RESUMO
G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Receptores Acoplados a Proteínas G/química , Fosforilação , Transdução de Sinais/fisiologia , Ligantes , Proteômica , Espectrometria de Massas , Proteínas de Transporte/metabolismoRESUMO
Establishment of functional synaptic connections in a selective manner is essential for nervous system operation. In mammalian retinas, rod and cone photoreceptors form selective synaptic connections with different classes of bipolar cells (BCs) to propagate light signals. While there has been progress in elucidating rod wiring, molecular mechanisms used by cones to establish functional synapses with BCs have remained unknown. Using an unbiased proteomic strategy in cone-dominant species, we identified the cell-adhesion molecule ELFN2 to be pivotal for the functional wiring of cones with the ON type of BC. It is selectively expressed in cones and transsynaptically recruits the key neurotransmitter receptor mGluR6 in ON-BCs to enable synaptic transmission. Remarkably, ELFN2 in cone terminals functions in synergy with a related adhesion molecule, ELFN1, and their concerted interplay during development specifies selective wiring and transmission of cone signals. These findings identify a synaptic connectivity mechanism of cones and illustrate how interplay between adhesion molecules and postsynaptic transmitter receptors orchestrates functional synaptic specification in a neural circuit.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Sinapses/metabolismo , Animais , Moléculas de Adesão Celular/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/análise , Proteoma/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Células Bipolares da Retina/metabolismoRESUMO
BACKGROUND: Leg length discrepancy (LLD) after total hip arthroplasty (THA) is a known source of complications and a leading cause of litigation (J Bone Joint Surg Br 87:155-157, 2005). There are limited studies investigating surgical approach combined with the use of fluoroscopy intraoperatively and their potential effects on LLD after THA. The purpose of this study was to compare the direct anterior (DA) approach utilizing a fluoroscopic overlay technique and anterolateral (AL) approach and their potential effect on LLD. METHODS: We retrospectively reviewed 121 patients who had undergone primary THA from September 1, 2016 to November 1, 2018 by either DA or AL approach by two separate surgeons. Leg length discrepancies were measured on pre-operative post-anesthesia care unit (PACU) and on post-operative low anterior/posterior (AP) pelvis plain radiographs by two investigators blinded to each other's measurements. To confirm inter-observer and intra-observer reliability between LLD measurements amongst investigators, a Pearson correlation test was performed. The primary outcome measurement was leg length discrepancy (LLD). RESULTS: We observed LLD > 1.0 cm and LLD > 1.5 cm in the DA and AL groups. The DA approach group showed a mean LLD of 4.5 mm against 7.76 mm in the AL group (p < 0.00001). There was a significantly higher rate of LLD in the AL group as compared to the DA group (LLD> 1 cm (28% vs. 8%, p = 0.0037) and LLD > 1.5 cm (7% vs. 0%, p = 0.0096). The LLD measurements showed strong correlation in terms of inter-observer (r = 0.95) and intra-observer reliability (r = 0.99) between the two investigators (p < 0.001). CONCLUSION: In our patient cohort, the DA approach with fluoroscopic overlay technique had less LLD in comparison with the AL approach, suggesting that intraoperative fluoroscopic use does have an impact on LLD.
