The family of adhesion G protein coupled receptors (aGPCRs) is the second largest class of GPCRs with increasingly recognized functions in development and disease. Structurally, aGPCRs are characterized by the presence of an extremely long N-terminal region that contains a GPCR autoproteolysis-inducing (GAIN) domain and seven transmembrane spanning regions. Most aGPCRs undergo GAIN domain-mediated autoproteolytic processing to generate an N- and a C-terminal fragment. GPR56/ADGRG1, a member of the adhesion GPCR family, was first cloned in 1999 by two independent groups, one searching for further members of the secretin family of GPCRs and the other screening for differentially expressed genes in a human melanoma metastasis model (Liu et al. 1999; Zendman et al. 1999). However, the function of GPR56 was not known until the discovery of its loss of function mutations linked to an autosomal recessive human brain malformation in 2004. Since then, GPR56 has been found to play several important roles in development and disease, most importantly in the central nervous system (CNS) and the hematopoietic system.
Structure and Signaling of GPR56
The first observation that GPR56 signals through RhoA pathway came from an in vitro study using an antibody against the extracellular domain of GPR56 (Iguchi et al. 2008). This finding was subsequently confirmed in a ligand-specific manner. The binding of collagen III to GPR56 activates RhoA, which can be attenuated by dominant negative Gα13, supporting the notion that GPR56 couples to Gα12/13 upon ligand binding. Mice lacking either GPR56 or collagen III display a malformed cerebral cortex, characterized by overmigration of neurons beyond the pial basement membrane (Luo et al. 2011). A very similar phenotype was found in mice lacking Gα12/13 (Moers et al. 2008), further supporting a model in which Gα12/13 is downstream of GPR56 signaling. The definitive evidence for GPR56 coupling to Gα13 came from acellular assays. Human GPR56 was expressed in insect cells, and prepared receptor membranes were reconstituted with purified G protein heterotrimers of defined composition before measurement of receptor-stimulated G protein activation. GPR56 robustly activated Gα13, only modestly coupled to Gi, and did not activated Gq or Gs (Stoveken et al. 2015). This signaling mechanism is not limited to cerebral cortical development. Recent work in mice and zebrafish demonstrated that GPR56 regulate oligodendrocyte proliferation by coupling Gα12/13 and activating RhoA (Ackerman et al. 2015; Giera et al. 2015).
The Ligands and Binding Partners of GPR56
Through work spurred by the discovery of GPR56 as an important mediator in cortical development, collagen III, a protein expressed by meningeal fibroblasts and endothelium of blood vessels in the developing brain, was identified as the primary CNS ligand of GPR56 (Luo et al. 2011). The GPR56-collagen III interaction activates the RhoA pathway by coupling to Gα12/13 and inhibits neuronal migration. Deleting mouse Col3a1 results in a cobblestone-like malformation similar to that seen in Gpr56 knockout mice, further validating collagen III as the primary CNS ligand for GPR56 (Luo et al. 2011). The binding of collagen III removes GPR56 NTF and thus exposes the tethered agonist and activates the receptor (Luo et al. 2014) (Fig. 2).
In the context of melanoma, GPR56 also binds specifically to tissue transglutaminase (TG2), a major cross-linking enzyme in the extracellular matrix that regulates cell adhesion (Xu et al. 2006). TG2 binds the NTF of GPR56 with its two C-terminal β-barrels. This interaction does not appear to induce canonical downstream signaling but instead leads to extracellular matrix remodeling. Recent experiments with a TG2 knockout model demonstrate that GPR56 antagonizes TG2’s inherent cross-linking activity (which promotes melanoma growth) and fibronectin deposition through receptor-mediated internalization and degradation (Yang et al. 2014a).
In an in vitro culture system, the NTF of GPR56 was found to bind heparin, where heparin binding reduces GPR56 receptor shedding without affecting membrane distribution of either the NTF or CTF (Fig. 1) (Chiang et al. 2016). Noting that both known ligands of GPR56, collagen III and TG2, are heparin-binding proteins, it is possible that heparin may influence how GPR56 interacts with its other protein ligands.
