RESUMO
GPR37 is an orphan G protein-coupled receptor (GPCR) implicated in several neurological diseases and important physiological pathways in the brain. We previously reported that its long N-terminal ectodomain undergoes constitutive metalloprotease-mediated cleavage and shedding, which have been rarely described for class A GPCRs. Here, we demonstrate that the protease that cleaves GPR37 at Glu167↓Gln168 is a disintegrin and metalloprotease 10 (ADAM10). This was achieved by employing selective inhibition, RNAi-mediated downregulation, and genetic depletion of ADAM10 in cultured cells as well as in vitro cleavage of the purified receptor with recombinant ADAM10. In addition, the cleavage was restored in ADAM10 knockout cells by overexpression of the wild type but not the inactive mutant ADAM10. Finally, postnatal conditional depletion of ADAM10 in mouse neuronal cells was found to reduce cleavage of the endogenous receptor in the brain cortex and hippocampus, confirming the physiological relevance of ADAM10 as a GPR37 sheddase. Additionally, we discovered that the receptor is subject to another cleavage step in cultured cells. Using site-directed mutagenesis, the site (Arg54↓Asp55) was localized to a highly conserved region at the distal end of the ectodomain that contains a recognition site for the proprotein convertase furin. The cleavage by furin was confirmed by using furin-deficient human colon carcinoma LoVo cells and proprotein convertase inhibitors. GPR37 is thus the first multispanning membrane protein that has been validated as an ADAM10 substrate and the first GPCR that is processed by both furin and ADAM10. The unconventional N-terminal processing may represent an important regulatory element for GPR37.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Encéfalo/metabolismo , Furina/metabolismo , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Furina/genética , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Domínios ProteicosRESUMO
Lung adenocarcinoma (LUAD) is associated with poor prognosis. Identifying novel cancer targets and helpful therapeutic strategies remains a serious clinical challenge. This study detected differentially expressed genes in The Cancer Genome Atlas (TCGA) LUAD data collection. We also identified a predictive DNA biomarker, G protein-coupled receptor 37 (GPR37), which was verified as a prognostic biomarker with a critical role in tumor progression. In human LUAD specimens and microarray analyses, we determined that GPR37 was significantly upregulated and associated with a poor prognosis. GPR37 downregulation markedly inhibited the proliferation and migration of LUAD both in vitro and in vivo. Mechanistically, GPR37 could bind to CDK6, thereby facilitating tumor progression in LUAD by inducing cell cycle arrest at the G1 phase. GPR37 also facilitates tumorigenesis in xenograft tumors in vivo. High-throughput screening for GPR37-targeted drugs was performed using the Natural Products Library, which revealed the potential of Hypocrellin B to inhibit GPR37 and cell growth in LUAD. We demonstrated that Hypocrellin B suppressed LUAD cell proliferation and migration both in vitro and in vivo via GPR37 inhibition. Collectively, our findings reveal the role of GPR37 in LUAD progression and migration and the potential of GPR37 as a target for the treatment of LUAD. Thus, the specific inhibition of GPR37 by the natural product Hypocrellin B may possess the potential for the treatment of LUAD.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Biomarcadores , Proliferação de Células/fisiologia , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Medicina de Precisão , Prognóstico , Receptores Acoplados a Proteínas GRESUMO
Recent studies have shed light on the diverse and complex roles of G-protein coupled receptors (GPCRs) in the pathophysiology of stroke. These receptors constitute a large family of seven transmembrane-spanning proteins that play an intricate role in cellular communication mechanisms which drive both tissue injury and repair following ischemic stroke. Orphan GPCRs represent a unique sub-class of GPCRs for which no natural ligands have been found. Interestingly, the majority of these receptors are expressed within the central nervous system where they represent a largely untapped resource for the treatment of neurological diseases. The focus of this review will thus be on the emerging roles of two brain-expressed orphan GPCRs, GPR37 and GPR37 L1, in regulating various cellular and molecular processes underlying ischemic stroke.
Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Encéfalo/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of vacuolating leukodystrophy (white matter disorder), which is mainly caused by defects in MLC1 or glial cell adhesion molecule (GlialCAM) proteins. In addition, autoantibodies to GlialCAM are involved in the pathology of multiple sclerosis. MLC1 and GLIALCAM genes encode for membrane proteins of unknown function, which has been linked to the regulation of different ion channels and transporters, such as the chloride channel VRAC (volume regulated anion channel), ClC-2 (chloride channel 2), and connexin 43 or the Na+/K+-ATPase pump. However, the mechanisms by which MLC proteins regulate these ion channels and transporters, as well as the exact function of MLC proteins remain obscure. It has been suggested that MLC proteins might regulate signalling pathways, but the mechanisms involved are, at present, unknown. With the aim of answering these questions, we have recently described the brain GlialCAM interactome. Within the identified proteins, we could validate the interaction with several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptors GPR37L1 and GPR37. In this review, we summarize new aspects of the pathophysiology of MLC disease and key aspects of the interaction between GPR37 receptors and MLC proteins.
Assuntos
Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , Megalencefalia , Malformações do Sistema Nervoso , Astrócitos/metabolismo , Canais de Cloreto/metabolismo , Cistos , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
GPR37L1 is an orphan G-protein-coupled receptor, which is implicated in neurological disorders, but its normal physiological role is poorly understood. Its close homologue, GPR37, is implicated in Parkinson's disease and affective disorders. In this study, we set out to characterize adult and middle-aged global GPR37L1 knock-out (KO) mice regarding emotional behaviors. Our results showed that GPR37L1KO animals, except adult GPR37L1KO males, exhibited impaired retention of aversive memory formation as assessed by the shorter retention latency in a passive avoidance task. Interestingly, the viral-mediated deletion of GPR37L1 in conditional knockout mice in the hippocampus of middle-aged mice also showed impaired retention in passive avoidance tasks, similar to what was observed in global GPR37L1KO mice, suggesting that hippocampal GPR37L1 is involved in aversive learning processes. We also observed that middle-aged GPR37L1KO male and female mice exhibited a higher body weight than their wild-type counterparts. Adult and middle-aged GPR37L1KO female mice exhibited a reduced level of serum corticosterone and middle-aged GPR37L1KO females showed a reduced level of epinephrine in the dorsal hippocampus in the aftermath of passive avoidance task, with no such effects observed in GPR37L1KO male mice, suggesting that lack of GPR37L1 influences behavior and biochemical readouts in age- and sex-specific manners.
Assuntos
Afeto , Transtornos da Memória , Animais , Camundongos , Masculino , Feminino , Camundongos Endogâmicos C57BL , Transtornos da Memória/genética , Camundongos Knockout , Hipocampo , Receptores Acoplados a Proteínas G/genéticaRESUMO
Inflammation is known to cause pain, and pain is of one of the cardinal signs of inflammation. Mounting evidence suggests that acute inflammation also resolves pain through specialized pro-resolving mediators (SPMs) and macrophage signaling. GPR37 is expressed by neurons and oligodendrocytes in the brain and has been implicated in multiple disorders, such as demyelination, Parkinson's disease, stroke, and cancer. Recent studies have demonstrated that GPR37 is expressed by macrophages and confers protection against infection by bacteria and parasites. Furthermore, GPR37 promotes the resolution of inflammatory pain and infection-induced pain, as the duration of pain after tissue injury and infection is prolonged in mice lacking Gpr37. Mechanistically, activation of GPR37 enhances macrophage phagocytosis, and Gpr37-deficient macrophages exhibit dysregulations of pro-inflammatory and anti-inflammatory cytokines, switching from M2- to M1-like phenotypes. We also discuss novel ligands of GPR37, including neuroprotectin D1 (NPD1), a SPM derived from docosahexaenoic acid (DHA), and bone-derived hormone osteocalcin (OCN), which can suppress oligodendrocyte differentiation and myelination. NPD1 stimulates macrophage phagocytosis via GPR37 and exhibits potent analgesic actions in various animal models of inflammatory and neuropathic pain. Targeting GPR37 may lead to novel therapeutics for treating inflammation, infection, pain, and neurological diseases.