RESUMO
G protein-coupled receptors (GPCRs) remain one of the most successful targets of U.S. Food and Drug Administration-approved drugs. GPCR research has predominantly focused on the characterization of the intracellular interactome's contribution to GPCR function and pharmacology. However, emerging evidence uncovers a new dimension in the biology of GPCRs involving their extracellular and transcellular interactions that critically impact GPCR function and pharmacology. The seminal examples include a variety of adhesion GPCRs, such as ADGRLs/latrophilins, ADGRBs/brain angiogenesis inhibitors, ADGRG1/GPR56, ADGRG6/GPR126, ADGRE5/CD97, and ADGRC3/CELSR3. However, recent advances have indicated that class C GPCRs that contain large extracellular domains, including group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, mGluR8), γ-aminobutyric acid receptors, and orphans GPR158 and GPR179, can also participate in this form of transcellular regulation. In this review, we will focus on a variety of identified extracellular and transcellular GPCR-interacting partners, including teneurins, neurexins, integrins, fibronectin leucine-rich transmembranes, contactin-6, neuroligin, laminins, collagens, major prion protein, amyloid precursor protein, complement C1q-likes, stabilin-2, pikachurin, dystroglycan, complement decay-accelerating factor CD55, cluster of differentiation CD36 and CD90, extracellular leucine-rich repeat and fibronectin type III domain containing 1, and leucine-rich repeat, immunoglobulin-like domain and transmembrane domains. We provide an account on the diversity of extracellular and transcellular GPCR complexes and their contribution to key cellular and physiologic processes, including cell migration, axon guidance, cellular and synaptic adhesion, and synaptogenesis. Furthermore, we discuss models and mechanisms by which extracellular GPCR assemblies may regulate communication at cellular junctions. SIGNIFICANCE STATEMENT: G protein-coupled receptors (GPCRs) continue to be the prominent focus of pharmacological intervention for a variety of human pathologies. Although the majority of GPCR research has focused on the intracellular interactome, recent advancements have identified an extracellular dimension of GPCR modulation that alters accepted pharmacological principles of GPCRs. Herein, we describe known endogenous allosteric modulators acting on GPCRs both in cis and in trans.
Assuntos
Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Humanos , Ligantes , Terapia de Alvo Molecular , Receptores Acoplados a Proteínas G/químicaRESUMO
The functional characterization of the GPCR interactome has predominantly focused on intracellular binding partners; however, the recent emergence of transsynaptic GPCR complexes represents an additional dimension to GPCR function that has previously been unaccounted for in drug discovery. Here, we characterize ELFN2 as a novel postsynaptic adhesion molecule with a distinct expression pattern throughout the brain and a selective binding with group III metabotropic glutamate receptors (mGluRs) in trans. Using a transcellular GPCR signaling platform, we report that ELFN2 critically alters group III mGluR secondary messenger signaling by directly altering G protein coupling kinetics and efficacy. Loss of ELFN2 in mice results in the selective downregulation of group III mGluRs and dysregulated glutamatergic synaptic transmission. Elfn2 knockout (Elfn2 KO) mice also feature a range of neuropsychiatric manifestations including seizure susceptibility, hyperactivity, and anxiety/compulsivity, which can be rescued by pharmacological augmentation of group III mGluRs. Thus, we conclude that extracellular transsynaptic scaffolding by ELFN2 in the brain is a cardinal organizational feature of group III mGluRs essential for their signaling properties and brain function.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Moléculas de Adesão de Célula Nervosa/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
ß-Arrestins are multifunctional adaptor proteins best know for their vital role in regulating G protein coupled receptor (GPCR) trafficking and signaling. ß-arrestin2 recruitment and receptor internalization of corticotropin-releasing factor receptor 1 (CRFR1), a GPCR whose antagonists have been shown to demonstrate both anxiolytic- and antidepressant-like effects, have previously been shown to be modulated by PDZ proteins. Thus, a structural characterization of the interaction between ß-arrestins and PDZ proteins can delineate potential mechanism of PDZ-dependent regulation of GPCR trafficking. Here, we find that the PDZ proteins PSD-95, MAGI1, and PDZK1 interact with ß-arrestin2 in a PDZ domain-dependent manner. Further investigation of such interaction using mutational analyses revealed that mutating the alanine residue at 175 residue of ß-arrestin2 to phenylalanine impairs interaction with PSD-95. Additionally, A175F mutant of ß-arrestin2 shows decreased CRF-stimulated recruitment to CRFR1 and reduced receptor internalization. Thus, our findings show that the interaction between ß-arrestins and PDZ proteins is key for CRFR1 trafficking and may be targeted to mitigate impaired CRFR1 signaling in mental and psychiatric disorders.
