Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Abstract

Summary Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology.

Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

“…Indeed, this was the case for the α5 monobody that we generated previously (36). Consequently, we decided to perform monobody selection separately for human and mouse GPR56 samples.…”

“…Although the single-domain constructs have exposed hydrophobic surfaces that would be sequestered in the interface between the PLL and GAIN domains, they were highly soluble and predominantly monomeric. Using a total of six samples as antigens, we carried out phage and yeast display selection of monobodies as previously described (36,37) and identified 19 clones (Table S1). These included human GPR56-specific clones that bind to the PLL domain but not the GAIN domain [e.g., Mb(hGPR56_β7)], to the GAIN domain but not the PLL domain [e.g., Mb(hGPR56_β6)], and to full-length ECR but not the PLL or GAIN domain in isolation [e.g., Mb(hGPR56_β1)] ( Fig.…”

“…Although it has been proposed that natural ligands may induce shedding on binding to N-terminal adhesion domains and thereby, activate the receptor, direct proof of ligand-induced shedding remains elusive. Several recent observations, including that some aGPCRs do not undergo autoproteolysis and therefore, cannot undergo shedding (20,34), have necessitated the introduction of a model, in which the ECR (i.e., associated NTF and Stachel) has a direct role in modulating the 7TM signaling (22,33,35,36). Regulation by this mechanism, which we term "Stachel-independent," is independent of Stachel-mediated activation, although the Stachel residues are present within the core of the GAIN domain (Fig.…”

“…It has a 377-residue ECR composed of two domains: an N-terminal pentraxin and laminin/neurexin/sex hormonebinding globulin-like (PLL) domain and a GAIN domain (36). Previously, we have shown that deletion of the PLL domain increases basal activity of the receptor (36).…”

“…It has a 377-residue ECR composed of two domains: an N-terminal pentraxin and laminin/neurexin/sex hormonebinding globulin-like (PLL) domain and a GAIN domain (36). Previously, we have shown that deletion of the PLL domain increases basal activity of the receptor (36). Additionally, we engineered a binding protein, termed monobody α5, that targets the ECR of mouse GPR56, bridges the PLL and GAIN domains, and functions as an allosteric inverse agonist of G-protein signaling.…”