Assuntos
Inflamação , Neuralgia , Animais , Camundongos , Inflamação/prevenção & controle , Fagocitose , Macrófagos , Anti-Inflamatórios/farmacologia , Receptores Acoplados a Proteínas GRESUMO
Renal luminal sodium transport is essential for physiological blood pressure control, and abnormalities in this process are strongly implicated in the pathogenesis of essential hypertension. Renal G protein-coupled receptors (GPCRs) are critical for the regulation of the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Recently, we showed that GPCR 37L1 (GPR37L1) is expressed on the apical membrane of renal proximal tubules (RPT) and regulates luminal sodium transport and blood pressure by modulating the function of the sodium proton exchanger 3 (NHE3). However, little is known about GPR37L1 intracellular signaling. Here, we show that GPR37L1 is localized to the nuclear membrane, in addition to the plasma membrane in human RPT cells. Furthermore, GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and enhance NHE3 transcription. Overall, we demonstrate the direct role of a nuclear membrane GPCR in the regulation of renal sodium through epigenetic gene regulation.
Assuntos
Fosfatidilinositol 3-Quinases , Trocadores de Sódio-Hidrogênio , Humanos , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sódio/metabolismo , Epigênese GenéticaRESUMO
Multiple mouse lines lacking the orphan G protein-coupled receptor, GPR37L1, have elicited disparate cardiovascular phenotypes. The first Gpr37l1 knockout mice study to be published reported a marked elevation in systolic blood pressure (SBP; â¼60 mmHg), revealing a potential therapeutic opportunity. The phenotype differed from our own independently generated knockout line, where male mice exhibited equivalent baseline blood pressure to wild type. Here, we attempted to reproduce the first study by characterizing the cardiovascular phenotype of both the original knockout and transgenic lines alongside a C57BL/6J control line, using the same method of blood pressure measurement. The present study supports the findings from our independently developed Gpr37l1 knockout line, finding that SBP and diastolic blood pressure (DBP) are not different in the original Gpr37l1 knockout male mice (SBP: 130.9 ± 5.3 mmHg; DBP: 90.7 ± 3.0 mmHg) compared with C57BL/6J mice (SBP: 123.1 ± 4.1 mmHg; DBP: 87.0 ± 2.7 mmHg). Instead, we attribute the apparent hypertension of the knockout line originally described to comparison with a seemingly hypotensive transgenic line (SBP 103.7 ± 5.0 mmHg; DBP 71.9 ± 3.7 mmHg). Additionally, we quantified myocardial GPR37L1 transcript in humans, which was suggested to be downregulated in cardiovascular disease. We found that GPR37L1 has very low native transcript levels in human myocardium and that expression is not different in tissue samples from patients with heart failure compared with sex-matched healthy control tissue. These findings indicate that cardiac GPR37L1 expression is unlikely to contribute to the pathophysiology of human heart failure.NEW & NOTEWORTHY This study characterizes systolic blood pressure (SBP) in a Gpr37l1 knockout mouse line, which was previously reported to have â¼60 mmHg higher SBP compared with a transgenic line. We observed only a â¼27 mmHg SBP difference between the lines. However, when compared with C57BL/6J mice, knockout mice showed no difference in SBP. We also investigated GPR37L1 mRNA abundance in human hearts and observed no difference between healthy and failing heart samples.
