Erythrocyte spectrin-actin junctions play a critical role in the maintenance of cell shape andmembrane properties. Their organization and membrane linkage is therefore of fundamentalimportance to erythrocyte physiology. Our recent evidence indicates that dematin performs apivotal function by linking these junctions to the membrane. Here, we propose that dematinperforms its biological function by linking the spectrin-actin junctions to the membrane viaglucose transporter-1 (GLUT1) in human erythrocytes. We will test this hypothesis under thefollowing three aims: A1: GLUT1 interaction with the spectrin-actin junctions in humanerythrocytes. We have identified GLUT1 as the primary membrane receptor for dematin andadducin in human erythrocytes. We propose to identify the interacting domains of GLUT1,dematin, and adducin, and disrupt the GLUT1-cytoskeletal bridge by biochemical means, thusassessing its impact on the membrane stability and shape of human erythrocytes. A2. Dematinand adducin binding receptor(s) in mouse erythrocytes and adipocytes. We propose thatdematin and adducin bind to a novel membrane receptor(s) in mouse erythrocytes. We willidentify this receptor using immunoprecipitation, mass spectrometry, and chemical crosslinkingassays. We will test the binding of dematin and adducin with several potential membraneproteins identified in our proteomics screen of mouse erythrocyte membrane vesicles. We willalso investigate the binding of dematin and adducin with GLUT4, and investigate their functionaleffects on GLUT4 recycling and glucose uptake in adipocytes. These experiments will clarify therole of alternate receptors that link the spectrin-actin junctions in mouse erythrocytes andadipocytes. A3. Physiological implications of complete dematin deficiency. Recently, weobserved an obesity phenotype in the dematin headpiece domain null mice. To elucidate thespecific role of the headpiece domain of dematin, we propose to generate a new mouse modelwith complete dematin deficiency using the conventional gene targeting techniques. We willcompare the anemia and obesity phenotypes in the headpiece and dematin null mice focusingparticularly on the impaired GLUT4 trafficking and diabetes. Together, these studies are likely tounveil a novel function of dematin, adducin, and glucose transporters in the regulation of anemiaand metabolic disorders with broader implications for other headpiece domain-containingproteins.
Erythrocyte membrane has served as a paradigm for discovering the function of its proteins in many non-erythroid cells. This project will investigate the role of dematin, adducin, and glucose transporters in the formation of a novel membrane-cytoskeletal bridge with functional implications in hemolytic anemia, diabetes, and obesity.
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