Receptor tyrosine kinase activation is incompletely understood. In the classic model, a ligand induces dimerization of the extracellular modules of two receptors to bring the cytoplasmic kinase domains into close proximity. This facilitates transphosphorylation of tyrosines to stabilize an active conformation. While this general model is valid, many additional layers of regulation exist. Recent studies indicate that different receptor tyrosine kinase families may have distinct modes of regulation. Specifically, the detailed mechanism that restricts PDGFR activation at the membrane is controversial. This family of receptor tyrosine kinases has five extracellular immunoglobulin-like modules, a transmembrane (TM) helix, and an intracellular module that consists of a juxtamembrane domain (JM), a kinase domain with a kinase insert segment, and a C-terminal tail. OBJECTIVE/HYPOTHESIS: The molecular details that depict how PDGFR-? is inhibited at the membrane and how it becomes activated upon ligand binding are limited. However, PDGFR-? does not appear to behave according to the classic model for receptor tyrosine kinase activation. Thus, I plan to systematically investigate the regulatory roles of the individual domains on PDGFR-?. Molecular characterization of this system will provide insight into how receptor tyrosine kinases prevent inadvertent transphosphorylation and activation from occurring spontaneously in the highly crowded environment at the plasma membrane. STUDY DESIGN: I plan to investigate the role of the individual components of PDGFR-? in its activation mechanism.
In Aim 1, I will investigate the role of the intracellular module in inhibiting and activating PDGFR-?. I will generate constructs with deletion of the JM domain, the C-terminal tail, and both. I will monitor the activiy of the isolated kinases in solution and on lipid vesicles to evaluate how molecular crowding influences their function.
In Aim 2, I will investigate the role of the extracellular module and th TM helix in coupling ligand binding to kinase activation in cells. I will study the full-length receptor in the presence and absence of ligand, PDGF-BB, as well as two mutant receptors with either the extracellular module deleted or a linker inserted between the extracellular module and the TM helix. I will evaluate how these variants influence both oligomerization of the receptor using a technique called two-color pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and autophosphorylation of the intracellular kinase domain using immunofluorescence assays.
In Aim 3, I will evaluate if the plasma membrane itself can restrict PDGFR-? activation in cells. Using the assays described in Aim 2, I will study the effect that the membrane has on just the intracellular module of the receptor when it is targeted to the plasma membrane with a localization motif. HEALTH RELATEDNESS: Abnormal activation of PDGFR due to mutation or overexpression is implicated in the pathogenesis of many human cancers. Understanding the molecular details that regulate its activation can provide insight into how oncogenic mutations affect the receptor and can lead to novel therapeutic approaches.
Aberrant activation of PDGFR and other related receptor tyrosine kinases due to overexpression or mutation is implicated in the progression of cancer, yet its mechanism of activation is incompletely understood. In this proposal, I aim to systematically interrogate the modes of regulation of PDGFR-? at the plasma membrane using innovative biophysical, biochemical, and structural techniques. This insight into the molecular details of activation will provide an understanding of both PDGFR's function in initiating signaling pathways as well as the effect that oncogenic mutations have on the receptor, and may lead to new therapeutic strategies.
|Kovacs, Erika; Zorn, Julie Anne; Huang, Yongjian et al. (2015) A structural perspective on the regulation of the epidermal growth factor receptor. Annu Rev Biochem 84:739-64|
|Zorn, Julie A; Wang, Qi; Fujimura, Eric et al. (2015) Crystal structure of the FLT3 kinase domain bound to the inhibitor Quizartinib (AC220). PLoS One 10:e0121177|
|Wang, Qi; Zorn, Julie A; Kuriyan, John (2014) A structural atlas of kinases inhibited by clinically approved drugs. Methods Enzymol 548:23-67|