Assuntos
Pressão Sanguínea , Insuficiência Cardíaca/metabolismo , Hipertensão/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Animais , Estudos de Casos e Controles , Feminino , Genótipo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fenótipo , Receptores Acoplados a Proteínas G/genética , Especificidade da EspécieRESUMO
G-protein-coupled receptors (GPCRs) are commonly pharmacologically modulated due to their ability to translate extracellular events to intracellular changes. Previously, studies have mostly focused on protein-protein interactions, but the focus has now expanded also to protein-lipid connections. GM1, a brain-expressed ganglioside known for neuroprotective effects, and GPR37, an orphan GPCR often reported as a potential drug target for diseases in the central nervous system, have been shown to form a complex. In this study, we looked into the functional effects. Endogenous GM1 was downregulated when stably overexpressing GPR37 in N2a cells (N2aGPR37-eGFP). However, exogenous GM1 specifically rescued N2aGPR37-eGFP from toxicity induced by the neurotoxin MPP+. The treatment did not alter transcription levels of GPR37 or the enzyme responsible for GM1 production, both potential mechanisms for the effect. However, GM1 treatment inhibited cAMP-dependent signaling from GPR37, here reported as potentially consecutively active, possibly contributing to the protective effects. We propose an interplay between GPR37 and GM1 as one of the many cytoprotective effects reported for GM1.
Assuntos
Gangliosídeo G(M1)/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Citoproteção , Regulação para Baixo , Células HEK293 , Humanos , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Receptores Acoplados a Proteínas G/genética , Transdução de SinaisRESUMO
G protein-coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormalities in renal epithelial ion transport play important roles in the pathogenesis of essential hypertension. The orphan G protein-coupled receptor 37L1 (GPR37L1), also known as endothelin receptor type B-like protein (ETBR-LP2), is expressed in several regions in the brain, but its expression profile and function in peripheral tissues are poorly understood. We found that GPR37L1 mRNA expression is highest in the brain, followed by the stomach, heart, testis, and ovary, with moderate expression in the kidney, pancreas, skeletal muscle, liver, lung, and spleen. Immunofluorescence analyses revealed the expression of GPR37L1 in specific regions within some organs. In the kidney, GPR37L1 is expressed in the apical membrane of renal proximal tubule cells. In human renal proximal tubule cells, the transient expression of GPR37LI increased intracellular sodium, whereas the silencing of GPR37LI decreased intracellular sodium. Inhibition of Na+/H+ exchanger isoform 3 (NHE3) activity abrogated the GPR37L1-mediated increase in intracellular sodium. Renal-selective silencing of Gpr37l1 in mice increased urine output and sodium excretion and decreased systolic and diastolic blood pressures. The renal-selective silencing of GPR37L1 decreased the protein expression of NHE3 but not the expression of Na+-K+-ATPase or sodium-glucose cotransporter 2. Our findings show that in the kidney, GPR37L1 participates in renal proximal tubule luminal sodium transport and regulation of blood pressure by increasing the renal expression and function of NHE3 by decreasing cAMP production. The role of GPR37L1, expressed in specific cell types in organs other than the kidney, remains to be determined.
Assuntos
Pressão Sanguínea/fisiologia , Transporte de Íons/fisiologia , Rim/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sódio/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Humanos , Túbulos Renais Proximais/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Receptores Acoplados a Proteínas G/genética , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Adenosine A2A receptors (A2A R) play a key role in modulating dopamine-dependent locomotor activity, as heralded by the sensitization of locomotor activity upon chronic A2A R blockade, which is associated with elevated dopamine levels and altered corticostriatal synaptic plasticity. Since the orphan receptor GPR37 has been shown to modulate A2A R function in vivo, we aimed to test whether the A2A R-mediated sensitization of locomotor activity is GPR37-dependent and involves adaptations of synaptic plasticity. To this end, we administered a selective A2A R antagonist, SCH58261 (1 mg/kg, i.p.), daily for 14 days, and the locomotor sensitization, striatum-dependent cued learning, and corticostriatal synaptic plasticity (i.e., long-term depression) were compared in wild-type and GPR37-/- mice. Notably, GPR37 deletion promoted A2A R-associated locomotor sensitization but not striatum-dependent cued learning revealed upon chronic SCH58261 treatment of mice. Furthermore, chronic A2A R blockade potentiated striatal long-term depression in corticostriatal synapses of GPR37-/- but not of wild-type mice, thus correlating well with neurochemical alterations of the adenosinergic system. Overall, these results revealed the importance of GPR37 regulating A2A R-dependent locomotor sensitization and synaptic plasticity in the basal ganglia circuitry. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
Assuntos
Locomoção/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Corpo Estriado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Wnt/ß-catenin signaling plays a key role in embryonic development, stem cell biology, and neurogenesis. However, the mechanisms of Wnt signal transmission, notably how the receptors are regulated, remain incompletely understood. Here we describe that the Parkinson's disease-associated receptor GPR37 functions in the maturation of the N-terminal bulky ß-propellers of the Wnt co-receptor LRP6. GPR37 is required for Wnt/ß-catenin signaling and protects LRP6 from ER-associated degradation via CHIP (carboxyl terminus of Hsc70-interacting protein) and the ATPase VCP GPR37 is highly expressed in neural progenitor cells (NPCs) where it is required for Wnt-dependent neurogenesis. We conclude that GPR37 is crucial for cellular protein quality control during Wnt signaling.
