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

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.

Functions of G protein‐coupled receptor 56 in health and disease

“…It comprises an N‐terminal pentraxin/laminin/neurexin/sex‐hormone‐binding‐globulin‐like (PLL) domain and a GPCR autoproteolysis‐inducing (GAIN) domain. The PLL domain plays a key role in regulating G protein signaling; deletion of this domain significantly enhances the basal activity of GPR56 and is considered as a splice variant of the receptor 10,19 . The GAIN domain is composed of subdomain A and subdomain B. Subdomain A is a triple α‐helix structure, while subdomain B is a highly conserved sequence containing a GPCR proteolysis site (GPS) and a tethered peptide directly next to the 7‐transmembrane (7TM) domain 20 .…”

Adhesion G protein-coupled receptors in glioblastoma

“…Different monobodies against GPR56 were shown to both increase and decrease GPR56-mediated signaling in HEK239T cells and hence modulate the receptor in vitro. 101 By targeting the PLL domain of GPR56 with monobodies, Salzman et al 124 were able to disrupt receptor interaction with TG2. The development of monobodies against other aGPCRs implicated in GBM and their testing in vivo would serve as an advanced tool in discovering new therapeutic options.…”

Role of ADGRG1/GPR56 in Tumor Progression

“…It was revealed that the GPR56 receptor in oligodendrocyte precursor cells (OPCs) responded to the microglia-derived TG2 in the presence of laminin. Recently, the PLL domain was identified as the specific binding site for collagen-III and TG2 on GPR56 [ 37 , 38 ].…”