UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Purification and mass spectrometric characterization of human CXCR4 Wong, Julie Patricia

Abstract

G protein- coupled receptors (GPCR5) are seven transmembrane receptors that comprise the largest superfamily of proteins in the body that are involved in a variety of fundamental processes including sight, smell, tactile as well as nervous responses. Tightly coordinated, control of these receptors is critical for normal physiology. Emphasis has been placed in pharmaceuticals to find ways to inhibit or accelerate these processes in GPCR implicated diseases. Although tertiary structural information would be beneficial for rational drug design, little is known about the structure of GPCRs despite extensive research in the field. Only two high resolution structures exist for any mammalian GPCR. Structural studies are challenged by the intrinsic difficulty associated with purifying these receptors in high quality and quantity. To overcome these challenges, we propose a mass spectrometric, sequence-based approach to characterize GPCRs allowing for the rapid characterization of an ectopically as well as endogenously expressed receptor employing a receptor tag. The sensitivity of the approach allows for the implementation of a mammalian expression system with the physiologically relevant post-translational modifications (PTMs). The sites of N-linked glycosylation has been mapped and receptor isoforms were identified for a model GPCR, CXCR4 and a related receptor, CCR5. This approach is versatile and applicable to other membrane proteins such as ATP binding cassette (ABC) transporters and has been adapted into an antibody screening platform. As proof- of- principle, we have developed a monoclonal anti-human CXCR4 antibody with broad applications and potential for clinical diagnostics. In view of the challenges preventing high resolution three-dimensional structure determination, photoaffinity crosslinking was combined with our sequence-based strategy for receptor binding site footprinting. CXCR4 ligand analogs containing a non-natural amino acid photocrosslinker, benzophenylalanine and a biotin tag for complex isolation were chemically synthesized and crosslinked to CXCR4 expressed on intact cells. Individual components of the receptor-ligand complex were identified by western blotting and tandem mass spectrometry. Regions on the bound receptor that were protected from protease treatment and consequently tandem MS/MS sequencing were identified as potential sites of ligand-receptor contact. These regions were identified as the receptor N-terminus and/or the first extracellular loop for ligand binding/docking.

Item Citations and Data

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International