Assuntos
Retículo Endoplasmático/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Chaperonas Moleculares/metabolismo , Doença de Parkinson/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização Wnt , Animais , Degradação Associada com o Retículo Endoplasmático , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Células-Tronco Neurais/metabolismo , Fosforilação , Proteólise , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Discovery of neuroprotective pathways is one of the major priorities for neuroscience. Astrocytes are natural neuroprotectors and it is likely that brain resilience can be enhanced by mobilizing their protective potential. Among G-protein coupled receptors expressed by astrocytes, two highly related receptors, GPR37L1 and GPR37, are of particular interest. Previous studies suggested that these receptors are activated by a peptide Saposin C and its neuroactive fragments (prosaptide TX14(A)), which were demonstrated to be neuroprotective in various animal models by several groups. However, pairing of Saposin C or prosaptides with GPR37L1/GPR37 has been challenged and presently GPR37L1/GPR37 have regained their orphan status. Here, we demonstrate that in their natural habitat, astrocytes, these receptors mediate a range of effects of TX14(A), including protection from oxidative stress. The Saposin C/GPR37L1/GPR37 pathway is also involved in the neuroprotective effect of astrocytes on neurons subjected to oxidative stress. The action of TX14(A) is at least partially mediated by Gi-proteins and the cAMP-PKA axis. On the other hand, when recombinant GPR37L1 or GPR37 are expressed in HEK293 cells, they are not functional and do not respond to TX14(A), which explains unsuccessful attempts to confirm the ligand-receptor pairing. Therefore, this study identifies GPR37L1/GPR37 as the receptors for TX14(A), and, by extension of Saposin C, and paves the way for the development of neuroprotective therapeutics acting via these receptors.
Assuntos
Astrócitos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Saposinas/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Animais Recém-Nascidos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Embrião de Mamíferos , Células HEK293 , Humanos , L-Lactato Desidrogenase/metabolismo , Fatores de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/química , Interferência de RNA/fisiologia , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Saposinas/química , Água/farmacologia , Ferimentos e Lesões/tratamento farmacológicoRESUMO
We show that the G protein-coupled receptor GPR37-like 1 (GPR37L1) is expressed in most astrocytes and some oligodendrocyte precursors in the mouse central nervous system. This contrasts with GPR37, which is mainly in mature oligodendrocytes. Comparison of wild type and Gpr37l1-/- mice showed that loss of GPR37L1 did not affect the input resistance or resting potential of astrocytes or neurons in the hippocampus. However, GPR37L1-mediated signalling inhibited astrocyte glutamate transporters and - surprisingly, given its lack of expression in neurons - reduced neuronal NMDA receptor (NMDAR) activity during prolonged activation of the receptors as occurs in ischemia. This effect on NMDAR signalling was not mediated by a change in the release of D-serine or TNF-α, two astrocyte-derived agents known to modulate NMDAR function. After middle cerebral artery occlusion, Gpr37l1 expression was increased around the lesion. Neuronal death was increased by â¼40% in Gpr37l1-/- brain compared to wild type in an in vitro model of ischemia. Thus, GPR37L1 protects neurons during ischemia, presumably by modulating extracellular glutamate concentration and NMDAR activation.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Infarto da Artéria Cerebral Média/terapia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciais de Ação/genética , Animais , Animais Recém-Nascidos , Ácido Aspártico/farmacologia , Astrócitos/efeitos dos fármacos , Encéfalo/patologia , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Hipocampo/citologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , N-Metilaspartato/farmacologia , Neurônios/fisiologia , Receptores Acoplados a Proteínas G/genética , Reconhecimento Psicológico/fisiologiaRESUMO