Assuntos
Domínios PDZ , Receptores de Hormônio Liberador da Corticotropina , beta-Arrestina 2 , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Proteína 4 Homóloga a Disks-Large/química , Proteína 4 Homóloga a Disks-Large/metabolismo , Guanilato Quinases/química , Guanilato Quinases/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Transporte Proteico , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/metabolismo , beta-Arrestina 2/química , beta-Arrestina 2/metabolismoRESUMO
Functional characterization of the GPCR interactome has been focused predominantly on intracellular interactions, yet GPCRs are increasingly found in complex with extracellular proteins. Extracellular leucine-rich repeat fibronectin type III domain containing 1 (ELFN1) was recently reported to physically anchor mGluR6 and mGluR7 across retinal and hippocampal synapses, respectively; however, the consequence of transsynaptic interactions on properties and pharmacology of these receptors are unknown. In the current study, we explore the effects of ELFN1 on mGluR signaling and pharmacology. First, we established the binding specificity of ELFN1 and found it to be recruited selectively to all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), but not other mGluR species. Using site-directed mutagenesis we mapped binding determinants of this interaction to two distinct sites on the ELFN1 ectodomain. To evaluate functional aspects of the interaction, we developed a transcellular signaling assay in reconstituted HEK293 cells which monitors changes in mGluR activity in one cell following its exposure to separate ELFN1-containing cells. Using this platform, we found that ELFN1 acts as an allosteric modulator of class III mGluR activity in suppressing cAMP accumulation: altering both agonist-induced and constitutive receptor activity. Using bioluminescence resonance energy transfer-based real-time kinetic assays, we established that ELFN1 alters the ability of mGluRs to activate G proteins. Our findings demonstrate that core properties of class III mGluRs can be altered via extracellular interactions with ELFN1 which serves as a transsynaptic allosteric modulator for these receptors. Furthermore, our unique assay platform opens avenues for exploring transcellular/transsynaptic pharmacology of other GPCR transcomplexes.
Assuntos
AMP Cíclico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sistemas do Segundo Mensageiro , Regulação Alostérica/fisiologia , Sítios de Ligação , AMP Cíclico/genética , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Receptores de Glutamato Metabotrópico/genéticaRESUMO
MAGI proteins are Membrane-Associated Guanylate Kinase Inverted proteins that belong to the MAGUK family. They are scaffolding proteins that were shown to mediate the trafficking and signaling of various G protein-coupled receptors (GPCRs). They contain PDZ domains in their structure and many GPCRs interact with these proteins via the PDZ motifs on the carboxyl terminal end of a receptor. In a PDZ overlay assay performed with the carboxyl terminal tail of 5-HT2AR, we were able to detect all three members of the MAGI subfamily, MAGI-1, MAGI-2 and MAGI-3 as interacting PDZ proteins. The PDZ motif of 5-HT2AR consists of three amino acids; serine (S), cysteine (C) and valine (V). In this study, we characterize these 5-HT2AR interactions with MAGI proteins. We first confirm the interaction using co-immunopricipitation and illustrate that the interaction is PDZ motif-dependent in human embryonic kidney (HEK 293) cells. We then assess the effects of overexpression and knockdown of the MAGI proteins on the internalization, trafficking and signaling of 5-HT2AR. We find that knockdown of either MAGI-1 or MAGI-3 using siRNA results in a significant reduction in the internalization of 5-HT2AR. As for signaling, we report here that MAGI proteins can modulate the signaling via the two transduction pathways that 5-HT2AR can activate. We illustrate a significant effect of modulating MAGI proteins expression on 5-HT-stimulated IP formation. We demonstrate an enhancement in 5-HT2AR-stimulated IP formation upon MAGI proteins overexpression. In addition, we show that knockdown of MAGI proteins with siRNA leads to a significant reduction in 5-HT2AR-stimulated IP formation. Furthermore, we illustrate a significant increase in 5-HT-stimulated ERK1/2 phosphorylation upon MAGI proteins knockdown. Interestingly, this effect on ERK1/2 activation is PDZ motif-independent. We also suggest two possible mechanisms of regulation for the effect of MAGI proteins on 5-HT2AR function. One mechanism involves the regulation of cell surface expression since we show that both MAGI-2 and MAGI-3 can enhance receptor trafficking to the plasma membrane when overexpressed in HEK 293 cells. The other mechanism points to regulation of second messengers in the signaling pathways. Specifically, we show that overexpression of any of the three MAGI proteins can enhance the recruitment of PLCß3 to 5-HT2AR. In addition, we report a negative effect for knocking down MAGI-3 on ß-arrestin recruitment to the receptor and this effect is PDZ motif-independent. Taken together, our findings document distinct roles for the three MAGI proteins in regulating 5-HT2AR trafficking and signaling and emphasize the importance of studying PDZ proteins and their interactions with GPCRs to regulate their function.
