Adhesion GPCR GPR56 Expression Profiling in Human Tissues.
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ABSTRACT:
SUBMITTER: Kaiser F
PROVIDER: S-EPMC8700376 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Adhesion G protein-coupled receptors (aGPCRs) comprise the second-largest class of GPCRs, the most common target for approved pharmacological therapies. aGPCRs play an important role in development and disease and have recently been associated with the kidney. Several aGPCRs are expressed in the kidney and some aGPCRs are either required for kidney development or their expression level is altered in diseased kidneys. Yet, general aGPCR function and their physiological role in the kidney are poorly understood. Here, we characterize in detail Gpr126 (Adgrg6) expression based on RNAscope® technology in zebrafish, mice, and humans during kidney development in adults. Gpr126 expression is enriched in the epithelial linage during nephrogenesis and persists in the adult kidney in parietal epithelial cells, collecting ducts, and urothelium. Single-cell RNAseq analysis shows that gpr126 expression is detected in zebrafish in a distinct ionocyte sub-population. It is co-detected selectively with slc9a3.2, slc4a4a, and trpv6, known to be involved in apical acid secretion, buffering blood or intracellular pH, and to maintain high cytoplasmic Ca2+ concentration, respectively. Furthermore, gpr126-expressing cells were enriched in the expression of potassium transporter kcnj1a.1 and gcm2, which regulate the expression of a calcium sensor receptor. Notably, the expression patterns of Trpv6, Kcnj1a.1, and Gpr126 in mouse kidneys are highly similar. Collectively, our approach permits a detailed insight into the spatio-temporal expression of Gpr126 and provides a basis to elucidate a possible role of Gpr126 in kidney physiology.
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Project description:In the vertebrate central nervous system, myelinating oligodendrocytes are postmitotic and derive from proliferative oligodendrocyte precursor cells (OPCs). The molecular mechanisms that govern oligodendrocyte development are incompletely understood, but recent studies implicate the adhesion class of G protein-coupled receptors (aGPCRs) as important regulators of myelination. Here, we use zebrafish and mouse models to dissect the function of the aGPCR Gpr56 in oligodendrocyte development. We show that gpr56 is expressed during early stages of oligodendrocyte development. In addition, we observe a significant reduction of mature oligodendrocyte number and myelinated axons in gpr56 zebrafish mutants. This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential. Finally, we show that these functions are mediated by G?12/13 proteins and Rho activation. Together, our data establish Gpr56 as a regulator of oligodendrocyte development.
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Project description:The androgen receptor (AR) is activated in patients with castration resistant prostate cancer (CRPC) despite low circulating levels of androgen, suggesting that intracellular signaling pathways and non-androgenic factors may contribute to AR activation. Many G-protein coupled receptors (GPCR) and their ligands are also activated in these cells indicating that they may play a role in development of Prostate Cancer (PCa) and CRPC. Although a cross talk has been suggested between the two pathways, yet, the identity of GPCRs which may play a role in androgen signaling, is not established yet. By using blast analysis of 826 GPCRs, we identified a GPCR, GPCR 205, which exhibited maximum similarity with the ligand binding domain of the AR. We demonstrate that adhesion GPCR 205, also known as GPR56, can be activated by androgens to stimulate the Rho signaling pathway, a pathway that plays an important role in prostate tumor cell metastasis. Testosterone stimulation of GPR56 also activates the cAMP/ Protein kinase A (PKA) pathway, that is necessary for AR signaling. Knocking down the expression of GPR56 using siRNA, disrupts nuclear translocation of AR and transcription of prototypic AR target genes such as PSA. GPR56 expression is higher in all twenty-five prostate tumor patient's samples tested and cells expressing GPR56 exhibit increased proliferation. These findings provide new insights about androgen signaling and identify GPR56 as a possible therapeutic target in advanced prostate cancer patients.
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Project description: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.
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Project description:GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.
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Project description:The diversity and near universal expression of G protein-coupled receptors (GPCR) reflects their involvement in most physiological processes. The GPCR superfamily is the largest in the human genome, and GPCRs are common pharmaceutical targets. Therefore, uncovering the function of understudied GPCRs provides a wealth of untapped therapeutic potential. We previously identified an adhesion-class GPCR, Gpr116, as one of the most abundant GPCRs in the kidney. Here, we show that Gpr116 is highly expressed in specialized acid-secreting A-intercalated cells (A-ICs) in the kidney using both imaging and functional studies, and we demonstrate in situ receptor activation using a synthetic agonist peptide unique to Gpr116. Kidney-specific knockout (KO) of Gpr116 caused a significant reduction in urine pH (i.e., acidification) accompanied by an increase in blood pH and a decrease in pCO2 compared to WT littermates. Additionally, immunogold electron microscopy shows a greater accumulation of V-ATPase proton pumps at the apical surface of A-ICs in KO mice compared to controls. Furthermore, pretreatment of split-open collecting ducts with the synthetic agonist peptide significantly inhibits proton flux in ICs. These data suggest a tonic inhibitory role for Gpr116 in the regulation of V-ATPase trafficking and urinary acidification. Thus, the absence of Gpr116 results in a primary excretion of acid in KO mouse urine, leading to mild metabolic alkalosis ("renal tubular alkalosis"). In conclusion, we have uncovered a significant role for Gpr116 in kidney physiology, which may further inform studies in other organ systems that express this GPCR, such as the lung, testes, and small intestine.
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Project description:ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways.
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Project description:To study growth and development of ocular tissues, gene expression patterns in normal human fetal versus adult eyes were compared. Human retina/retinal pigment epithelium, choroid, sclera, optic nerve* and cornea* tissues were dissected from fetal (24 week gestational age) (N = 9; *N = 6), and adult (N = 6) normal donor eyes. The Illumina(®) whole genome expression microarray platform was used to assess differential expression. Statistical significance for all comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Significant gene expression fold changes > 1.5 were found in adult versus fetal retina/RPE (N = 1185), choroid (N = 6446), sclera (N = 1349), and cornea (N = 3872), but not optic nerve. Genes showing differential expression were assessed using Ingenuity Pathway Analysis (IPA) for enriched functions and canonical pathways. In all tissues, development, cell death/growth, cancer functions, and signaling canonical pathways were enriched. There was also a general trend of down-regulation of collagen genes in adult tissues.
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