Type

Text

Type

Dissertation

Advisor

Miller, W. Todd | London, Erwin | Seeliger, Jessica | Garcia-Diaz, Miguel | Hubbard, Stevan

Date

2017-12-01

Keywords

Biochemistry

Department

Department of Biochemistry and Structural Biology

Language

en_US

Source

This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.

Identifier

http://hdl.handle.net/11401/78225

Publisher

The Graduate School, Stony Brook University: Stony Brook, NY.

Format

application/pdf

Abstract

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are receptor tyrosine kinases (RTKs) involved in the regulation of many important cellular processes. Hyperactivity of IR and IGF1R is associated with cancer, whereas low IR activity and insulin resistance are linked to diabetes. The current proposed models of activation are derived from structural studies using either soluble extracellular domains or cytoplasmic tyrosine kinase domains. Although these structures have provided a wealth of information about the IR, there is still a significant disconnect in our understanding of the transmembrane signaling mechanism. Only studies on intact, membrane-spanning, full-length, holoreceptors can provide an opportunity to elucidate the transmembrane signaling process. We have stably expressed and purified full-length IR and IGF1R from HEK 293T mammalian cells at quantities suitable for biochemical and structural characterization. We screened a panel of structurally diverse detergents for optimal ligand activation. Biochemical assays of n-dodecyl-β-D-maltoside(DDM) solubilized IR and IGF1R showed ligand-stimulated autophosphorylation and kinase activity, suggesting intact transmembrane signaling capabilities and a native-like conformation. We examined the effects of cholesterol depletion and sterol substitution in the plasma membrane of mammalian cells on the activation of IR and IGF1R. This demonstrated a dependence of IR and IGF1R on lipid rafts for proper receptor function. IR and IGF1R autophosphorylation in vivo were inhibited by cholesterol depletion by treatment with methyl-ß-cyclodextrin (MßCD) and autophosphorylation was restored with the replenishment of cholesterol. Additionally, we screened sterols of varying characteristics for effects on IR activation. IR autophosphorylation in mammalian cells was strongly correlated with the replenishment of sterols with lipid-rafts supporting propensities. These data support the necessity of lipid rafts for proper ligand-induced activation of IR and IGF1R in HEK 293T cells Lastly, we have utilized single particle electron microscopy for structural studies of the IR holoreceptor. Full-length IR was purified from mammalian cells and fixed to carbon-coated grids for analysis. Class averaging and 3D reconstruction of more than five thousand particles generated a structure that correlated with the electron density of the crystallized IR ectodomain. Future work will aim in producing structural images of full-length IR and IGF1R in its basal state and insulin-simulated conformations. | 159 pages

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