Assuntos
Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipase C beta/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Moléculas de Adesão Celular , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/genética , Endocitose/efeitos dos fármacos , Guanilato Quinases , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Domínios PDZ , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor 5-HT2A de Serotonina/química , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , beta-Arrestina 1/metabolismoRESUMO
Activation of Group I metabotropic glutamate receptors (mGluRs) activates signaling cascades, resulting in calcium release from intracellular stores, ERK1/2 activation, and long term changes in synaptic activity that are implicated in learning, memory, and neurodegenerative diseases. As such, elucidating the molecular mechanisms underlying Group I mGluR signaling is important for understanding physiological responses initiated by the activation of these receptors. In the current study, we identify the multifunctional scaffolding protein spinophilin as a novel Group I mGluR-interacting protein. We demonstrate that spinophilin interacts with the C-terminal tail and second intracellular loop of Group I mGluRs. Furthermore, we show that interaction of spinophilin with Group I mGluRs attenuates receptor endocytosis and phosphorylation of ERK1/2, an effect that is dependent upon the interaction of spinophilin with the C-terminal PDZ binding motif encoded by Group I mGluRs. Spinophilin knock-out results in enhanced mGluR5 endocytosis as well as increased ERK1/2, AKT, and Ca(2+) signaling in primary cortical neurons. In addition, the loss of spinophilin expression results in impaired mGluR5-stimulated LTD. Our results indicate that spinophilin plays an important role in regulating the activity of Group I mGluRs as well as their influence on synaptic activity.
Assuntos
Sinalização do Cálcio/fisiologia , Endocitose/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Células HEK293 , Humanos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Glutamato Metabotrópico/genéticaRESUMO
Corticotropin-releasing factor (CRF) is a neuropeptide commonly associated with the hypothalamic-pituitary adrenal axis stress response. Upon release, CRF activates two G protein-coupled receptors (GPCRs): CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2). Although both receptors contribute to mood regulation, CRFR1 antagonists have demonstrated anxiolytic and antidepressant-like properties that may be exploited in the generation of new pharmacological interventions for mental illnesses. Previous studies have demonstrated CRFR1 capable of heterologously sensitizing serotonin 2A receptor (5-HT2AR) signaling: another GPCR implicated in psychiatric disease. Interestingly, this phenomenon was dependent on Postsynaptic density 95 (PSD-95)/Disc Large/Zona Occludens (PDZ) interactions on the distal carboxyl termini of both receptors. In the current study, we demonstrate that endogenous PSD-95 can be co-immunoprecipitated with CRFR1 from cortical brain homogenate, and this interaction appears to be primarily via the PDZ-binding motif. Additionally, PSD-95 colocalizes with CRFR1 within the dendritic projections of cultured mouse neurons in a PDZ-binding motif-dependent manner. In HEK 293 cells, PSD-95 overexpression inhibited CRFR1 endocytosis, whereas PSD-95 shRNA knockdown enhanced CRFR1 endocytosis. Although PSD-95 does not appear to play a significant role in CRF-mediated cAMP or ERK1/2 signaling, PSD-95 was demonstrated to suppress ß-arrestin2 recruitment: providing a potential mechanism for PSD-95's inhibition of endocytosis. In revisiting previously documented heterologous sensitization, PSD-95 shRNA knockdown did not prevent CRFR1-mediated enhancement of 5-HT2AR signaling. In conclusion, we have identified and characterized a novel functional relationship between CRFR1 and PSD-95 that may have implications in the design of new treatment strategies for mental illness.