Recently, dihydromunduletone (DHM), a rotenoid derivative, was found to be a selective antagonist specifically for adhesion GPCRs GPR56 and GPR114. Its mechanism of action targets GPR56’s CTF directly, acting as a neutral antagonist and competing with the tethered-peptide agonist for its orthosteric binding site, although allosteric modulation is also a possibility. DHM inhibits peptide agonist-stimulated activity, but not basal receptor activity, and its discovery opens the doors to the recognition of further small-molecule modulators of aGPCRs (Stoveken et al. 2016).
GPR56 has also been shown to interact with membrane proteins. In 2004, biochemical approaches first suggested an interaction between GPR56 and Gαq/11, with CD 9 and CD81 acting as scaffolding proteins for signal transduction, although the downstream signaling targets remain unknown (Little et al. 2004). A synergistic function between integrin α3β1 and GPR56 has been demonstrated, in which more severe neuronal overmigration through breached pial basement membranes was observed in a double knockout model than in Gpr56 single knockout (Jeong et al. 2013).
Role in Central Nervous System
GPR56 also plays an integral role in the myelination of the CNS. Magnetic resonance imaging of BFPP brains displays reduced white matter volume and T2-weighted signal changes, suggestive of defective myelination (Bahi-Buisson et al. 2010; Piao et al. 2005; Piao et al. 2004). The expression of Gpr56 is restricted to oligodendrocyte (OL) precursor cells (OPC) and immature OLs and then downregulated before terminal differentiation (Giera et al. 2015; Zhang et al. 2014). Work from zebrafish and mouse models demonstrates that GPR56 is required for OPC proliferation by activating RhoA pathway (Ackerman et al. 2015; Giera et al. 2015).
Role in Oncologic Disorders
GPR56 was found to be significantly downregulated in high metastatic melanoma cells (Xu et al. 2006; Zendman et al. 1999). Its relation with a variety of oncologic processes has been progressively explored since that initial discovery. Its function as a modulator of cell adhesion and its relationship with the extracellular matrix play an important role in tumor growth and spread. In searches for tumor prognostic factors, GPR56 was found to be differentially expressed or translated in a number of cancers, including overexpression in glioblastomas/astrocytomas (Shashidhar et al. 2005), overexpression in esophageal squamous cell carcinomas, decreased expression in pancreatic cancer, overexpression in childhood T-cell acute lymphoblastic leukemia, overexpression in hepatocellular carcinoma , and overexpression in colorectal cancers, especially in proximal adenocarcinomas. It has also recently been shown that GPR56 is an unfavorable prognostic factor in osteosarcoma patients (Xu et al. 2010).
In primary acute myeloid leukemia (AML) patients, high level GPR56 expression is significantly associated with poor outcomes and can be used as a marker for subpopulations of AML cells with different stem cell-like properties, especially those with high engraftment capacity (Pabst et al. 2016). GPR56 has furthermore been shown to contribute to the development of AML in a mouse model, accelerating myeloid leukemogenesis in vivo, and overexpression of Gpr56 leads to a differential expression of almost 900 genes. Additionally, blocking GPR56 with a monoclonal antibody led to impairment of engraftment of human AML cells into a mouse model (Daria et al. 2016), which may be secondary to impaired cell adhesion and increased cellular migration of AML cells (Saito et al. 2013). GPR56 is also implicated in apoptosis, with increased rates of apoptosis in those AML cells with GPR56 knocked down (Saito et al. 2013).
Through further exploration of the melanoma model, several important functions of GPR56 have been discovered. The internalization and degradation of TG2 was found to be important in the inhibition of melanoma progression (Yang et al. 2014a). Furthermore, GPR56 was shown to regulate VEGF production through protein kinase C activation, which is activated by the CTF (or GPR56 lacking the TG2 binding segment) and inhibited by full-length GPR56, implying the delicate balance GPR56 plays in melanoma progression (Yang et al. 2011).
