Specific and direct modulation of the interaction between adhesion GPCR GPR56/ADGRG1 and tissue transglutaminase 2 using synthetic ligands

Shu Zhang,Demet Araç,Celia G. Fernandez,Shohei Koide,Gabriel S. Salzman

doi:10.1038/s41598-020-74044-6

Abstract

Blocking the interaction between cell-surface receptors and their ligands is a proven therapeutic strategy. Adhesion G protein-coupled receptors (aGPCRs) are key cell-surface receptors that regulate numerous pathophysiological processes, and their large extracellular regions (ECRs) mediate ligand binding and function. The aGPCR GPR56/ADGRG1 regulates central nervous system myelination and melanoma progression by interacting with its ligand, tissue transglutaminase 2 (TG2), but the molecular basis for this interaction is largely undefined. Here, we show that the C-terminal portion of TG2 directly interacted with the GPR56 ECR with high-nanomolar affinity, and used site-directed mutagenesis to identify a patch of conserved residues on the pentraxin/laminin-neurexin-sex-hormone-binding-globulin-like (PLL) domain of GPR56 as the TG2 binding site. Importantly, we also show that the GPR56-TG2 interaction was blocked by previously-reported synthetic proteins, termed monobodies, that bind the GPR56 ECR in a domain- and species-specific manner. This work provides unique tools to modulate aGPCR-ligand binding and establishes a foundation for the development of aGPCR-targeted therapeutics.

Highlights

Cell-surface receptors communicate signals across the plasma membrane
We have shown that the GPR56 extracellular regions (ECRs) is composed of an N-terminal pentraxin and laminin/neurexin/sex hormone-binding globulin (PLL) domain and a juxtamembrane GPCR autoproteolysis-inducing (GAIN) domain and that the ECR plays a receptor-autonomous regulatory role in G protein signaling[12]
As experiments were not performed with purified proteins, it remained unclear whether the GPR56-transglutaminase 2 (TG2) interaction was a direct, binary interaction that might be suitable for targeting by pharmaceutical agents

Summary

Introduction

Cell-surface receptors communicate signals across the plasma membrane. Vital to this process is the extracellular interaction between a signaling molecule and its receptor, which initiates a multi-step cascade, and results in a cellular response. Recent work has shown that aGPCR ECRs play a direct role in the regulation of receptor function and downstream signaling[11,12,13,14,15,16,17,18,19]. Though most aGPCRs are still orphan receptors, the ligands of several aGPCRs have been identified as other cell-surface proteins or soluble extracellular matrix (ECM) proteins[14,20,21,22]. TG2 is an enzyme that plays an important role in cross-linking proteins in the extracellular matrix (ECM). It has four domains (D1-D4), the second of which (D2) catalyzes a Ca2+-dependent transamidation reaction that crosslinks proteins in the ECM46,47. As experiments were not performed with purified proteins, it remained unclear whether the GPR56-TG2 interaction was a direct, binary interaction that might be suitable for targeting by pharmaceutical agents

Methods

Results

Conclusion