RESUMO
Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.
Assuntos
Proteínas de Membrana/genética , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fosfatidilserinas/farmacologia , Receptores Acoplados a Proteínas G/genética , Sinapses/efeitos dos fármacos , Animais , Regulação da Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Microglia/metabolismo , Plasticidade Neuronal , Neurônios/citologia , Neurônios/metabolismo , Fosfatidilserinas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sinapses/genética , Sinapses/metabolismo , Transmissão Sináptica , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato , Proteína Vermelha FluorescenteRESUMO
Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer, and the early detection and diagnosis of this disease are crucial in reducing mortality rates. The timely diagnosis of LUAD is essential for controlling tumour development and enabling early surgical treatment. GPR56 is a vital G protein-coupled receptor and its role in T lymphocytes has received considerable attention. However, its function in B cells remains unclear. This study aimed to investigate the significance of GPR56 in LUAD. We found that GPR56 exhibited a significant increase in circulating plasmablasts and a decrease in new memory B cells. GPR56 expression in B cells was significantly reduced after LPS stimulation and the proportion of HLA-DR+ and CD40+ proportions were also decreased in GPR56+ B cells after stimulation. Additionally, GPR56 exhibited significant down-regulation in circulating B cell subsets of early-stage LUAD patients, and there were significant correlations between GPR56+ B cell subsets and tumour markers. In conclusion, GPR56 could reflect the hypoactivation state of B cells and the decreased proportion of GPR56+ B cell subset in LUAD patients can signify the active humoral immunity in vivo. The expression of GPR56 in B cells could potentially hold value in the early diagnosis of LUAD.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Ativação Linfocitária , Regulação para Baixo , Estadiamento de Neoplasias , Imunidade Humoral , Biomarcadores Tumorais/metabolismoRESUMO
Developmental synaptic remodeling is important for the formation of precise neural circuitry, and its disruption has been linked to neurodevelopmental disorders such as autism and schizophrenia. Microglia prune synapses, but integration of this synapse pruning with overlapping and concurrent neurodevelopmental processes, remains elusive. Adhesion G protein-coupled receptor ADGRG1/GPR56 controls multiple aspects of brain development in a cell type-specific manner: In neural progenitor cells, GPR56 regulates cortical lamination, whereas in oligodendrocyte progenitor cells, GPR56 controls developmental myelination and myelin repair. Here, we show that microglial GPR56 maintains appropriate synaptic numbers in several brain regions in a time- and circuit-dependent fashion. Phosphatidylserine (PS) on presynaptic elements binds GPR56 in a domain-specific manner, and microglia-specific deletion of Gpr56 leads to increased synapses as a result of reduced microglial engulfment of PS+ presynaptic inputs. Remarkably, a particular alternatively spliced isoform of GPR56 is selectively required for microglia-mediated synaptic pruning. Our present data provide a ligand- and isoform-specific mechanism underlying microglial GPR56-mediated synapse pruning in the context of complex neurodevelopmental processes.
Assuntos
Processamento Alternativo , Microglia/metabolismo , Fosfatidilserinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sinapses/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Microglia/citologia , Fosfatidilserinas/genética , Ligação Proteica , Isoformas de Proteínas , Receptores Acoplados a Proteínas G/genética , Sinapses/genéticaRESUMO
Adhesion GPCRs (aGPCRs) are a subfamily of GPCRs that are involved in cell adhesion, cell proliferation, and cell migration in various tissues. G protein-coupled receptor proteolytic site (GPS) of aGPCR is required to cleave the extracellular domain autocatalytically, generating two fragments; a N-terminal fragment (NTF) and a C-terminal fragment (CTF) containing seven transmembrane structure. NTF can interact with CTF non-covalently after cleavage, however the physiological significance of the cleavage of aGPCR at GPS, and also the interaction between NTF and CTF have not been fully clarified yet. In this study, we first investigated the expression profiles of two aGPCRs, GPR56/ADGRG1, and LPHN1/ADGRL1 in mouse brain, and found that the NTF and CTF of GPR56 independently expressed in different brain region at different developmental stages. Immunoprecipitation of GPR56CTF co-immunoprecipitated LPHN1NTF from mouse brain and HEK293T cells expressing both fragments. Stimulation with LPHN1 ligand, α-Latrotoxin N4C (αLTXN4C), to cells expressing LPHN1NTF and GPR56CTF increased intracellular Ca2+ concentration ([Ca2+ ]i). We also demonstrated that GPR56KO mouse neurons attenuated their Ca2+ response to αLTXN4C. These results suggest the possibility of functional and chimeric complex containing LPHN1NTF and GPR56CTF in neuronal signal transduction.
