Resolving the interaction network of cell surface receptors

PROJECT SUMMARY/ABSTRACT Cell signaling is initiated at the plasma membrane (PM), but our understanding of the chemical interactions that regulate membrane protein function is still incomplete. The overall theme of my research group is to resolve functional, molecular interactions in the complex environment of the PM. These efforts produce fundamental

insights into structure-function relationships of cell surface receptors and provide a crucial link between structural biology and cell signaling. My work is aimed at the two largest families of membrane proteins: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Both of these protein families are heavily targeted in

disease. GPCRs are the targets of over 30% of all FDA approved drugs. RTK inhibitors are the most successful targeted therapies in cancer treatment. RTK function is coupled to dimerization, but the degree of heterodimerization between receptors has not been investigated in a systematic way with a quantitative, live cell

methodology. Our objective is to quantify these interactions in live cells and determine the effects of ligands and the plasma membrane environment. Chemokine receptors are class A GPCRs that regulate cell movement like migration and infiltration in a broad range of cell types. Consequently, they are important in diseases ranging

from asthma and arthritis to psoriasis and cancer. CXCR4, for example, is the target of an FDA approved compound for mobilizing hematopoietic stem cells in cancer. Many studies have demonstrated that chemokine receptors can assemble into homodimers and heterodimers that regulate cell signaling and can be targeted in

drug development. These dimerization interactions are non-covalent and thus dynamic in nature, although the precise thermodynamic and kinetic principles governing the interactions are not yet resolved. Furthermore, the prevalence and stability of putative dimeric complexes has only been well characterized for a small number of

receptors, leaving open many questions about the importance of dimerization for other chemokine receptors. My lab will use an innovative approach called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to resolve and quantify membrane protein interactions in live cells. Our future research

plans are to: (1) Investigate how ligand binding and cellular environment affect the local network of RTK interactions, (2) Resolve the role of heteromerization on chemokine receptor GPCRs, and (3) Develop a 3-color- PIE-FCCS instrument to resolve competition in the local interaction network of membrane proteins in live cells.

The outcomes of this work will be a quantitative description of membrane protein interaction networks, which will provide fundamental insight into cell signaling pathways involving these receptors. This insight will in turn create new opportunities for drug development and translational research.