Assuntos
Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , beta-Arrestina 2/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteína 4 Homóloga a Disks-Large , Endocitose , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Camundongos , Densidade Pós-Sináptica/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Receptores de Hormônio Liberador da Corticotropina/química , Transdução de SinaisRESUMO
Corticotropin-releasing factor (CRF) receptor1 (CRFR1) is associated with psychiatric illness and is a proposed target for the treatment of anxiety and depression. Similar to many G protein-coupled receptors (GPCRs), CRFR1 harbors a PDZ (PSD-95/Disc Large/Zona Occludens)-binding motif at the end of its carboxyl-terminal tail. The interactions of PDZ proteins with GPCRs are crucial for the regulation of receptor function. In the present study, we characterize the interaction of all members of the membrane-associated guanylate kinase with inverted orientation PDZ (MAGI) proteins with CRFR1. We show using co-immunoprecipitation that CRFR1 interacts with MAGI-1 and MAGI-3 in human embryonic kidney (HEK293) cells in a PDZ motif-dependent manner. We find that overexpression as well as knockdown of MAGI proteins result in a significant reduction in CRFR1 endocytosis. This effect is dependent on an intact PDZ binding motif for MAGI-2 and MAGI-3 but not MAGI-1. We show that the alteration in expression levels of MAGI-1, MAGI-2 or MAGI-3 can interfere with ß-arrestin recruitment to CRFR1. This could explain the effects observed with receptor internalization. We also find that knockdown of endogenous MAGI-1, MAGI-2 or MAGI-3 in HEK293 cells can lead to an enhancement in ERK1/2 signaling but has no effect on cAMP formation. Interestingly, we observe a compensation effect between MAGI-1 and MAGI-3. Taken together, our data suggest that the MAGI proteins, MAGI-1, MAGI-2 and MAGI-3 can regulate ß-arrestin-mediated internalization of CRFR1 as well as its signaling and that there is a compensatory mechanism involved in regulating the function of the MAGI subfamily.
RESUMO
The piriform cortex (PC) is richly innervated by corticotropin-releasing factor (CRF) and serotonin (5-HT) containing axons arising from central amygdala and Raphe nucleus. CRFR1 and 5-HT2A/2CRs have been shown to interact in manner where CRFR activation subsequently potentiates the activity of 5-HT2A/2CRs. The purpose of this study was to determine how the activation of CRFR1 and/or 5-HT2Rs modulates PC activity at both the circuit and cellular level. Voltage sensitive dye imaging showed that CRF acting through CRFR1 dampened activation of the Layer II of PC and interneurons of endopiriform nucleus. Application of the selective 5-HT2A/CR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) following CRFR1 activation potentiated this effect. Blocking the interaction between CRFR1 and 5-HT2R with a Tat-CRFR1-CT peptide abolished this potentiation. Application of forskolin did not mimic CRFR1 activity but instead blocked it, while a protein kinase A antagonist had no effect. However, activation and antagonism of protein kinase C (PKC) either mimicked or blocked CRF modulation, respectively. DOI had no effect when applied alone indicating that the prior activation of CRFR1 receptors was critical for DOI to show significant effects similar to CRF. Patch clamp recordings showed that both CRF and DOI reduced the synaptic responsiveness of Layer II pyramidal neurons. CRF had highly variable effects on interneurons within Layer III, both increasing and decreasing their excitability, but DOI had no effect on the excitability of this group of neurons. These data show that CRF and 5-HT, acting through both CRFR1 and 5-HT2A/CRs, reduce the activation of the PC. This modulation may be an important blunting mechanism of stressor behaviors mediated through the olfactory cortex.