Assuntos
Neurônios , Receptores Acoplados a Proteínas G , Transdução de Sinais , Animais , Humanos , Camundongos , Adesão Celular , Movimento Celular , Células HEK293 , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
OBJECTIVE: This study aimed to investigate the expression of GPR56 in the T cells of early-stage lung adenocarcinoma (LUAD) patients and clarify its diagnostic significance. METHODS: Blood samples were collected from 32 patients with stage IA LUAD and 31 healthy controls. GPR56 and perforin were analysed in circulating T-cell subsets by flow cytometry. In addition, a correlation between perforin and GPR56 expression was detected. Changes in GPR56+ cells in early LUAD patients were analysed, and the diagnostic significance of GPR56+ T cells for early LUAD was studied by receiver operating characteristic (ROC) curve analysis. RESULTS: The expression of GPR56 in CD8+ T cells from early-stage LUAD patients was significantly greater than that in CD4+ T cells. The percentage of perforin-positive GPR56+ cells in early-stage LUAD patients was high. GPR56 levels in the T cells of LUAD patients were significantly lower than those in healthy controls. ROC analysis revealed that the area under the curve for the percentage of GPR56-positive CD8+ TEMRA cells to distinguish early-stage LUAD patients from healthy individuals- reached 0.7978. CONCLUSION: The decreased expression of GPR56 in the peripheral blood of early-stage LUAD patients correlated with perforin levels, reflecting compromised antitumor immunity and aiding early-stage LUAD screening.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Estadiamento de Neoplasias , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Masculino , Feminino , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Pessoa de Meia-Idade , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Idoso , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Perforina/metabolismo , Perforina/genética , Curva ROC , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Biomarcadores Tumorais/metabolismo , AdultoRESUMO
Adhesion G protein-coupled receptors (aGPCRs) have extracellular regions (ECRs) containing GPCR autoproteolysis-inducing (GAIN) domains. The GAIN domain enables the ECR to self-cleave into N- and C-terminal fragments. However, the impact of force on the GAIN domain's conformation, critical for mechanosensitive aGPCR activation, remains unclear. Our study investigated the mechanical stability of GAIN domains in three aGPCRs (B, G, and L subfamilies) at a loading rate of 1 pN/s. We discovered that forces of a few piconewtons can destabilize the GAIN domains. In autocleaved aGPCRs ADGRG1/GPR56 and ADGRL1/LPHN1, these forces cause the GAIN domain detachment from the membrane-proximal Stachel sequence, preceded by partial unfolding. In noncleavable aGPCR ADGRB3/BAI3 and cleavage-deficient mutant ADGRG1/GPR56-T383G, complex mechanical unfolding of the GAIN domain occurs. Additionally, GAIN domain detachment happens during cell migration. Our findings support the mechanical activation hypothesis of aGPCRs, emphasizing the sensitivity of the GAIN domain structure and detachment to physiological force ranges.
Assuntos
Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/química , Modelos Moleculares , Adesão CelularRESUMO
A wound healing model was developed to elucidate the role of mesenchymal-matrix-associated transglutaminase 2 (TG2) in keratinocyte re-epithelialisation. TG2 drives keratinocyte migratory responses by activation of disintegrin and metalloproteinase 17 (ADAM17). We demonstrate that epidermal growth factor (EGF) receptor ligand shedding leads to EGFR-transactivation and subsequent rapid keratinocyte migration on TG2-positive ECM. In contrast, keratinocyte migration was impaired in TG2 null conditions. We show that keratinocytes express the adhesion G-protein-coupled receptor, ADGRG1 (GPR56), which has been proposed as a TG2 receptor. Using ADAM17 activation as a readout and luciferase reporter assays, we demonstrate that TG2 activates GPR56. GPR56 activation by TG2 reached the same level as observed with an agonistic N-GPR56 antibody. The N-terminal GPR56 domain is required for TG2-regulated signalling response, as the constitutively active C-GPR56 receptor was not activated by TG2. Signalling required the C-terminal TG2 ß-barrel domains and involved RhoA-associated protein kinase (ROCK) and ADAM17 activation, which was blocked by specific inhibitors. Cell surface binding of TG2 to the N-terminal GPR56 domain is rapid and is associated with TG2 and GPR56 endocytosis. TG2 and GPR56 represent a ligand receptor pair causing RhoA and EGFR transactivation. Furthermore, we determined a binding constant for the interaction of human TG2 with N-GPR56 and show for the first time that only the calcium-enabled "open" TG2 conformation associates with N-GPR56.
