SR-B1 Sorting Signals in HDL Metabolism

描述(申请人提供)：血浆高密度脂蛋白(高密度脂蛋白)是血浆胆固醇分泌胆汁胆固醇的主要载体。高密度脂蛋白受体清道夫受体B-L(SR-BI)介导高密度脂蛋白和胆固醇酯摄取排泄到胆汁中，其表达与胆汁胆固醇分泌有关。SR-BI基因敲除小鼠的胆汁胆固醇分泌显著减少，表明SR-BI在这一过程中起着重要作用。然而，关于SR-BI介导肝胆胆固醇分泌的分子机制还知之甚少。SR-BI在肝窦和肝小管膜上均有表达，并可跨细胞进入肝小管膜。SR-BI基侧靶向和跨细胞进入小管膜所需的分选信号尚不清楚。我们定义了三个重要的因素，可能调节SR-BI在肝脏中的亚细胞分类，从而调节SR-BI依赖的胆汁胆固醇分泌。其中一个因素是PDZK1，它是一种PDZ结构域蛋白，已被证明与SR-BI相互作用，并对维持肝脏SR-BI水平至关重要。第二个因子是SR-BI C末端的基侧靶向信号，第三个因子是SR-BI C末端跨膜结构域的胆固醇结合结构域。SR-BI的C端跨膜结构域可以直接结合胆固醇，但对SR-BI介导的选择性摄取或胆固醇外流并不重要。肝脏中SR-BI与胆固醇的结合可能在SR-BI跨细胞反应中起一定作用。具体目标1将确定PDZK1在SR-BI分选中的作用。我们已经建立了PDZK1基因敲除小鼠模型，并将使用PDZK1缺陷小鼠和体外极化细胞模型来检测PDZK1在SR-BI分选中的作用。特异性靶点2将界定SR-BI的底侧靶向信号，并测试该序列在体内和体外SR-BI分选以及体内胆汁胆固醇分泌中的作用。特定目标3将检验SR-BI胆固醇结合结构域在SR-BI跨细胞作用中发挥作用的假设。我们将测试该结构域在体内胆汁胆固醇分泌和体外SR-BI细胞转运中的作用。这些研究将为调节SR-BI分选和胆汁胆固醇分泌的机制提供基本的见解。

DESCRIPTION (provided by applicant): Plasma high density lipoprotein (HDL) is the principal carrier of plasma cholesterol for biliary cholesterol secretion. The HDL receptor Scavenger Receptor B-l (SR-BI) mediates the uptake of HDL cholesterol and cholesteryl ester for excretion into bile and its expression correlates with biliary cholesterol secretion. SR-BI knockout mice have a significant decrease in biliary cholesterol secretion, indicating an important role of SR-BI in that process. However, little is known regarding the molecular mechanisms by which SR-BI mediates hepatobiliary secretion of cholesterol. SR-BI is expressed on both sinusoidal and canalicular membranes in liver, and undergoes transcytosis to the canalicular membrane. The sorting signals necessary for SR-BI basolateral targeting and transcytosis to the canalicular membrane are unknown. We have defined three important factors that may regulate SR-BI subcellular sorting in liver and thus SR-BI- dependent biliary cholesterol secretion. One factor is PDZK1, a PDZ domain protein that has been shown to interact with SR-BI and to be essential to maintain hepatic SR-BI levels. A second factor is a basolateral targeting signal in the C-terminus of SR-BI, and a third factor is a cholesterol binding domain in the C- terminal transmembrane domain of SR-BI. The C-terminal transmembrane domain of SR-BI can directly bind cholesterol but is not important for SR-BI-mediated selective uptake or cholesterol efflux. Cholesterol binding by SR-BI in liver may play a role in SR-BI transcytosis. Specific Aim 1 will determine the role of PDZK1 in SR-BI sorting. We have generated a PDZK1 knockout mouse model and will use both PDZK1 deficient mice and in vitro polarized cells models to examine the role of PDZK1 in SR-BI sorting. Specific Aim 2 will delimit the basolateral targeting signal of SR-BI and test the role of this sequence in SR-BI sorting in vivo and in vitro as well as in biliary cholesterol secretion in vivo. Specific Aim 3 will test the hypothesis that the SR-BI cholesterol binding domain plays a role in SR-BI transcytosis. We will test the role of this domain in biliary cholesterol secretion in vivo and SR-BI transcytosis in vitro. These studies will provide fundamental insights into the mechanisms regulating SR-BI sorting and biliary cholesterol secretion.