Archives
The zinc transporter proteins are members of the SLC superfa
The zinc transporter proteins are members of the SLC superfamily of metal transporters, and comprise two structurally and functionally different groups, the zinc-importer (ZIP; Zrt-, Irt-like proteins) family (SLC39A), which regulate zinc transport into the cytoplasm from outside the cell and from intracellular stores, and the (ZnT) family (SLC30), which controls the flux of zinc from the cell (Eide, 2004). These transporter proteins have been shown to be important not only for normal cell function, but also for proper neuronal synapse function and neuronal development (Eide, 2004, Wu et al., 2011). Fourteen ZIPs with different functions are known in humans. These have been grouped into four subfamilies, Subfamily I and II, gufA and the LIV-1 subfamily. The LIV-1 subfamily is the largest, containing 9 of the 14 known ZIPs, while the subfamilies I and gufA consist of only single ZIPs (Table 1). Members of the LIV-1 family are predicted to have seven transmembrane domains by hydrophobicity analysis (Begum et al., 2002), whereas most of the other ZIPs are predicted to have eight transmembrane domains (Kambe et al., 2004, Table 1). The various ZIP transporters display diverse patterns of gene and protein regulation in different tissues and cell types and in their subcellular location, which probably reflect their different roles in zinc homeostasis. ZIP9 is the sole member of ZIP subfamily I and is expressed in most tissues. ZIP9 is localized to the plasma, nuclear, and mitochondrial membranes, as well as on the endoplasmic reticulum (Thomas et al., 2014). The functions of ZIP9 have not been described thoroughly, but it has been suggested that ZIP9 is involved in regulating intracellular zinc homeostasis (Matsuura et al., 2009, Taniguchi et al., 2013).
Localization and androgen binding characteristics of croaker ZIP9
In order to establish that ZIP9 is a mAR it is first necessary to demonstrate it is expressed on the plasma membranes of target cells and binds steroids with the characteristics of a specific membrane androgen receptor. Croaker ZIP9 mRNA and protein were found to be highly expressed in ovarian, testicular and rp gift card synthesis tissues, with lower expression in hepatic tissues and no detectable expression in the gills. Ovarian croaker ZIP9 transcripts are abundant in the follicle cells, with only minor expression in the oocytes (A). Western blot analysis using the murine antibody shows ZIP9 is localized to plasma membrane preparations of croaker ovaries and immunocytochemical analysis shows it is primarily expressed on the plasma membranes of granulosa cells (B), with no detectable expression in theca cells. A similar plasma membrane expression of croaker ZIP9 was observed with the recombinant protein stably expressed in a human nAR-negative breast cancer (SKBR-3) cell line.
The [3H]-T binding characteristics of the recombinant croaker ZIP9 were investigated in the transfected SKBR3 cells. Specific [3H]-T binding to plasma membranes of ZIP9-transfected cells was increased several-fold compared to that of cell membranes transfected with vector (C). Saturation binding and Scatchard analysis demonstrated the presence of a high affinity (Kd 14nM), limited capacity, single binding site for T (D). The kinetics of association/dissociation of [3H]-T binding to recombinant ZIP9 are rapid with t1/2s of 1–2min, which is characteristic of steroid membrane receptors. (Berg et al., 2014). Competitive binding assays showed steroid binding to the receptor is highly specific for T, whereas estradiol-17β, the maturation-inducing steroid, 17,20β,21-trihydroxy-4-pregene-3-one (20β-S) and cortisol showed no displacement of [3H]-T. The steroid specificity of croaker ZIP9 differed from that of the croaker nAR in that the nAR agonists mibolerone and R1881 were ineffective competitors for [3H]-T binding (Berg et al., 2014, Sperry and Thomas, 1999). Thus, mibolerone and R1881 are potentially useful pharmacological agents to distinguish androgen actions mediated by the nAR from those mediated through ZIP9. The natural androgens 5α-dihydrotestosterone (DHT) and androstenedione displayed low relative binding affinities for ZIP9 (<1% that of T), and surprisingly the teleost androgen, 11-ketotestosterone is an ineffective competitor. Specific [3H]-T binding was also demonstrated to wild type croaker ZIP9 in cultured granulosa/theca (G/T) cells which was abrogated by transfection with croaker ZIP9 siRNA, but not by transfection with croaker nAR siRNA, indicating the [3H]-T binding on croaker G/T cell membranes is solely mediated by ZIP9 (Berg et al., 2014). Finally, a single peak of [3H]-T binding was detected at ∼40kDa on a ligand blot of plasma membranes from croaker ZIP9-transfected cells that coincides with the position of immunoreactive ZIP9, suggesting that the [3H]-T binding is an intrinsic property of ZIP9 and that it does not require any protein partner for its androgen binding activity. Collectively, these results clearly demonstrate that croaker ZIP9 is localized on the plasma membrane and has all the steroid binding characteristics of a specific mAR. The croaker ZIP9 protein is the first novel androgen receptor unrelated to nuclear receptors identified in vertebrates and the first ZIP protein shown to have the characteristics of a steroid receptor.