Assuntos
Proteína 2 Glutamina gama-Glutamiltransferase , Receptores Acoplados a Proteínas G , Humanos , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Receptores ErbB/metabolismo , Ligantes , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
ADGRG1 (also called GPR56) plays critical roles in brain development and wiring, including cortical lamination, central nervous system (CNS) myelination, and developmental synaptic refinement. However, the underlying mechanism(s) in mediating such diverse functions is not fully understood. Here, we investigate the function of one specific alternative splicing isoform, the GPR56 splice variant 4 (S4), to test the hypothesis that alternative splicing variants of GPR56 in part support its different functions. We created a new transgenic mouse line, Gpr56∆S4 , using CRISPR/Cas9, in which GPR56 S4 was deleted. Detailed phenotype analyses show that Gpr56∆S4 mice manifest no deficits in cortical architecture and CNS myelination compared to controls. Excitingly, they present significantly increased synapse densities, decreased synapse engulfment by microglia, and impaired eye-segregation. Taken together, our findings support that the GPR56 S4 variant is dispensable for cortical development and CNS myelination but is essential for microglia-mediated synaptic pruning.
Assuntos
Microglia , Receptores Acoplados a Proteínas G , Camundongos , Animais , Receptores Acoplados a Proteínas G/genética , Camundongos Transgênicos , Isoformas de Proteínas , SinapsesRESUMO
The adhesion G-protein-coupled receptor is a seven-transmembrane receptor protein with a complex structure. Impaired GPR56 has been found to cause developmental damage to the human brain, resulting in intellectual disability and motor dysfunction. To date, studies on gpr56 deficiency in zebrafish have been limited to the nervous system, and there have been no reports of its systemic effects on juvenile fish at developmental stages. In order to explore the function of gpr56 in zebrafish, the CRISPR/Cas9 gene-editing system was used to construct a gpr56-knockout zebrafish. Subsequently, the differentially expressed genes (DEGs) at the transcriptional level between the 3 days post fertilization (dpf) homozygotes of the gpr56 mutation and the wildtype zebrafish were analyzed via RNA-seq. The results of the clustering analysis, quantitative PCR (qPCR), and in situ hybridization demonstrated that the expression of innate immunity-related genes in the mutant was disordered, and multiple genes encoding digestive enzymes of the pancreatic exocrine glands were significantly downregulated in the mutant. Motor ability tests demonstrated that the gpr56-/- zebrafish were more active, and this change was more pronounced in the presence of cold and additional stimuli. In conclusion, our results revealed the effect of gpr56 deletion on the gene expression of juvenile zebrafish and found that the gpr56 mutant was extremely active, providing an important clue for studying the mechanism of gpr56 in the development of juvenile zebrafish.
Assuntos
Transcriptoma , Peixe-Zebra , Animais , Humanos , Mutação , Receptores Acoplados a Proteínas G/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Neoplasias Colorretais/genética , Quinase 1 de Adesão Focal/genética , Receptores Acoplados a Proteínas G/genética , Fator de Resposta Sérica/genética , Proteína rhoA de Ligação ao GTP/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Carcinogênese/genética , Adesão Celular/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Quinase 1 de Adesão Focal/imunologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Paxilina/genética , Paxilina/imunologia , Fosforilação/genética , Receptores Acoplados a Proteínas G/imunologia , Fator de Resposta Sérica/imunologia , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/imunologia , Quinases da Família src/genética , Quinases da Família src/imunologiaRESUMO
OBJECTIVE: This study aims to elucidate the changes in the percentage of GPR56 and/or granzyme B (GZMB) positive cells in rheumatoid arthritis (RA) CD4 and CD8 T lymphocytes, and to explore their clinical value in diagnosing and reflecting the progression of RA. METHODS: The percentages of GPR56 and/or GZMB positive cells were analyzed in peripheral blood (PB) and spleen T cells in a collagen-induced arthritis (CIA) model established in DBA/1 mice. The percentages of GPR56+ and/or GZMB+ cells were further analyzed in PBs from RA patients and healthy controls. Correlation analysis was performed between clinical indicators and GPR56+, GZMB+, and GPR56+ GZMB+ T cells. Receiver operating characteristic (ROC) curves were used to evaluate the value of GPR56 and GZMB in differentiating active and stable remitting RA. RESULTS: GPR56+ levels were increased in CD4 and CD8 T cells in the PB of CIA mice. The percentages of GPR56+ and GZMB+ cells were increased in both CD4 and CD8 T cell subsets in patients with active RA. GPR56+, GZMB+, and GPR56+ GZMB+ cells were positively correlated with rheumatoid factor and DAS28. ROC analysis revealed that AUCs for GPR56+, GZMB+, and GPR56+ GZMB+ cell percentages to distinguish active RA from stable remission RA were 0.7106, 0.6941, 0.7024, with cut-off values of 16.35, 16.40, 14.80 in CD4 + T cells, and 0.8031, 0.8086, 0.8196 with cut-off values 60.25, 62.15, 40.15 in CD8 + T cells, respectively. CONCLUSIONS: GPR56+ and/or GZMB+ T cells are up-regulated in patients with active RA and reflect their condition. The detection of GPR56 and GZMB is helpful for RA disease assessment.
