Date of Award

12-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Biochemistry

Research Advisor

Jie J. Zheng, Ph.D.

Committee

Martha M. Howe, Ph.D. David R. Nelson, Ph.D. Stacey K. Ogden, Ph.D. Stephen White, DPhil.

Keywords

Cancer, Drug, GPCR, Hedgehog, NMR, Smoothened

Abstract

The Hedgehog (Hh) signal transduction pathway functions as one of the key developmental pathways and deranged Hh signalling is associated with numerous cancer and tumor conditions. The Smoothened (Smo) G protein coupled receptor (GPCR) functions as the signal transducer of the Hh pathway and is the most attractive drug target of the pathway. The structure of the Smo receptor includes seven membrane spanning domains, extracellular and intracellular loops connecting the membranous domains and the extracellular cysteine rich domain (CRD). The extracellular CRD of the Smo receptor is homologous to the Frizzled (FzD) CRD. The FzD CRD interacts with the physiological ligand of the FzD receptor: Wnt, but the function of the Smo CRD is not clearly elucidated.

In the present dissertation we determined the first structure of the Drosophila Smo CRD by solution Nuclear Magnetic Resonance (NMR) spectroscopy. The structure of the Smo CRD comprises primarily of four helices and is stabilized by disulfide bonds between the conserved cysteine residues. The tertiary structure of the Smo CRD is similar to the structure of the FzD CRD.

Additionally, we showed that both the Drosophila and the human Smo CRD interact with the glucocorticoid: Budesonide (Bud), albeit with different binding affinities. We identified the key residues in both species that are important for this interaction and generated the complex structure of the Drosophila Smo CRD with Bud using computational methodologies. The binding pocket in the Smo CRD can be targeted for the development of novel anti-cancer therapeutics.

The Smo receptor possesses a binding cavity located in the transmembrane domains and we determined that the Smo CRD also possesses ligand binding capabilities. This finding enabled us to propose a novel mechanism of Hh signalling. We speculate that certain Smo ligands bind to the Smo CRD and membrane spanning domains simultaneously to regulate intracellular signalling. Phosphatidylinositol-4-phosphate (PI(4)P) has been shown to regulate Smo activation. We determined that the fatty-acid side chains of the phosphatidylinositols interact with Smo CRD, and PI(4)P specifically enhances activator induced Hh signalling. Therefore, we speculated that PI(4)P may function to bridge the Smo extracellular and the membrane spanning domains, wherein the fatty-acid side chains of PI(4)P docks in the Smo CRD and the inositol head with the fourth position phosphate is necessary for anchoring in the cavity in the transmembrane domains; however, PI(4)P may not necessarily function as endogenous Smo ligand because in the cell-based luciferase reporter assays PI(4)P could only enhance signalling in the presence of an activator of Hh signalling. This suggests that PI(4)P binding to Smo is either a prerequisite and renders Smo active to further induce downstream signalling or PI(4)P acts as an allosteric activator of Hh signalling.

DOI

10.21007/etd.cghs.2014.0257

Comments

Six month embargo expired June 2015

Download

Share

COinS