Molecular Mechanisms of Signaling Regulation in the Adhesion Family of G Protein- Coupled Receptors

Abstract

G protein-coupled receptors (GPCRs) comprise the largest family of human cell surface proteins and regulate nearly every facet of mammalian physiology, while also serving as the targets of approximately one third of all approved pharmaceutical drugs. The 33 members of the Adhesion Family of GPCRs (aGPCRs, or Family B2) comprise the second-largest evolutionary subgrouping of GPCRs in the human genome and control development, tissue homeostasis, and cell-cell communication in a wide range of tissues. While the relationships between many GPCRs and their activating ligands, known as agonists, are well understood from binding to physiological response, signaling mechanisms for aGPCRs are more complex due to the unique extracellular structures of these receptors. These ectodomains typically include large adhesion protein modules and a membrane-proximal regulatory domain called the GPCR Autoproteolysis-INducing (GAIN) domain. Within the GAIN domain resides a tethered agonist (TA) peptide ligand, which is thought to activate the corresponding receptor domain when the GAIN domain is physically disrupted, possibly through the application of force to the adhesion module. This dissertation presents significant experimental progress in elucidating how the complex ectodomain of any given aGPCR influences signaling. In Chapter 2, I describe the development of a system for measuring TA-induced aGPCR signaling with high signal-to-noise. I show that this assay system is modular, can be applied to aGPCRs in parallel, and accurately reports on structure-function relationships among the TA, GAIN, and receptor sequences. In Chapter 3, I describe the application of this signaling platform to the full family of 33 human aGPCRs. Across several orthogonal metrics of GPCR activation, from ligand binding to receptor desensitization, I show that, strikingly, only about one half of aGPCRs signal through their TA peptide. In addition, I profile the effectors activated by TA-driven aGPCR signaling for each family member. These studies reveal a substantial preference of the aGPCRs to couple to G12/13 heterotrimers, which are the least preferred G protein partners for proteins of other GPCR families. The activation of G12/13 proteins leads to actin cytoskeletal rearrangement, which is a key feature of many biological processes governed by aGPCRs, such as platelet activation, planar cell polarity, and synapse formation. In Chapter 4, I adapt my signaling platform to evaluate the effects of the GAIN domain on TA-dependent and TA-independent signaling by aGPCRs. These studies show that basal, TA- independent signaling of some, but not all, aGPCRs is autoinhibited by the GAIN domain. The work described in this dissertation thus provides a comprehensive analysis of aGPCR signal regulation at the molecular level, and should serve as a reference for the design and interpretation of in vivo studies and for the analysis and development of compounds that modulate aGPCR activity.