Assuntos
Artrite Reumatoide , Linfócitos T Citotóxicos , Animais , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos , Progressão da Doença , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos DBA , Receptores Acoplados a Proteínas GRESUMO
Glycoprotein (GP)VI and integrin αIIbß3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbß3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbß3 activation and signaling.
Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Trombose , Plaquetas/metabolismo , Colágeno/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Peptídeos/metabolismo , Peptídeos/farmacologia , Ativação Plaquetária , Adesividade Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombose/metabolismoRESUMO
Myelination of axons in the central nervous system (CNS) is a concerted effort between many cell types, resulting in significant cross-talk and communication among cells. Adhesion G protein-coupled receptor ADGRG1 (GPR56) is expressed in all major glial cells and regulates a wide variety of physiological processes by mediating cell-cell and cell-matrix communications. Previous literature has demonstrated the requirement of ADGRG1 in oligodendrocyte precursor cells (OPCs) during developmental myelination. However, it is unknown if ADGRG1 is responsible for myelin formation in a cell-type-specific manner. To that end, here we profiled myelin status in response to deletion of Adgrg1 specifically in OPCs, microglia, astrocytes, and neurons. Interestingly, we find that knocking out Adgrg1 in OPCs significantly decreases OPC proliferation and reduced number of myelinated axons. However, deleting Adgrg1 in microglia, astrocytes, and neurons does not impact developmental myelination. These data support an autonomous functional role for Adgrg1 in OPCs related to myelination.
Assuntos
Sistema Nervoso Central , Animais , Camundongos , Camundongos Knockout , Bainha de Mielina , Oligodendroglia , Receptores Acoplados a Proteínas G/genéticaRESUMO
From a mouse triple-negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G-protein-coupled receptor (ADGR) family, G-protein-coupled receptor 56 (GPR56)/adhesion G-protein-coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1-transduced 4T1, TS/A (mouse breast cancer cell line), or MDA-MB-231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1-induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.
Assuntos
Neoplasias Ósseas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Receptores Acoplados a Proteínas G/genética , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Proliferação de Células/genética , Colágeno Tipo III/metabolismo , Feminino , Deleção de Genes , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos Endogâmicos BALB C , Camundongos SCID , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Carga Tumoral/genéticaRESUMO
Adhesion G protein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members.
Assuntos
Limoninas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Ligantes , Limoninas/química , Masculino , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Obesity-associated conditions represent major global health and financial burdens and understanding processes regulating adipogenesis could lead to novel intervention strategies. This study shows that adhesion G-protein coupled receptor 56 (GPR56) gene transcripts are reduced in abdominal visceral white adipose tissue derived from obese Zucker rats versus lean controls. Immunostaining in 3T3-L1 preadipocytes reveals both mitotic cell restricted surface and low level general expression patterns of Gpr56. Gpr56 transcripts are differentially expressed in 3T3-L1 cells during adipogenesis. Transient knockdown (KD) of Gpr56 in 3T3-L1 cells dramatically inhibits differentiation through reducing the accumulation of both neutral cellular lipids (56%) and production of established adipogenesis Pparγ 2 (60-80%), C/ebpα (40-78%) mediator, and Ap2 (56-80%) marker proteins. Furthermore, genome editing of Gpr56 in 3T3-L1 cells created CW2.2.4 and RM4.2.5.5 clones (Gpr56 -/- cells) with compound heterozygous deletion frameshift mutations which abolish adipogenesis. Genome edited cells have sustained levels of the adipogenesis inhibitor ß-catenin, reduced proliferation, reduced adhesion, altered profiles, and or abundance of extracellular matrix component gene transcripts for fibronectin, types I, III, and IV collagens and loss of actin stress fibers. ß-catenin KD alone is insufficient to restore adipogenesis in Gpr56 -/- cells. Together these data show that Gpr56 is required for adipogenesis in 3T3-L1 cells. This report is the first demonstration that Gpr56 participates in regulation of the adipogenesis developmental program. Modulation of the levels of this protein and/or its biological activity may represent a novel target for development of therapeutic agents for the treatment of obesity.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Animais , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Obesidade/metabolismo , Ratos , Ratos ZuckerRESUMO
AIMS: G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on ß-cell function. METHODS: GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on ß-cell proliferation, apoptosis, intracellular calcium ([Ca2+]i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56-/- mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56-/- mice. RESULTS: Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet ß-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected ß-cells from cytokine-induced apoptosis, triggered increases in [Ca2+]i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56-/- islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation. CONCLUSION: We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.
