Structural And Functional Studies Of FGFR1c In Nanodisc Membranes

As members of the receptor tyrosine kinases(RTKs)family,fibroblast growth factor receptors(FGFRs)play important roles as the regulators of proliferation,differentiation,migration,and survival in various cell types.Accordingly,the deregulation of FGFR signaling contributes to various pathological conditions and developmental syndromes.FGF ligands bind to the extracellular domain of FGFR with various facilitating cofactors,such as heparin sulfate and klothos,and induce receptor homo-dimerization and subsequent activation of cytoplasmic tyrosine kinase domains by tyrosine autophosphorylation at multiple sites.Tyrosine autophosphorylation plays a crucial regulatory role in the kinase activities of FGFRs,and in the recruitment and activation of downstream intracellular signaling pathways.At least seven tyrosine autophosphorylation sites have been described in the intracellular kinase domain(FGFR1K).The phosphorylation of the A-loop residues Y653 and Y654 have been reported to be critical for upregulation of kinase activity;while the phosphorylation sites of tyrosine residues in other parts of the cytoplasmic region serve as specific binding sites for downstream signaling molecules phospho-tyrosine binding domains,resulting in the activation of specific signaling pathways.Most of previous in-vitro studies of tyrosine kinases were performed using isolated kinase domains in aqueous solution,but failed to reproduce the in vivo observations.This largely reflects the importance of cell membrane localization of kinase domains,which is essential for promoting dimerization and cooperative cross-receptor autophosphorylation.Nanodiscs are discoidal nanomembrane particles with a planar phospholipid bilayer enwrapped by proteins such as apolipoprotein A-I or membrane scaffolding proteins(MSP).Peptide nanodisc,is based on the use of synthetic peptides mimicking the amphipathic helices of Apolipoprotein I,whereas the nanodisc sizes can be conveniently controlled by varying the lipid/peptide ratio.In addition,the lipid compositions of nanodiscs can be controlled precisely providing a nanoscale membrane surface for investigating various membrane recognition events.Nanodiscs have been widely used for analyzing structures and functions of membrane proteins by dispersing them in solution.Our works focus on the application of the nanodiscs system in studying the structure and function of FGFRlc,a FGF receptor highly related with cancer pathology and drug development.The thesis consists two parts,1)the embedding of full length FGFRlc and/or FGFRlc-cofactor complex into the MSP nanodiscs,and 2)the FGFR1 kinase domain activity in the presence of nanoscale 2D-membrane mimics.Below are the main results:1.We constructed a Baculovirus Expression System for full length FGFRlc.then the bFGF/heparin/FGFR complex were successfully assembled into the MSPAH5 nanodiscs system.The function of FGFRlc was characterized and a preliminary structural study was performed using Cryo-EM.2.An NTA(Ni)-nanodiscs system was constructed using a 22-residue peptide and the nanodisc size can be finely adjusted.An N-terminal HiS6-tagged FGFR1K anchored spontaneously onto the nickel-chclating lipids on the membrane with orientations that mimic those in plasma membranes.The 2D membrane surfaces provided by peptide nanodiscs signmcantly rahanced the phosphorylation rates of Y6S3 and Y6S4 in FGFRIKA-loop.