The goal of this proposal is to map the protein-structure-functional relationships of the rapidly recycling Fcg- receptor FcRn; and explain its biology. Highly conserved sequences in the TM and cytosolic tail of FcRn indicate structural interactions with the membrane necessary for FcRn function. We propose studies utilizing advances in large-scale oligonucleotide synthesis, multi-parameter FACS, and next-generation sequencing to quantitatively determine the contribution of FcRn structure to IgG recycling. We will utilize a lentiviral FcRn-expression library that is enabled for Illumina deep sequencing. The library includes non-biased mutations in the TM or cytosolic FcRn domains that incorporate 19 different amino acid substitutions for every native residue (67 residues in total) - while otherwise keeping the rest of the protein as wild type. We will have several thousand cells expressing each mutation and comparison of the mutants ability to recycle IgG, as compared to WT, will determine the significance of the mutation. We will use this novel method to test hypothesis-driven mutations to delineate structural features in the TM region that contribute to efficient endosome recycling and transcytosis. Localization of membrane proteins can be influenced by properties such as TM length. We will also determine if FcRn use different dimerization motifs, to switch between functional dimers, as has been shown for other proteins. We will use this technology to delineate individual residues, motifs, or linkages in the highly conserved cytoplasmic domain that assists efficient endosome recycling and transcytosis. Research suggests an juxtamembrane amphipathic helix may sense/induce curvature. There are also several small likely motifs (YXX?, acidic di-leucine, CaM-binding, phosphorylation sites) that will be probed by systematic helix insertions/deletions/scrambling. We will perform this lentiviral screen across cell types; the comparison of those results will provide information on the generality or specificity of the mutational hits. Both the hypothesis-driven mutations, and the discoveries from the non- biased mutations will be further characterized with recycling and transcytosis assays used in our lab. The high- resolution map produced will provide broad utility for the field, serving as a template by which to interrogate the membrane-structure/function relationships that govern in any protein where function is amenable to FACS-based single-cell readout and sorting. As importantly, this project will expand my training in cell and membrane biology relevant to human diseases and put me in a position to meaningfully extend my career in the life sciences.
The goal of this application is to use single cell FACS to functionally test the IgG recycling ability of FcRn to in cells with single, but exhaustive, mutations for each position in the transmembrane (TM) and cytoplasmic tail. The structure/function relationships from this experiment will provide insight, not only on FcRn trafficking, but also on the endosome network and membrane trafficking within the cell. Comparisons between different cell types, coupled with more traditional cell-based assays of selected mutants will determine the function of the particular WT sequences.