RESUMO
Corticotropin releasing factor (CRF) receptor1 (CRFR1) is associated with psychiatric illness and is a proposed target for the treatment of anxiety and depression. Like many G protein-coupled receptors (GPCRs), CRFR1 harbors a PDZ (PSD95/Disc Large/Zona Occludens 1)-binding motif at the end of its carboxyl terminal tail. The interactions of PDZ proteins with GPCRs are crucial for the regulation of their receptor function. In the present study, we characterize the interaction of the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) with CRFR1. We show using co-immunoprecipitation that the two proteins interact in human embryonic kidney (HEK293) cells in a PDZ motif-dependent manner. We find that the interaction occurs at the Golgi apparatus and that overexpression of CAL retains a proportion of CRFR1 in the intracellular compartment and prevents trafficking to the cell surface. We also demonstrate a significant reduction in the levels of receptor at the plasma membrane upon CAL overexpression, as well as a reduction in internalization. We find that the overexpression of CAL in HEK293 cells resulted in a significant decrease in CRF-stimulated extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation, but has no effect on cAMP signaling mediated by the receptor. This effect was dependent on an intact PDZ motif and knockdown of CAL expression using CAL siRNA results in a significant enhancement in ERK1/2 signaling. We show that CAL contributes to the regulation of CRFR1 glycosylation and utilize glycosylation-deficient CRFR1 mutants to further examine the role of glycosylation in the cell surface trafficking of CRFR1. We find that the mutation of Asn residues 90 and 98 results in a reduction in cell surface CRFR1 that is comparable to the effect of CAL overexpression and that these mutants are retained in the Golgi apparatus. Mutation of Asn residues 90 and 98 also results in a decrease in the efficacy for CRF-stimulated cAMP formation mediated by CRFR1. Taken together, our data suggest that CAL can regulate the anterograde trafficking, the internalization as well as the signaling of CRFR1 via modulating the post-translational modifications that the receptor undergoes at the Golgi apparatus.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , AMP Cíclico/metabolismo , Endocitose , Glicosilação , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana Transportadoras , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Sistemas do Segundo MensageiroRESUMO
G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and leukemia-associated RhoGEF), RGS3 and RGS12, spinophilin and neurabin-1, SRC homology 3 domain and multiple ankyrin repeat domain (Shank) proteins (Shank1, Shank2, and Shank3), partitioning defective proteins 3 and 6, multiple PDZ protein 1, Tamalin, neuronal nitric oxide synthase, syntrophins, protein interacting with protein kinase C α 1, syntenin-1, and sorting nexin 27.
Assuntos
Domínios PDZ/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Ligação Proteica/fisiologia , Transporte Proteico/fisiologiaRESUMO
The corticotropin-releasing factor receptor 1 (CRFR1) and serotonin 2A receptor (5-HT2AR) are linked to cellular mechanisms underlying stress anxiety and depression. Both receptors are members of the G protein-coupled receptor (GPCR) superfamily and encode class I PSD-95/DiscsLarge/Zona Occludens 1 (PDZ) binding motifs (-S/T-x-V/I/L) at the end of their carboxyl-terminal tails. We have identified PDZK1, also referred to as Na(+)/H(+) exchange regulatory cofactor 3 (NHERF3) as both a CRFR1- and 5-HT2AR-interacting protein. We have examined whether PDZK1 plays a role in regulating both CRFR1 and 5-HT2AR activity. We find that while PDZK1 interactions with CRFR1 are PDZ binding motif-dependent, PDZK1 associates with 5-HT2AR in a PDZ binding motif-independent manner and CRFR1 expression, but not 5-HT2AR expression, redistributes PDZK1 to the plasma membrane in PDZ binding motif-dependent manner. PDZK1, negatively regulates 5-HT2AR endocytosis and has no effect upon 5-HT2AR-mediated ERK1/2 phosphorylation. In contrast, PDZK1 overexpression does not affect CRFR1 endocytosis, but selectively increases CRFR1-stimulated ERK1/2 phosphorylation. Similar to what has been previously reported for PSD-95 and SAP97, PDZK1 positively influences 5-HT2AR-stimulated inositol phosphate formation, but does not contribute to the regulation of CRFR1-mediated cAMP signaling. Taken together, these results indicate that PDZK1 differentially regulates the signaling and trafficking of CRFR1 and 5-HT2AR via PDZ-dependent and -independent mechanisms, respectively.
