Structural and Functional Characterization of CELSR-subfamily adhesion G protein-coupled receptors - Project Summary/Abstract Cellular adhesion is a process critical for animal development and is mediated by the adhesion family of G protein-coupled receptors (aGPCRs), an understudied group of cell-surface receptors that link cell adhesion to intracellular signaling. Cadherin EGF Laminin G seven-pass G-type receptors (CELSRs or ADGRCs) are conserved aGPCRs which are essential for animal development. CELSRs are involved in the process of planar cell polarity (PCP), where they are key for neural tube closure and the organization of several tissues including the nervous system. Mutations in CELSRs are strongly associated with developmental pathologies and Tourette syndrome. However, there is a lack of molecular-level insight into CELSR function, and this hinders understanding of CELSR-mediated pathophysiology and future therapeutic development. CELSRs have large extracellular regions (ECRs) containing 23 domains which mediate cell adhesion and modulate intracellular signaling. In addition, CELSRs have large intracellular regions (ICRs) that mediate intracellular events, yet the ICR interactome is undefined. As preliminary data for this proposal, I determined the 4.3 Å cryo-EM reconstruction of the mCELSR1 ECR with 14 domains resolved in a compact conformation. I have also optimized assays to examine CELSR-mediated adhesion and signaling in cells. Finally, I have conducted bioinformatic analysis which identifies protein-protein interaction motifs in the ICR and begun cloning CELSR-APEX constructs to conduct a proximity labeling screen. My central hypotheses are that the compact CELSR ECR conformation regulates cell adhesion and signaling, and that the CELSR ICR mediates non-canonical events. This proposal aims to determine the structural basis for regulation of CELSR function by its ECR, and to identify non- canonical binding partners of the ICR. I propose three specific aims: First, I will improve my cryo-EM reconstruction of the mCELSR1 ECR in order to build an experimentally derived atomic model, with help from cryo-EM expert Dr. Minglei Zhao. Second, I will test disease associated variants for their ability to alter the functions of CELSR1. This aim will grow my experience in cell-based assays. Third, I will discover binders of the CELSR1 ICR using a proximity labeling screen, and in parallel, pursue likely targets such as RhoGEFs. I will be trained by my co-mentor and GPCR-APEX expert Dr. Andrew Kruse, and I will learn to interrogate the results of the screens from GPCR signaling expert Dr. Silvio Gutkind. This proposal will result in a mechanistic description of ECR-dependent regulation of CELSR function, explaining how dysregulation of this protein can contribute to disease. I will also identify the CELSR ICR interactome; this will give me the material to study the structural biology of non-canonical signaling downstream of aGPCRs. In the Araç laboratory, I will be provided the opportunity to work with the world leader in aGPCR structural biology; I will continue to develop my skills as a scientist in the world-class environment that the University of Chicago provides, and with my mentoring team I will develop the material and skillsets to start my own independent laboratory at a research-intensive institution.
