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1.
Cell ; 182(3): 770-785.e16, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32634377

RESUMO

Heterotrimeric G-proteins (Gαßγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gßγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/instrumentação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Guanosina Trifosfato/química , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Neurônios/química , Neurônios/metabolismo , Neurônios/fisiologia , Transdução de Sinais , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo
2.
Proc Natl Acad Sci U S A ; 114(48): E10319-E10328, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29133411

RESUMO

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gßγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.


Assuntos
Proteínas Ativadoras de GTPase/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Nucleares/genética , Engenharia de Proteínas/métodos , Receptores Acoplados a Proteínas G/genética , Proteínas de Transporte Vesicular/genética , Motivos de Aminoácidos , Animais , Clonagem Molecular , Embrião não Mamífero , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
3.
J Biol Chem ; 293(51): 19586-19599, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30352874

RESUMO

The causative role of G protein-coupled receptor (GPCR) pathway mutations in uveal melanoma (UM) has been well-established. Nearly all UMs bear an activating mutation in a GPCR pathway mediated by G proteins of the Gq/11 family, driving tumor initiation and possibly metastatic progression. Thus, targeting this pathway holds therapeutic promise for managing UM. However, direct targeting of oncogenic Gαq/11 mutants, present in ∼90% of UMs, is complicated by the belief that these mutants structurally resemble active Gαq/11 WT. This notion is solidly founded on previous studies characterizing Gα mutants in which a conserved catalytic glutamine (Gln-209 in Gαq) is replaced by leucine, which leads to GTPase function deficiency and constitutive activation. Whereas Q209L accounts for approximately half of GNAQ mutations in UM, Q209P is as frequent as Q209L and also promotes oncogenesis, but has not been characterized at the molecular level. Here, we characterized the biochemical and signaling properties of Gαq Q209P and found that it is also GTPase-deficient and activates downstream signaling as efficiently as Gαq Q209L. However, Gαq Q209P had distinct molecular and functional features, including in the switch II region of Gαq Q209P, which adopted a conformation different from that of Gαq Q209L or active WT Gαq, resulting in altered binding to effectors, Gßγ, and regulators of G-protein signaling (RGS) proteins. Our findings reveal that the molecular properties of Gαq Q209P are fundamentally different from those in other active Gαq proteins and could be leveraged as a specific vulnerability for the ∼20% of UMs bearing this mutation.


Assuntos
Carcinogênese/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Mutação , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Transdução de Sinais/genética
4.
Anal Biochem ; 583: 113363, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31310737

RESUMO

Long-term storage of DNA is a routine practice in biomedical research, diagnostics and drug discovery. Periodic monitoring is important for early detection of changes in DNA quality and quantity. Existing methods include agarose gel, ultraviolet (UV) absorbance, fluorometric reading and qPCR. However, these methods are either limited by sensitivity or depend on DNA standards, which face the same storage challenges. In this paper, we tested the state-of-the-art droplet digital PCR (ddPCR) technology that can quantify the absolute DNA copy number with no need of a standard curve. We found that ddPCR was very accurate in determining the level of a plasmid DNA standard and was sensitive to DNA loss due to degradation or adsorption. With the ddPCR technology, we found a gradual process of DNA adsorption to several types of low binding tubes, which was unnoticed before. Although modest, adsorption significantly affected recovery of highly diluted DNA (<0.2 µg/mL), which could be rescued by addition of carrier DNA. In conclusion, this paper not only demonstrated that ddPCR is an ideal method for monitoring DNA storage, but also provided an effective approach to improving recovery of highly diluted DNA, which may have broad implications in assay development, diagnostics and forensic sciences.


Assuntos
DNA/análise , Reação em Cadeia da Polimerase/métodos , Plasmídeos/análise , Manejo de Espécimes/métodos
5.
Proc Natl Acad Sci U S A ; 113(39): E5721-30, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27621449

RESUMO

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK-ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV-Gαi complexes and activates GIV's GEF function; then a second phosphomodification terminates GIV's GEF function, triggers the assembly of GIV-Gαs complexes, and activates GIV's GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK-ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/química , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Quinase C-theta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/química
6.
Proc Natl Acad Sci U S A ; 112(35): E4874-83, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26286990

RESUMO

Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development.


Assuntos
Movimento Celular , Proliferação de Células , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Receptores ErbB/metabolismo , Humanos , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Morfogênese , Fosforilação , Transporte Proteico , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas de Transporte Vesicular/química , Cicatrização
7.
J Biol Chem ; 291(15): 8269-82, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-26887938

