Search

The EGFR family of receptor tyrosine kinases (RTKs) includes EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB. The EGFR family members play crucial roles in development and in regulating normal cellular processes. However, these receptors are found to be overexpressed or mutated in various cancers. The complete mechanism by which the EGFR family is autoregulated is still unknown. This work focuses on the role of the extracellular domain of EGFR family members on receptor activation, dimerization/oligomerization, and downstream signaling. We generated a system using designed ankyrin repeat proteins (DARPins) to study EGFR extracellular domain dimers of varying separation distances. We biochemically, cellularly, and structurally characterize interactions between HER3 and A30, an RNA that binds to the extracellular domain of HER3 and reduces HER2 phosphorylation. This work also includes preliminary efforts to characterize HER2-HER3 oligomers using single molecule photobleaching experiments. This work lays the groundwork for future studies involving EGFR family receptor regulation

It has been shown that the amino terminus and second extracellular loop (EC2) of CXCR2 are crucial for ligand binding and receptor activation. The lack of an ionic lock motif in the third intracellular loop of CXCR2 focuses an investigation of the mechanism by which these two extracellular regions contribute to receptor recognition and activation. The first objective of this investigation was to predict the structure of CXCR2 based on known structures of crystallized GPCRs. Rhodopsin, β2-adrenergic receptor, CXCR4 were used for homology modeling of CXCR2 structure. Highly conserved motifs found in sequence alignments of the template GPCRs were helpful to generate CXCR2 models. We also studied solvent accessibility of residues in the EC2 of CXCR2 in the inactive state. Most of the residues in the EC2 were found to be solvent accessible in the inactive state, suggesting the residues might be involved in ligand recognition. Second, we studied the role of charged residues in the EC2 of CXCR2 in ligand binding and receptor activation using constitutively active mutants (CAM) of CXCR2, D9K and D9R. Combinatorial mutations consisting of the CAM in the amino terminus and single mutations of charged residues in the EC2 were generated to study two concepts including “attraction” and “repulsion” models. The mutant receptors were used to test their effects on cell surface expression, ligand binding, receptor activation through PLC-β3, and cellular transformation. All the mutations in the repulsion model result in CXCR2 receptors that are unable to bind ligand, suggesting that each of the Arg residues in the EC2 are important for ligand recognition. Interestingly, mutations in the attraction model partially inhibited receptor activation by the CAM D9K, suggesting that Glu198 and Asp199 residues in the EC2 are associated with receptor activation. Furthermore, a novel CAM, E198A/D199A, was identified in this study. These negatively charged residues are very close to a conserved disulfide bond linking the EC2 and the third transmembrane. In this sense, these current discoveries concerning the structural basis of CXCR2 and interdisciplinary approaches would provide new insights to investigate unknown mechanisms of interaction with its cognate ligands and receptor activation.