Marine Science Institute (A.H.B., M.S.R., J.D., P.T.), The University of Texas at Austin, Port Aransas, Texas 78373; Department of Science and Technology (A.H.B.), Örebro University, SE-701 82 Örebro, Sweden SE-70182; and Department of Biology (C.D.R.), Clemson University, Pendleton, South Carolina 29670.
Endocrinology. 2014 Nov;155(11):4237-49. doi: 10.1210/en.2014-1198. Epub 2014 Jul 11.
Rapid, cell surface-initiated, pregenomic androgen actions have been described in various vertebrate cells, but the receptors mediating these actions remain unidentified. We report here the cloning and expression of a cDNA from Atlantic croaker (Micropogonias undulatus) ovaries encoding a 33-kDa, seven-transmembrane protein with binding and signaling characteristics of a membrane androgen receptor that is unrelated to any previously described steroid receptor. Instead, croaker membrane androgen receptor has 81-93% amino acid sequence identity with zinc transporter ZIP9 (SLC39A9) subfamily members, indicating it is a ZIP9 protein. Croaker ZIP9 is expressed in gonadal tissues and in brain and is up-regulated in the ovary by reproductive hormones. Croaker ZIP9 protein is localized to plasma membranes of croaker granulosa cells and human breast cancer (SKBR-3) cells stably transfected with ZIP9. Recombinant croaker ZIP9 has a high affinity (dissociation constant, Kd, 12.7 nM), limited capacity (maximal binding capacity 2.8 nM/mg protein), displaceable, single binding site-specific for androgens, characteristic of steroid receptors. Testosterone activates a stimulatory G protein coupled to ZIP9, resulting in increased cAMP production. Testosterone promotes serum starvation-induced cell death and apoptosis in transfected cells and in croaker ovarian follicle cells that is associated with rapid increases in intracellular free zinc concentrations, suggesting an involvement of zinc in this nonclassical androgen action to promote apoptosis. These responses to testosterone are abrogated by treatment with ZIP9 small interfering RNA. The results provide the first evidence that zinc transporter proteins can function as specific steroid membrane receptors and indicate a previously unrecognized signaling pathway mediated by steroid receptors involving alterations in intracellular zinc.
已在各种脊椎动物细胞中描述了快速、细胞表面起始的前基因组雄激素作用，但介导这些作用的受体仍未被鉴定。我们在此报告从大西洋鲱鱼（Micropogonias undulatus）卵巢中克隆和表达的 cDNA，该 cDNA 编码一种 33kDa 的七跨膜蛋白，具有膜雄激素受体的结合和信号转导特征，与任何先前描述的类固醇受体均不相关。相反，鲱鱼膜雄激素受体与锌转运蛋白 ZIP9（SLC39A9）亚家族成员具有 81-93%的氨基酸序列同一性，表明它是一种 ZIP9 蛋白。鲱鱼 ZIP9 在性腺组织和大脑中表达，并被生殖激素上调。鲱鱼 ZIP9 蛋白定位于鲱鱼颗粒细胞和稳定转染 ZIP9 的人乳腺癌（SKBR-3）细胞的质膜上。重组鲱鱼 ZIP9 具有高亲和力（解离常数，Kd，12.7 nM）、有限的容量（最大结合容量 2.8 nM/mg 蛋白）、可置换、雄激素特异性的单结合位点，这是类固醇受体的特征。睾酮激活与 ZIP9 偶联的刺激性 G 蛋白，导致 cAMP 产生增加。睾酮促进转染细胞和鲱鱼卵巢滤泡细胞中血清饥饿诱导的细胞死亡和凋亡，这与细胞内游离锌浓度的快速增加有关，表明锌在此促进凋亡的非经典雄激素作用中发挥作用。用 ZIP9 小干扰 RNA 处理可消除这些对睾酮的反应。这些结果首次证明锌转运蛋白可作为特定的类固醇膜受体发挥作用，并表明涉及细胞内锌变化的类固醇受体介导的以前未被认识的信号通路。