RESUMO

Activation of the tyrosine kinase focal adhesion kinase (FAK) upon cell stimulation by the extracellular matrix initiates integrin outside-in signaling. FAK is directly recruited to active integrins, which enhances its kinase activity and triggers downstream signaling like activation of PI3K. We recently described that Gα-interacting, vesicle-associated protein (GIV), a protein up-regulated in metastatic cancers, is also required for outside-in integrin signaling. More specifically, we found that GIV is a non-receptor guanine nucleotide exchange factor that activates trimeric G proteins in response to integrin stimulation to enhance PI3K signaling and tumor cell migration. In contrast, previous reports have established that GIV is involved in phosphotyrosine (Tyr(P))-based signaling in response to growth factor stimulation;i.e.GIV phosphorylation at Tyr-1764 and Tyr-1798 recruits and activates PI3K. Here we show that phosphorylation of GIV at Tyr-1764/Tyr-1798 is also required to enhance PI3K-Akt signaling and tumor cell migration in response to integrin stimulation, indicating that GIV functions in Tyr(P)-dependent integrin signaling. Unexpectedly, we found that activation of FAK, an upstream component of the integrin Tyr(P) signaling cascade, was diminished in GIV-depleted cells, suggesting that GIV is required to establish a positive feedback loop that enhances integrin-FAK signaling. Mechanistically, we demonstrate that this feedback activation of FAK depends on both guanine nucleotide exchange factor and Tyr(P) GIV signaling as well as on their convergence point, PI3K. Taken together, our results provide novel mechanistic insights into how GIV promotes proinvasive cancer cell behavior by working as a signal-amplifying platform at the crossroads of trimeric G protein and Tyr(P) signaling.


Assuntos
Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Ativação Enzimática , Quinase 1 de Adesão Focal/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias/patologia , Fosforilação
8.
J Biol Chem ; 291(53): 27098-27111, 2016 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-27864364

RESUMO

GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gαi3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function.


Assuntos
Membrana Celular/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células HeLa , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Immunoblotting , Proteínas dos Microfilamentos/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética
9.
J Biol Chem ; 289(32): 21818-27, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-24982418

RESUMO

There is an increasing number of disease-associated Gα mutations identified from genome-wide sequencing campaigns or targeted efforts. Albright's Hereditary Osteodystrophy (AHO) was the first inherited disease associated with loss-of-function mutations in a G protein (Gαs) and other studies revealed gain-of-function Gα mutations in cancer. Here we attempted to solve the apparent quandary posed by the fact that the same mutation in two different G proteins appeared associated with both AHO and cancer. We first confirmed the presence of an inherited Gαs-R265H mutation from a previously described clinical case report of AHO. This mutation is structurally analogous to Gαo-R243H, an oncogenic mutant with increased activity in vitro and in cells due to rapid nucleotide exchange. We found that, contrary to Gαo-R243H, Gαs-R265H activity is compromised due to greatly impaired nucleotide binding in vitro and in cells. We obtained equivalent results when comparing another AHO mutation in Gαs (D173N) with a counterpart cancer mutation in Gαo (D151N). Gαo-R243H binds nucleotides efficiently under steady-state conditions but releases GDP much faster than the WT protein, suggesting diminished affinity for the nucleotide. These results indicate that the same disease-linked mutation in two different G proteins affects a common biochemical feature (nucleotide affinity) but to a different grade depending on the G protein (mild decrease for Gαo and severe for Gαs). We conclude that Gαs-R265H has dramatically impaired nucleotide affinity leading to the loss-of-function in AHO whereas Gαo-R243H has a mild decrease in nucleotide affinity that causes rapid nucleotide turnover and subsequent hyperactivity in cancer.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Pseudo-Hipoparatireoidismo/genética , Pseudo-Hipoparatireoidismo/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Cromograninas , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/genética , Linhagem , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
10.
Traffic ; 13(11): 1532-46, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22882974

RESUMO

ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.


Assuntos
Proteínas ADAM/metabolismo , Clatrina/metabolismo , Endocitose , Proteína Adaptadora GRB2/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/química , Proteína ADAM12 , Neoplasias da Mama/química , Neoplasias da Mama/enzimologia , Carcinoma/química , Endossomos/metabolismo , Feminino , Proteína Adaptadora GRB2/análise , Células HEK293 , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/química , Domínios Proteicos Ricos em Prolina , Domínios e Motivos de Interação entre Proteínas
11.
Hepatology ; 54(6): 2173-84, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21826695

RESUMO

UNLABELLED: During chronic liver disease, tissue remodeling leads to dramatic changes and accumulation of matrix components. Matrix metalloproteases and their inhibitors have been involved in the regulation of matrix degradation. However, the role of other proteases remains incompletely defined. We undertook a gene-expression screen of human liver fibrosis samples using a dedicated gene array selected for relevance to protease activities, identifying the ADAMTS1 (A Disintegrin And Metalloproteinase [ADAM] with thrombospondin type 1 motif, 1) gene as an important node of the protease network. Up-regulation of ADAMTS1 in fibrosis was found to be associated with hepatic stellate cell (HSC) activation. ADAMTS1 is synthesized as 110-kDa latent forms and is processed by HSCs to accumulate as 87-kDa mature forms in fibrotic tissues. Structural evidence has suggested that the thrombospondin motif-containing domain from ADAMTS1 may be involved in interactions with, and activation of, the major fibrogenic cytokine, transforming growth factor beta (TGF-ß). Indeed, we observed direct interactions between ADAMTS1 and latency-associated peptide-TGF-ß (LAP-TGF-ß). ADAMTS1 induces TGF-ß activation through the interaction of the ADAMTS1 KTFR peptide with the LAP-TGF-ß LKSL peptide. Down-regulation of ADAMTS1 in HSCs decreases the release of TGF-ß competent for transcriptional activation, and KTFR competitor peptides directed against ADAMTS1 block the HSC-mediated release of active TGF-ß. Using a mouse liver fibrosis model, we show that carbon tetrachloride treatment induces ADAMTS1 expression in parallel to that of type I collagen. Importantly, concurrent injection of the KTFR peptide prevents liver damage. CONCLUSION: Our results indicate that up-regulation of ADAMTS1 in HSCs constitutes a new mechanism for control of TGF-ß activation in chronic liver disease.