Assuntos
Proteínas de Transporte/metabolismo , Endocitose/genética , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Transdução de Sinais/genética , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Domínios PDZ , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor 5-HT2A de Serotonina/genética , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
Serotonin (5-HT) interacts with a wide variety of 5-HT receptors (5-HTR) of which 5-HT2AR plays an important target for antidepressant and atypical antipsychotic drugs. The carboxyl-terminal tail of 5-HT2AR encodes a motif that mediates interactions with PSD-95/disc large/zona occludens (PDZ) domain-containing proteins. In the present study, we found that 5-HT2AR interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by coimmunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates. We found that 5-HT2AR expression results in the recruitment of SAP97 from the cytosol to the plasma membrane and that this recruitment is dependent on an intact 5-HT2AR PDZ binding motif. We also show that 5-HT2AR interacts with SAP97 using bioluminescence energy transfer and that overexpression of SAP97 retards 5-HT2AR endocytosis, while single hairpin RNA knockdown facilitates 5-HT2AR internalization. The knockdown of SAP97 in HEK 293 cells results in a reduction in the maximum efficacy for 5-HT2AR-stimulated inositol phosphate formation and that the deletion of the 5-HT2AR PDZ motif also impairs 5-HT2AR signaling. Similarly to what has been observed for the corticotropin-releasing factor receptor 1 (CRFR1), SAP97 expression is essential for 5-HT2AR-stimulated extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation by a PDZ interaction-independent mechanism. Moreover, we find that SAP97 is not responsible for CRFR1-mediated sensitization of 5-HT2AR signaling. Taken together, our studies show that SAP97 plays a conserved role in regulating 5-HT2AR endocytosis and ERK1/2 signaling, but plays a novel role in regulating 5-HT2AR G protein coupling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Membrana/fisiologia , Receptor 5-HT2A de Serotonina/fisiologia , Membrana Celular/metabolismo , Proteína 1 Homóloga a Discs-Large , Endocitose , Ativação Enzimática , Humanos , Fosfatos de Inositol/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Transdução de SinaisRESUMO
The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Guanilato Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Proteína 1 Homóloga a Discs-Large , Endocitose/fisiologia , Endossomos/genética , Endossomos/metabolismo , Guanilato Quinases/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Ratos , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
GPCRs represent the largest family of integral membrane proteins and were first identified as receptor proteins that couple via heterotrimeric G-proteins to regulate a vast variety of effector proteins to modulate cellular function. It is now recognized that GPCRs interact with a myriad of proteins that not only function to attenuate their signalling but also function to couple these receptors to heterotrimeric G-protein-independent signalling pathways. In addition, intracellular and transmembrane proteins associate with GPCRs and regulate their processing in the endoplasmic reticulum, trafficking to the cell surface, compartmentalization to plasma membrane microdomains, endocytosis and trafficking between intracellular membrane compartments. The present review will overview the functional consequence of ß-arrestin, receptor activity-modifying proteins (RAMPS), regulators of G-protein signalling (RGS), GPCR-associated sorting proteins (GASPs), Homer, small GTPases, PSD95/Disc Large/Zona Occludens (PDZ), spinophilin, protein phosphatases, calmodulin, optineurin and Src homology 3 (SH3) containing protein interactions with GPCRs.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Animais , Arrestinas/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Transporte Proteico , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1α (HIF-1α) to drive transcription of various genes. Therefore, we have used a HIF-1α reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1α bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1α binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1α bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.