancer cells indicate it is dependent on G protein activation (Thomas et al., 2017) and results in upregulation of proapoptotic genes (Bax, p53, and JNK) which are discussed in Section 9 (Thomas et al., 2014). Consistent with these findings, knockdown of ZIP9 in G/T cells results in loss of [3H]-T binding and is accompanied with a complete loss of the apoptotic and zinc responses to T (B, C, D). On the other hand, the apoptotic response to T was not impaired after knockdown of the croaker nAR. Taken together, these results demonstrate that croaker ZIP9 is the intermediary in T-induced apoptosis and that it is dependent upon an increase in intracellular zinc.
    Hormonal regulation of ZIP9 in croaker ovaries Ubiquitous characteristics of steroid hormones receptors, including novel membrane receptors, are that their expression and activity are hormonally regulated (Pang and Thomas, 2010, Zhu et al., 2003). ZIP9 is no exception because croaker ZIP9 expression and [3H]-T binding in ovarian tissues were shown to be upregulated 4 to 6-fold after four hours in vitro treatment with a gonadotropin (human chorionic gonadotropin, hCG). Treatments with testosterone and estradiol-17β also increase ZIP9 expression and [3H]-T binding (), which suggests the action of gonadotropin may be indirect through stimulation of steroidogenesis. The fact that ovarian ZIP9 expression and T binding are upregulated by ovarian steroids suggests that ZIP9 receptor activity may change during the ovarian cycle and regulate ovarian functions in a follicle stage-dependent manner. Knowledge of ovarian follicle stage-dependent changes in ZIP9 expression should indicate some potential functions of the receptor in croaker ovaries, in particular its involvement in upregulation of apoptosis. Extensive remodeling of the ovaries occurs post-ovulation in teleost species such as croaker that spawn many thousands of eggs and also during atresia induced by environmental stressors. Thus, ZIP9 may have an important role in ovarian remodeling by regulating breakdown of post-ovulatory follicles and atresia of vitellogenic follicles.