Research — Ferguson Lab
We are interested in extracellular control of receptor tyrosine kinases (RTKs) in both normal and neoplastic environments. The 58 RTKs in the human proteome fall into 20 classes based on the domains of their extracellular regions (ECRs). Ligand-induced dimerization is a central component in the activation of most RTKs, but it is increasingly clear that there is great diversity in the mechanisms of regulation of receptor activation across the RTK superfamily. RTKs can be subject to complex allosteric regulation by their ligands, by co-receptors and by other modulators. We seek molecular understanding of these diverse mechanisms, and of how receptor activity can be modulated by disease linked mutations or regulated by therapeutic agents. We apply a combination of biophysical, structural, biochemical and cellular approaches.
1. Mechanisms of activation of RTKs that are dimeric in the unliganded state:
For most (if not all) RTKs, regulation involves more than simple ligand-induced dimerization. In some cases, RTKs form dimers in the absence of ligand so activation must proceed by some alternate mechanism. The insulin receptor (IR), for example, is a disulfide-bonded dimer that is regulated by ligand induced conformational changes. We are interested in the regulation of RTKs that form non-covalent inactive dimers, such at Tie2, that forms an unliganded dimer mediated by its membrane proximal FNIII domains, and the invertebrate epidermal growth factor receptors (EGFRs) that form dimers of varying stability and poorly characterized structure. How ligand induces activation in these cases is not well understood and may involve conformational rearrangement in a dimer or formation of higher order oligomers (or both).
For the invertebrate EGFRs, we are particularly interested in understanding whether their activation mechanisms share features with the IR family of RTKs. The EGFR and IR families (and only these two RTK families) share a common ligand binding module in the N-terminal region of their ECRs. Domains I, II and III of these two receptor families are not only structurally related, but the domain I-II-III module of EGFR and IR undergo analogous ligand-induced conformational changes, in both cases breaking autoinhibitory interactions and promoting formation of active states. The human EGFR is, however, monomeric in the unliganded state. Ligand binding drives receptor dimerization to activate the receptor - a very different situation than the constitutively dimeric IR, where receptor conformational changes drive activation. Interestingly, unlike their mammalian counterparts, the homologs of the EGFR in Drosophila and C. elegans form dimers in the absence of their ligands. These dimeric, unliganded EGFR molecules may offer a window to unify the mechanisms of activation of the EGFR and IR families. We are using cryoEM approaches to gain new insights into the nature of the unliganded dimers of the invertebrate EGFRs and an understanding of how these are altered upon ligand binding. We are also investigating the structures of full length receptors, which has been informative for the mammalian systems. Our structure derived mechanistic hypotheses are tested with biochemical, cellular and in vivo assays.
Adapted from Ferguson et al. (2020) Prot. Sci. 29, 1331-1344
2. Understanding how the membrane environment directs RTK structure and function:
As part of a new NIH-funded interdisciplinary team science project, we and several other laboratories in the Departments of Pharmacology and Cell Biology are working to understand how membrane composition directs membrane protein structure and function. We seek to define the components (lipid & protein) of functional complexes isolated from native membranes, to study the role of the local membrane environment in the function and regulation of the integral membrane proteins, and to determine the 3-dimensional structures of functional complexes. Members of the team focus on different biological systems, combinding their complementary expertise in cryo-electron microscopy, mass spectrometry, multi-omic analysis, optical imaging, biochemistry and cellular signaling. In the Ferguson group, we focus on select RTKs where modulation of function by lipid components is well characterized, and the potential to alter receptor function with drugs that modulate the local membrane environment has been suggested.
3. Antibody modulation of RTK regulation
Our laboratory has a long-standing interest in the mechanisms of inhibition of receptor tyrosine kinases (RTKs) by therapeutic antibodies, most notably those that bind the epidermal growth factor receptor (EGFR) - one of the first targets of antibody-based drugs to treat cancer. Most therapeutic antibodies to EGFR family members were developed before there was any structural understanding of the activation mechanism of these receptors. Whereas most therapeutic EGFR antibodies block ligand binding, inhibition of EGFR activity contributes little to the clinical effects. We seek to understand how select existing antibodies may alter receptor conformation to modulate function and whether somatic mutations in EGFR receptors may alter antibody binding. We are also developing new mechanism-based antibody therapeutics, drawing on the rich understanding of EGFR family receptor structure and dynamics. We combine X-ray crystallography, cryoEM, biochemistry, computational analysis, and cellular studies to address these questions.
A gallery of Fabs and nanobodies in complex wild type and variants of the EGFR ECR.