The G-protein-coupled receptor 37 ( GPR37) has been implicated in the juvenile form of Parkinson's disease, in dopamine signalling and in the survival of dopaminergic cells in animal models. The structure and function of the receptor, however, have remained enigmatic. Here, we demonstrate that although GPR37 matures and is exported from the endoplasmic reticulum in a normal manner upon heterologous expression in HEK293 and SH-SY5Y cells, its long extracellular N-terminus is subject to metalloproteinase-mediated limited proteolysis between E167 and Q168. The proteolytic processing is a rapid and efficient process that occurs constitutively. Moreover, the GPR37 ectodomain is released from cells by shedding, a phenomenon rarely described for GPCRs. Immunofluorescence microscopy further established that although full-length receptors are present in the secretory pathway until the trans-Golgi network, GPR37 is expressed at the cell surface predominantly in the N-terminally truncated form. This notion was verified by flow cytometry and cell surface biotinylation assays. These new findings on the GPR37 N-terminal limited proteolysis may help us to understand the role of this GPCR in the pathophysiology of Parkinson's disease and in neuronal function in general.
Assuntos
Metaloproteases/metabolismo , Doença de Parkinson/patologia , Proteólise , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Dipeptídeos/farmacologia , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Metaloproteases/antagonistas & inibidores , Estrutura Terciária de ProteínaRESUMO
Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.
Assuntos
Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Cílios/genética , Cílios/patologia , Meduloblastoma/genética , Meduloblastoma/patologia , Animais , Animais Recém-Nascidos , Cerebelo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genéticaRESUMO
Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4+ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4+ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4+ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4+ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4+ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4+ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Exossomos/fisiologia , HIV-1/fisiologia , Ativação Viral/fisiologia , Adulto , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/uso terapêutico , Linhagem Celular , Técnicas de Cocultura , Quimioterapia Combinada , Infecções por HIV/virologia , Humanos , MasculinoRESUMO
GPR37 is an orphan G protein-coupled receptor mostly enriched in brain areas such as the cerebellum, striatum, and hippocampus. Identified as a substrate of parkin, GPR37 has been suggested to play a role in Parkinson's disease. Distributed throughout the brain, the function of GPR37, however, remains unknown. We now provide the first mapping of GPR37 within the hippocampus, where GPR37 is widely expressed and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts, and axon terminals. GPR37 per se does not appear to play a role in learning and memory, since knocking out GPR37 (GPR37-KO) did not alter the performance in different hippocampal-related memory tasks. This is in agreement with slice electrophysiology experiments showing no differences both in short-term plasticity paired-pulse facilitation and long-term potentiation between WT and GPR37-KO mice. However, we report a potential functional interaction between GPR37 and adenosine A2A receptors (A2 A R) in the hippocampus, with A2 A R modulating the GPR37-associated phenotype. Thus, the absence of GPR37 appeared to sensitize mice to hippocampal A2 A R-mediated signaling, as observed by the effect of the A2 A R antagonist SCH58261 increasing synaptic depotentiation, reducing novel object recognition memory and reverting the anxiolytic effect of GPR37 deletion. Collectively, these findings afford insight into the localization and role of the orphan GPR37 within the hippocampus with potential involvement in A2 A R function (i.e., A2 A R sensitization). GPR37 is an orphan G protein-coupled receptor widely expressed in the hippocampus and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts and axon terminals. This orphan receptor per se does not appear to directly control the learning and memory processes; however knocking-out GPR37 triggers anxiolytic-like effects and sensitizes mice to hippocampal A2A R-mediated signalling.
