Adhesion GPCR GPR56 Expression Profiling in Human Tissues

Despite the immense functional relevance of GPR56 (gene ADGRG1) in highly diverse (patho)physiological processes such as tumorigenesis, immune regulation, and brain development, little is known about its exact tissue localization. Here, we validated antibodies for GPR56-specific binding using cells with tagged GPR56 or eliminated ADGRG1 in immunotechniques. Using the most suitable antibody, we then established the human GPR56 tissue expression profile. Overall, ADGRG1 RNA-sequencing data of human tissues and GPR56 protein expression correlate very well. In the adult brain especially, microglia are GPR56-positive. Outside the central nervous system, GPR56 is frequently expressed in cuboidal or highly prismatic secreting epithelia. High ADGRG1 mRNA, present in the thyroid, kidney, and placenta is related to elevated GPR56 in thyrocytes, kidney tubules, and the syncytiotrophoblast, respectively. GPR56 often appears in association with secreted proteins such as pepsinogen A in gastric chief cells and insulin in islet β-cells. In summary, GPR56 shows a broad, not cell-type restricted expression in humans.

CSIG-25. BAI1/ADGRB1 REGULATES TGFβ1-INDUCED MESENCHYMAL SWITCH IN MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant brain tumor in children. MB tends to metastasize to the brain meninges and subarachnoid space and the spinal cord. Leptomeningeal metastasis is frequently found at initial diagnosis and leads to tumor relapse after standard treatment. Leptomeningeal metastasis remains a major challenge and is related with poor outcome. Acquiring a better knowledge of molecular defects underlying metastatic disease is essential for the development of effective therapies. Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in normal brain, but its expression is lost in the majority of medulloblastoma through epigenetic silencing. We reported that BAI1 protects p53 from Mdm2-mediated degradation and regulate tumor growth in medulloblastoma (Zhu D. et al, Cancer Cell, 2018). However, it is unclear whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in MB. Microarray analysis of the published MB dataset revealed that low BAI1 mRNA expression correlates with poor outcome and with expression of many key mesenchymal genes, including Fibronectin1, SLUG, and TWIST1. Restoration of BAI1 expression in human MB cells suppresses mesenchymal gene expression in culture, and dramatically decreases brain tumor invasion. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in MB cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically reduced tumor cell invasion of MB cells. These experiments demonstrate that epigenetic silencing of BAI1 is important for activation of the MB invasive phenotype through TGFβ1 pathway activation. Epigenetic targeting of this process by KCC-07 can reduce MB invasion.

Activation of the adhesion GPCR GPR133 (ADGRD1) by antibodies targeting the N-terminus

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor (aGPCR) whose canonical signaling raises cytosolic cAMP, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We showed that dissociation of autoproteolytically generated N-terminal and C-terminal fragments (NTF and CTF) of GPR133 at the plasma membrane promotes receptor activation and increases signaling. Toward developing biologics modulating GPR133 function, we tested antibodies against the N-terminus of GPR133 for effects on receptor signaling. Treatment of HEK293T cells overexpressing GPR133 with such antibodies increased cAMP levels in a concentration-dependent manner. Analysis of supernatants following antibody treatment revealed complexes of the antibodies with the autoproteolytically cleaved NTF of GPR133. Cells expressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. The antibody-mediated stimulation of wild-type GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that NTF-CTF dissociation promotes receptor activation and signaling.

Adhesion Class GPCRs in GtoPdb v.2021.3

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [9] containing a GPCR proteolytic site. The N-terminus often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [101, 403]. Several receptors have been suggested to function as mechanosensors [309, 280, 383, 35]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [122].

Duale Rezeptorsignale: Wie setzen Adhäsions-GPCR Signale in Funktion um?

Adhesion GPCR are exceptional receptors due to their functional and structural diversity. A key to their function/signalling, setting them apart from other GPCR, is their extraordinarily large, complex N terminus, via which they mediate different molecular mechanisms and integrate diverse biological functions. Here, we discuss dual modes of adhesion GPCR action and how they translate into physiological functions: activation of G protein pathways and signals solely elicited by the N terminus.

Constitutive signaling by the C-terminal fragment of polycystin-1 is mediated by a tethered peptide agonist

Mutation of the PKD1 gene, encoding polycystin-1 (PC1), is the primary cause of autosomal dominant polycystic kidney disease. PC1 is an 11-transmembrane domain protein that binds and modulates the activity of multiple heterotrimeric G protein families and is thought to function as a non-canonical G protein-coupled receptor (GPCR). PC1 shares a conserved GPCR autoproteolysis inducing (GAIN) domain with the adhesion family of GPCRs, that promotes an auto-catalytic, cis-cleavage at the GPCR proteolysis site (GPS) located proximal to the first transmembrane domain. GPS cleavage divides these receptors into two associated subunits, the extracellular N-terminal (NTF) and transmembrane C-terminal (CTF) fragments. For the adhesion GPCRs, removal of the NTF leads to activation of G protein signaling as a result of the exposure and subsequent intramolecular binding of the extracellular N-terminal stalk of the CTF, i.e., the tethered cryptic ligand or tethered agonist model. Here, we test the hypothesis that PC1-mediated signaling is regulated by an adhesion GPCR-like, tethered agonist mechanism. Using cell-based reporter assays and mutagenesis of PC1 expression constructs, we show that the CTF form of PC1 requires the stalk for signaling activation and synthetic peptides derived from the PC1 stalk sequence can re-activate signaling by a stalk-less CTF. In addition, we demonstrate that ADPKD-associated missense mutations within the PC1 stalk affect signaling and can inhibit GPS cleavage. These results provide a foundation for beginning to understand the molecular mechanism of G protein regulation by PC1 and suggest that a tethered agonist-mediated mechanism can contribute to PKD pathogenesis.

Orphan GPR116 mediates the insulin sensitizing effects of the hepatokine FNDC4 in adipose tissue

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.