De Toni Luca, Guidolin Diego, De Filippis Vincenzo, Tescari Simone, Strapazzon Giacomo, Santa Rocca Maria, Ferlin Alberto, Plebani Mario, Foresta Carlo
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Endocrinology. 2016 Nov;157(11):4473-4486. doi: 10.1210/en.2016-1312. Epub 2016 Sep 27.
The undercarboxylated form of osteocalcin (ucOC) regulates male fertility and energy metabolism, acting through the G protein-coupled receptor (GPRC)6A, thus forming a new pancreas-bone-testis axis. Recently, GPRC6A has also been suggested to mediate the nongenomic responses of free testosterone (T). However, these data did not consider the physiological scenario, where circulating T is mainly bound to sex hormone-binding globulin (SHBG) and only a small percentage circulates freely in the blood. Here, by the use of computational modelling, we document the existence of similar structural moieties between ucOC and SHBG that are predicted to bind to GPRC6A at docking analysis. This hypothesis of competition was assessed by binding experiments on human embryonic kidney-293 cells transfected with human GPRC6A gene. Unliganded SHBG specifically bound the membrane of human embryonic kidney-293 cells transfected with GPRC6A and was displaced by ucOC when coincubated at 100-fold molar excess. Furthermore, specific downstream Erk1/2 phosphorylation after stimulation of GPRC6A with ucOC was significantly blunted by 100-fold molar excess of unliganded SHBG. Intriguingly previous incubation with unliganded SHBG, followed by incubation with T, induced Erk1/2 phosphorylation in a dose-dependent manner. Neither binding nor stimulating activities were shown for SHBG saturated with T. Experiments on mutation constructs of GPRC6A strengthened the hypothesis of a common binding site of ucOC and SHBG. Given the role of GPRC6A on energy metabolism, these data agree with epidemiological association between SHBG levels and insulin sensitivity, suggest GPRC6A as a likely SHBG receptor, and add bases for the possible regulation of androgen activity in a nonsteroidal manner.
骨钙素的未羧化形式（ucOC）通过G蛋白偶联受体（GPRC）6A调节雄性生育能力和能量代谢，从而形成一条新的胰腺-骨骼-睾丸轴。最近，GPRC6A也被认为介导游离睾酮（T）的非基因组反应。然而，这些数据并未考虑生理情况，即循环中的T主要与性激素结合球蛋白（SHBG）结合，只有一小部分在血液中自由循环。在这里，通过计算建模，我们证明了ucOC和SHBG之间存在相似的结构部分，在对接分析中预测它们会与GPRC6A结合。通过对转染了人GPRC6A基因的人胚肾-293细胞进行结合实验，评估了这种竞争假说。未结合配体的SHBG特异性结合转染了GPRC6A的人胚肾-293细胞的膜，当以100倍摩尔过量共同孵育时，会被ucOC取代。此外，用ucOC刺激GPRC6A后，100倍摩尔过量的未结合配体的SHBG会显著减弱特定的下游Erk1/2磷酸化。有趣的是，先用未结合配体的SHBG孵育，然后用T孵育，会以剂量依赖的方式诱导Erk1/2磷酸化。与T饱和的SHBG既没有显示结合活性也没有显示刺激活性。对GPRC6A突变体构建体的实验强化了ucOC和SHBG存在共同结合位点的假说。鉴于GPRC6A在能量代谢中的作用，这些数据与SHBG水平和胰岛素敏感性之间的流行病学关联相符，表明GPRC6A可能是SHBG受体，并为以非甾体方式调节雄激素活性增加了依据。