Role in Hematopoiesis and Lymphocyte Function
GPR56 is required for the maintenance of hematopoietic stem cells (HSCs) during embryonic and adult hematopoiesis (Saito et al. 2013) and in facilitating the process known as endothelial to hematopoietic cell transition (EHT) for generation of the first HSCs, a function evolutionarily conserved between mouse and zebrafish (Solaimani Kartalaei et al. 2015). In Gpr56 −/− mice, both short-term and long-term HSCs were significantly reduced in the bone marrow. However, evidence indicates that some of these HSCs migrate from the bone marrow to peripheral organs where they retain their differentiation capacity, as opposed to those who remain in the bone marrow, which correlates with findings of enlarged thymuses in knockout mice (Saito et al. 2013). Knockdown of Gpr56 results in loss of the repopulating potential of hematopoietic stem cells as well as impaired cellular adhesion (Saito et al. 2013). Its highest expression patterns are within primitive lineages, and this expression declines sharply as these HSCs undergo differentiation into mature blood types, with the absence of GPR56 not significantly affecting steady-state hematopoiesis (Rao et al. 2015).
GPR56 also exerts its function in lymphoid lineage. Human cytotoxic lymphocytes, including NK cells, express GPR56, which is downregulated upon NK cellular activation (Chang et al. 2016; Peng et al. 2011). This expression in lymphocytes is driven by the recently discovered transcription factor Hobit (Chang et al. 2016). Cytomegalovirus infection induces GPR56 expression, starting at the peak of infection and then steadily increasing for up to 1-year postinfection (Peng et al. 2011). Not required for NK cell development, GPR56 is rather a differentiation marker (Chang et al. 2016). It functions as an inhibitory receptor that inhibits immediate effector functions, including inflammatory cytokine and cytolytic protein production, degranulation, and target cell killing. Association with CD81 (but not any G proteins) appears to be critical to these inhibitory functions of GPR56 (Chang et al. 2016). Consisting with those experimental findings with cell line, NK cells isolated from BFPP patients with confirmed GPR56 mutations present with elevated cytotoxic function (Chang et al. 2016).
Role in Other Developmental Processes
GPR56 has a variety of other roles during development and disease. In gonadal development, GPR56 was found to have increased expression during gonadal sex differentiation in female chicken embryo (Ayers et al. 2015) and is required for testis development/male fertility in the mouse (Chen et al. 2010). GPR56 is expressed in skeletal muscle cells and mediates overload-induced muscle hypertrophy and associated anabolic signaling (White et al. 2014). In cardiomyocytes, GPR56 promotes a hypertrophic response that is regulated by a RNA-binding protein, PCBP2 (Zhang et al. 2015). GPR56 displays a decreased expression profile in patients with idiopathic pulmonary fibrosis (Yang et al. 2014b).
GPR56 is an adhesion G protein coupled receptor with a number of important implications in development and disease. It functions through interactions with multiple ligands/binding partners. Some of these have been identified (TG2 and collagen III), but work remains to be continued to characterize the full range of binding partners for GPR56. It works through a diverse range of downstream signaling pathways, including those utilizing canonical G proteins and those dependent on lateral interactions with other membrane proteins such as integrin and tetraspanin proteins. To date, the most notable functions of GPR56 are in the central nervous system development, oncologic disorders, and hematopoiesis. Studies continue to find the importance of GPR56 throughout other developmental processes, from gonadal differentiation to skeletal muscle hypertrophy and pulmonary fibrosis. As further associations to human disease and development are revealed, GPR56 becomes more compelling as an attractive drug target. As GPR56’s structure and function are more thoroughly investigated, light will be also shed on the general family of adhesion GPCRs, many of which remain orphan receptors with unclear mechanisms of action. Unlocking the mysteries of this family may open a field rich with novel therapeutic targets for human disease.
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