Project Summary/Abstract Cellular adhesion is a process critical for the development of all organs and is mediated by the adhesion family of cell-surface G protein-coupled receptors (aGPCRs). aGPCRs are a large, understudied group of receptors that link cell adhesion to cell signaling. aGPCRs regulate organ development, nervous system development and function; they are dysregulated in developmental disease, neurological disease, and cancer. aGPCRs stand as the next generation of drug targets for these indications, yet drug development is hindered by an incomplete understanding of the molecular basis for aGPCR function. aGPCRs are defined by their enigmatic multidomain extracellular regions (ECRs) which are hypothesized to mediate ligand binding and receptor activity. Our hypothesis is that the ECR can affect basal receptor signaling activity by occupying conformations that modulate the seven-transmembrane region of the aGPCR. This proposal aims to discover the structural basis of the regulation of an aGPCR by its ECR, the key gap in aGPCR biology. This will be done using a model receptor, GPR126. GPR126 is linked with peripheral nerve myelination and spine development. GPR126 dysregulation is associated with developmental disease and cancer. GPR126 has a large multidomain ECR with two splice variants that promote low and high signaling activity. Our lab previously determined the structure of one GPR126 splice variant which represents the low activity conformation of this ECR. I have further built the groundwork for this proposal by purifying the high activity GPR126 splice variant and obtaining protein crystals. I have also optimized a signaling assay that will allow me to interrogate the effect of point variations in the ECR on GPR126 signaling activity. I propose three Specific Aims that will help interrogate the structural basis GPR126 ECR function: First, I aim to determine a high-resolution structure of the high activity GPR126 splice variant. Second, I will test disease-associated GPR126 variations for their ability to alter activity using functional assays and I will collaborate with the laboratory of Dr. Kelly Monk to study the variants in zebrafish. Third, I will discover antibodies targeting the ECR of GPR126 and characterize them in functional assays. This proposal will result in a mechanistic description of ECR-dependent regulation of GPR126 signaling, thus explaining how dysregulation of this protein can contribute to disease. I will also produce antibodies which can be used as tools to probe GPR126 function in disease models. This proposal is a multi-disciplinary and collaborative one, with great training potential due to the span of techniques proposed, from structural biology to cell-based assays and antibody discovery. In the Araç laboratory, I will be provided the opportunity to work with a world leader in aGPCRs and to perform high-impact research on the molecular basis of adhesion GPCR signaling. I will expand my skillset into...