Assuntos
Colágeno/genética , Diabetes Mellitus Tipo 2/genética , Receptores Acoplados a Proteínas G/genética , Animais , Apoptose/genética , Cálcio/metabolismo , Proliferação de Células/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/patologiaRESUMO
GPR56 is an adhesion-class G-protein-coupled receptor responsible for bilateral frontoparietal polymicrogyria (BFPP), a severe disorder of cortical formation. Additionally, GPR56 is involved in biological processes as diverse as hematopoietic stem cell generation and maintenance, myoblast fusion, muscle hypertrophy, immunoregulation and tumorigenesis. Collagen III and tissue transglutaminase 2 (TG2) have been revealed as the matricellular ligands of GPR56 involved in BFPP and melanoma development, respectively. In this study, we identify heparin as a glycosaminoglycan interacting partner of GPR56. Analyses of truncated and mutant GPR56 proteins reveal two basic-residue-rich clusters, R(26)GHREDFRFC(35) and L(190)KHPQKASRRP(200), as the major heparin-interacting motifs that overlap partially with the collagen III- and TG2-binding sites. Interestingly, the GPR56-heparin interaction is modulated by collagen III but not TG2, even though both ligands are also heparin-binding proteins. Finally, we show that the interaction with heparin reduces GPR56 receptor shedding, and enhances cell adhesion and motility. These results provide novel insights into the interaction of GPR56 with its multiple endogenous ligands and have functional implications in diseases such as BFPP and cancer.
Assuntos
Adesão Celular , Movimento Celular , Heparina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Células HEK293 , Heparina/química , Humanos , Ligantes , Microdomínios da Membrana/metabolismo , Invasividade Neoplásica , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Mapeamento de Interação de Proteínas , Proteína Quinase C-alfa/metabolismo , Receptores Acoplados a Proteínas G/química , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Collagens are one of the major constituents of the pial membrane, which plays a crucial role in neuronal migration and cortical lamination during brain development. Type III procollagen, the chains of which are encoded by COL3A1, is the ligand of the G protein-coupled receptor 56 (GPR56), also known as adhesion G protein-coupled receptor G1. Bi-allelic mutations in GPR56 give rise to cobblestone-like malformation, white matter changes and cerebellar dysplasia. This report shows that bi-allelic mutations in COL3A1 are associated with a similar phenotype. METHODS: Exome analysis was performed in a family consisting of two affected and two non-affected siblings. Brain imaging studies of this family and of two previously reported individuals with bi-allelic mutations in COL3A1 were reviewed. Functional assays were performed on dermal fibroblasts. RESULTS: Exome analysis revealed a novel homozygous variant c.145C>G (p.Pro49Ala) in exon 2 of COL3A1. Brain MRI in the affected siblings as well as in the two previously reported individuals with bi-allelic COL3A1 mutations showed a brain phenotype similar to that associated with mutations in GPR56. CONCLUSION: Homozygous or compound heterozygous mutations in COL3A1 are associated with cobblestone-like malformation in all three families reported to date. The variability of the phenotype across patients suggests that genetic alterations in distinct domains of type III procollagen can lead to different outcomes. The presence of cobblestone-like malformation in patients with bi-allelic COL3A1 mutations emphasises the critical role of the type III collagen-GPR56 axis and the pial membrane in the regulation of brain development and cortical lamination.
Assuntos
Colágeno Tipo III/genética , Cistos/genética , Malformações do Desenvolvimento Cortical/genética , Receptores Acoplados a Proteínas G/genética , Substância Branca/patologia , Adulto , Alelos , Células Cultivadas , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , Criança , Pré-Escolar , Cistos/patologia , Exoma/genética , Éxons/genética , Feminino , Fibroblastos/patologia , Humanos , Ligantes , Imageamento por Ressonância Magnética/métodos , Masculino , Malformações do Desenvolvimento Cortical/patologia , Mutação/genética , Fenótipo , Adulto JovemRESUMO
The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease.