Structural and Biophysical Regulation of the Receptor Tyrosine Kinases dROS1 and ErbB3
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Pharmacology
First Advisor
Klein, Daryl
Abstract
Receptor tyrosine kinases (RTKs) are cell-surface protein receptors involved in the regulation of many critical cellular activities. They are known oncogenic drivers, and a broad spectrum of diseases, including cancers, are driven by abnormality and dysfunction of RTKs. Over the past 3 decades, numerous drugs including small molecule kinase inhibitors and monoclonal antibodies, have been developed to attenuate or inhibit RTK activities. Understanding the mechanistic and quantitative details of RTK regulation and activation is critical to develop new effective therapeutic strategies that target diseases driven by RTKs. Here in this dissertation, we describe structural and biophysical analyses of two important RTKs – dROS1 and ErbB3. ROS1 is one of the least well-studied receptors in the human RTK family. It is also an orphan RTK with an unknown ligand. Previous literature on the Drosophila homolog of human ROS1 (dROS1 or Sevenless), revealed both an unusual regulatory ligand (BOSS) and a unique activation mechanism. These differences point to an activation mechanism distinct from the canonical RTK paradigm. However, the structural basis for how dROS1 interacts with the G-Protein Coupled Receptor (GPCR) BOSS has long remained unknown. Here we provide the first structure, using cryogenic electron microscopy (Cryo-EM), of the amino (N)-terminal region of dROS1’s extracellular region (ECR), which mediates the binding interaction with BOSS. We show that the N-terminal region of dROS1 adopts a folded-over conformation harboring a novel structural domain that might function as a ligand for BOSS. We further narrowed down the interacting binding epitopes on both dROS1 and BOSS. This includes a beta-strand in dROS1’s third fibronectin type III (FN3) domain and BOSS’ C-terminal ECR. Our mutagenesis studies, coupled with AlphaFold complex predictions, support a binding interaction mediated by a hydrophobic interaction and a beta-strand augmentation between these regions. Our findings contribute to the understanding of the regulatory function of dROS1 and further provide mechanistic insight into the human ortholog and oncogene ROS1. ErbB3, part of the ErbB receptor family, is also an oncogenic RTK involved in a variety of cancers. The activation mechanism of ErbB receptors has been well-established. However, unlike other ErbB receptors, ErbB3 has little kinase activity and is only activated by forming a heterodimer with ErbB2. The regulatory control that physically prevents ErbB3 from homodimerization upon ligand binding– while providing heterodimerization with ErbB2 – has not yet been firmly established. Here we provide structural analysis together with biophysical investigations on ligand-bound ErbB3. My structure suggests domain II of ErbB3 plays a unique role in regulating ErbB3 homo and hetero dimerization. These findings help to establish a fundamental understanding of the molecular mechanism for ErbB receptor regulation.
Recommended Citation
Zhang, Jianan, "Structural and Biophysical Regulation of the Receptor Tyrosine Kinases dROS1 and ErbB3" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1491.
https://elischolar.library.yale.edu/gsas_dissertations/1491
