The N Terminus of the Adhesion G Protein-coupled Receptor GPR56 Controls Receptor Signaling Activity

Abstract

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of ␤-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with ␤-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.During the development of the cerebral cortex, neuronal precursors proliferate in the ventricular and subventricular zones that line the cerebral cavity and then migrate outward to make connections with other neurons. Given the billions of cells involved and the requirements for temporal and spatial precision, it is perhaps not surprising that many different types of problems can arise during this process. Abnormalities in cortical development can lead to a range of distinct neurodevelopmental disorders, some of which are caused by mutations to a single gene. For example, bilateral frontoparietal polymicrogyria is a condition in which patients exhibit profound cognitive abnormalities and seizures because of disordered cortical connectivity in the frontoparietal area. Bilateral frontoparietal polymicrogyria is an autosomal recessive syndrome that results from mutations in the orphan receptor GPR56 (1). Thus, insights into the natural function of GPR56 might shed light on the specific pathology underlying bilateral frontoparietal polymicrogyria and also lead to new insights about the fundamental mechanisms controlling cortical development.GPR56 is a member of the adhesion family of G proteincoupled receptors (GPCRs) 2 , which are characterized by extremely large extracellular N termini (NT) exhibiting homology to adhesion proteins (2). There are approximately 30 adhesion GPCRs, all of which are still considered to be orphan receptors. Almost all members of the adhesion GPCR family possess an N-terminal region known as a "GPCR proteolytic site" or GPS dom...

Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist

“…3 B and C), corroborating previous work and our finding that active urea-mediated ECD dissociation from full-length aGPCRs resulted in receptor activation ( Fig. 2 C and D) (11,12). Serial deletion of single amino acids from the GPR56 or GPR110 7TM domain N termini sequentially reduced the ability of the receptors to activate G proteins.…”

“…The means of ligand action resulting in aGPCR activation is unknown. Transfection of aGPCRs with deleted N-terminal ECDs (expressed 7TM domains only) enhanced cell-based signaling outputs (11,12). Therefore, aGPCR ECDs are suspected to impart an inhibitory influence upon the 7TM domains, and ligands are proposed to bind the ECD and alter its orientation with respect to the 7TM domain to relieve this inhibition.…”

Agonistic Antibodies Reveal the Function of GPR56 in Human Glioma U87-MG Cells

“…It was reported that the GPR56ECD may have an inhibitory effect on GPR56 signaling. 16) Therefore, there was one possibility that agonistic antibodies weaken the interaction between the ECD and TM. To investigate this possibility, we performed immunoprecipitation experiments using agonistic or non-functional antibodies.…”

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

“…1). After cleavage, the NTF and CTF remain noncovalently but tightly associated throughout trafficking and localization to the plasma membrane (20,22). The conservation of the GAIN domain suggests that it plays a role in aGPCR function.…”

“…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.…”