We have described the structures of the full-length LDL, and insulin receptors in both their detergent and vesicle-reconstituted forms. The objectives now are to extend our structural studies of both receptors to higher resolution and to examine the interactions with their ligands, LDL and insulin. For the LDL receptor, we will use the purified bovine receptor, as well as the expressed human LDL receptor and constructs of its extracellular domain(s). Our research is directed to the following goals: (1) use gold-labeled LDL receptor (bovine and human) and LDL receptor extracellular domains (human) to localized the receptor-binding domains of the LDL receptor; (3) use the C-terminal cysteine to bind extracellular domains of the LDL receptor to lipid monolayer surfaces; (4) perform structural studies of oriented LDL receptor and 2-D arrays by electron microscopy; (4) use oriented extracellular domains to bind and oreint LDL for structural studies by electron microscopy (with Project 3); (5) using CD and calorimetry, to define the secondary structure, conformation and unfolding of the human LDL receptor and its expressed extracellular sub-domains. We will continue our structural studies of the insulin receptor and its extracellular domain with the following goals: (1) to engineer terminal cysteine sites at the C-terminal of the extracellular domain of the insulin receptor for gold labeling and binding to lipid monolayer surfaces; (2) to use supported lipid monolayers to orient the extracellular insulin-binding domain of the insulin receptor; (3) to produce oriented 2-D arrays of the extracellular domaisn of the insulin receptor; (4) to use electron microscopy to define the structure of insulin receptor extracellular domain oriented at lipid surfaces; and (5) to study the effect of insulin binding on insulin receptor extracellular domain structure. Improved structural descriptions of the LDL and insulin receptors, as well as their interaction with their ligands LDL and insulin, should result.
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