METABOLIC AND ENDOCRINE CONTROL OF SUGAR TRANSPORT

这一建议代表了我们实验室的持续努力， 了解内分泌和代谢的分子基础 调节细胞糖的运输。 代谢耗竭 似乎刺激肌肉和鸟类红细胞（ARBC）运输 通过增加细胞表面糖载体的内在活性 (i.e.通过载波激活）。 这与胰岛素刺激相反 脂肪细胞和肌肉运输的细胞表面增加 号码 本提案的总体目标是， 载体活化的生物化学及其与胰岛素的关系 糖的运输。 这些研究将有助于我们长期 理解蛋白质分子基础的目标 介导的糖转运，并最终可能在 管理紊乱状态，如糖尿病。 葡萄糖转运蛋白含有转运调节蛋白， ATP结合位点。 该网站最近被特别标记为 使用叠氮ATP。 特定的AIM 1是通过以下方式识别该站点： 对标记肽水解后释放的标记肽进行测序 载体 具体目标2问：由于承运人最近 显示对激活/抑制非常敏感， 特异性膜双层溶血磷脂（LPLs）， 由改变的双层脂质引起的代谢消耗激活 组成？ 我们采用重组系统和磷脂 分析和操作对照和贫化ARBC， 回答这个问题 具体目标3：由于刺激性LPL降低 和抑制性LPL增加了葡萄糖载体对 ATP和LPL之间的这种协同作用是否会放大 对交通运输的影响？ 我们进行配体结合， 运输研究与重组载体回答这个问题。 具体目标4直接测试激活和招募 运输监管的假设，使用最近开发的 抗血清（仅对细胞外结构域具有反应性， 载体）以定量对照中的细胞表面载体数目 和代谢耗尽的ARBC。 具体目标5扩展目标 2至4对胰岛素刺激脂肪细胞糖转运的影响。 如果 如果成功，这些研究将为未来的研究奠定基础。 运输中载体ATP结合位点作用的评估 法规和理解承运人之间的关系 活性和细胞代谢状态。

This proposal represents a continuing effort by our laboratory to understand the molecular basis of endocrine and metabolic regulation of cellular sugar transport. Metabolic depletion appears to stimulate muscle and avian red cell (ARBC) transport by increasing the intrinsic activity of cell surface sugar carriers (i.e. by carrier activation). This contrasts with insulin stimulation of adipocyte and muscle transport by increased cell surface numbers. The broad goal of this proposal is to characterize the biochemistry of carrier activation and its relationship to insulin regulation of sugar transport. These studies will assist in our long term goal of understanding the molecular basis of protein mediated sugar transport and could ultimately be of value in the management of disordered states such as diabetes. The glucose transport protein contains a transport modulating ATP-binding site. The site has recently been specifically labeled using azido-ATP. Specific AIM 1 is to identify this site by sequencing labelled peptides released on hydrolysis of labelled carrier. Specific Aim 2 asks: since the carrier has recently been shown to be exquisitely susceptible to activation/inhibition by specific membrane bilayer lysophospholipids (LPLs), does carrier activation by metabolic depletion result from altered bilayer lipid composition? We employ reconstituted system and phospholipid analyses and manipulation of control and depleted ARBCs to answer this. Specific Aim 3 asks: since stimulatory LPLs reduce and inhibitory LPLs increase the affinity of glucose carrier for ATP, does this synergism between ATP and LPLs act to amplify their effects on transport? We perform ligand binding and transport studies with reconstituted carrier to answer this. Specific Aim 4 directly tests activation and recruitment hypotheses for transport regulation using recently developed antisera (exclusively reactive against an extracellular domain of the carrier) to quantitate cell surface carrier numbers in control and metabolically depleted ARBCs. Specific Aim 5 extends Aims 2 to 4 to insulin stimulation of adipocyte sugar transport. If successful, these studies will provide the groundwork for future assessment of the role of the carrier ATP-binding site in transport regulation and for understanding the relationship between carrier activity and cellular metabolic status.