Assuntos
Hipocampo/metabolismo , Doença de Parkinson/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Ansiedade/metabolismo , Células Cultivadas , Células HEK293 , Hipocampo/química , Humanos , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A2A de Adenosina/análise , Receptores Acoplados a Proteínas G/análiseRESUMO
Autism spectrum disorder (ASD) is a developmental brain disorder. Mutations in synaptic components including synaptic adhesion molecules have been found in ASD patients. Contactin-associated protein-like 2 (CASPR2) is one of the synaptic adhesion molecules associated with ASD. CASPR2 forms a complex with receptors via interaction with multiple PDZ domain protein 1 (MUPP1). Little is known about the relationship between impaired CASPR2-MUPP1-receptor complex and the pathogenesis of ASD. GPR37 is a receptor for survival factors. We recently identified mutations including R558Q in the G-protein-coupled receptor 37 (GPR37) gene in ASD patients. The mutated GPR37s accumulate in the endoplasmic reticulum. In this study, we show that GPR37 is a component of the CASPR2-MUPP1 receptor complex in the mouse brain. CASPR2 and GPR37 mainly interacted with the PDZ3 and PDZ11 domains of MUPP1, respectively. Compared to GPR37, GPR37(R558Q) slightly interacted with MUPP1 and caused dendritic alteration. GPR37, but not GPR37(R558Q) nor GPR37-deltaC which lacks its PDZ binding domain, was transported to the cell surface by MUPP1. In primary hippocampal neurons, GPR37 co-localized with MUPP1 and CASPR2 at the synapse, but not GPR37(R558Q). Thus, ASD-related mutation of GPR37 may cause the impaired CASPR2-MUPP1-GPR37 complex on the dendrites associated with one of the pathogenesis of ASD. In this study, we identified that GPR37 is a component of the MUPP1 and CASPR2 receptor complex. Autism deleterious mutated GPR37(R558Q) slightly interacts with MUPP1 and retains in ER, resulting in dendritic alteration. In neuron, GPR37, but not GPR37(R558Q), is transported to the dendrite and synapse by MUPP1. Thus, ASD-related mutation of GPR37 may cause the impaired CASPR2-MUPP1-GPR37 complex on the dendrites associated with one of the pathogenesis of ASD.
Assuntos
Proteínas de Transporte/metabolismo , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Proteínas de Membrana/metabolismo , Mutação/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Células Cultivadas , Transtornos Globais do Desenvolvimento Infantil/genética , Chlorocebus aethiops , Hipocampo/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Ligação Proteica/fisiologia , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genéticaRESUMO
The parkin-associated endothelial-like receptor (PAELR, GPR37) is an orphan G protein-coupled receptor that interacts with and is degraded by parkin-mediated ubiquitination. Mutations in parkin are thought to result in PAELR accumulation and increase neuronal cell death in Parkinson's disease. In this study, we find that the protein interacting with C-kinase (PICK1) interacts with PAELR. Specifically, the Postsynaptic density protein-95/Discs large/ZO-1 (PDZ) domain of PICK1 interacted with the last three residues of the c-terminal (ct) located PDZ motif of PAELR. Pull-down assays indicated that recombinant and native PICK1, obtained from heterologous cells and rat brain tissue, respectively, were retained by a glutathione S-transferase fusion of ct-PAELR. Furthermore, coimmunoprecipitation studies isolated a PAELR-PICK1 complex from transiently transfected cells. PICK1 interacts with parkin and our data showed that PICK1 reduces PAELR expression levels in transiently transfected heterologous cells compared to a PICK1 mutant that does not interact with PAELR. Finally, PICK1 over-expression in HEK293 cells reduced cell death induced by PAEALR over-expression during rotenone treatment and these effects of PICK1 were attenuated during inhibition of the proteasome. These results suggest a role for PICK1 in preventing PAELR-induced cell toxicity.