Assuntos
Proteínas ADAM/fisiologia , Cirrose Hepática/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína ADAMTS1 , Idoso , Motivos de Aminoácidos/fisiologia , Animais , Tetracloreto de Carbono , Colágeno Tipo I/biossíntese , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Células Estreladas do Fígado/fisiologia , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Regulação para Cima
12.
J Cell Biol ; 218(5): 1743-1763, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30948426

RESUMO

Heterotrimeric G proteins are signaling switches that control organismal morphogenesis across metazoans. In invertebrates, specific GPCRs instruct G proteins to promote collective apical cell constriction in the context of epithelial tissue morphogenesis. In contrast, tissue-specific factors that instruct G proteins during analogous processes in vertebrates are largely unknown. Here, we show that DAPLE, a non-GPCR protein linked to human neurodevelopmental disorders, is expressed specifically in the neural plate of Xenopus laevis embryos to trigger a G protein signaling pathway that promotes apical cell constriction during neurulation. DAPLE localizes to apical cell-cell junctions in the neuroepithelium, where it activates G protein signaling to drive actomyosin-dependent apical constriction and subsequent bending of the neural plate. This function is mediated by a Gα-binding-and-activating (GBA) motif that was acquired by DAPLE in vertebrates during evolution. These findings reveal that regulation of tissue remodeling during vertebrate development can be driven by an unconventional mechanism of heterotrimeric G protein activation that operates in lieu of GPCRs.


Assuntos
Embrião não Mamífero/citologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Morfogênese , Placa Neural/citologia , Receptores Acoplados a Proteínas G/metabolismo , Actomiosina/metabolismo , Animais , Células Cultivadas , Constrição , Embrião não Mamífero/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas dos Microfilamentos/genética , Placa Neural/metabolismo , Neurulação , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia
13.
Nat Commun ; 8: 15163, 2017 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-28516903

RESUMO

Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif. We combine NMR spectroscopy, computational modelling and biochemistry to map changes in Gα caused by binding of GBA proteins with residue-level resolution. We find that the GBA motif binds to the SwitchII/α3 cleft of Gα and induces changes in the G-1/P-loop and G-2 boxes (involved in phosphate binding), but not in the G-4/G-5 boxes (guanine binding). Our findings reveal that G-protein-binding and activation mechanisms are fundamentally different between GBA proteins and GPCRs, and that GEF-mediated perturbation of nucleotide phosphate binding is sufficient for Gα activation.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos/fisiologia , Linhagem Celular , Ativação Enzimática/fisiologia , Células HEK293 , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia
14.
Sci Signal ; 9(423): ra37, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-27072656

RESUMO

Auriculo-condylar syndrome (ACS), a rare condition that impairs craniofacial development, is caused by mutations in a G protein-coupled receptor (GPCR) signaling pathway. In mice, disruption of signaling by the endothelin type A receptor (ET(A)R), which is mediated by the G protein (heterotrimeric guanine nucleotide-binding protein) subunit Gα(q/11) and subsequently phospholipase C (PLC), impairs neural crest cell differentiation that is required for normal craniofacial development. Some ACS patients have mutations inGNAI3, which encodes Gα(i3), but it is unknown whether this G protein has a role within the ET(A)R pathway. We used a Xenopus model of vertebrate development, in vitro biochemistry, and biosensors of G protein activity in mammalian cells to systematically characterize the phenotype and function of all known ACS-associated Gα(i3) mutants. We found that ACS-associated mutations in GNAI3 produce dominant-negative Gα(i3) mutant proteins that couple to ET(A)R but cannot bind and hydrolyze guanosine triphosphate, resulting in the prevention of endothelin-mediated activation of Gα(q/11) and PLC. Thus, ACS is caused by functionally dominant-negative mutations in a heterotrimeric G protein subunit.


Assuntos
Otopatias/genética , Orelha/anormalidades , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Mutação , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Otopatias/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Immunoblotting , Microscopia de Fluorescência , Modelos Genéticos , Ligação Proteica , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevis
15.
J Cell Biol ; 210(7): 1165-84, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26391662

RESUMO

Signal transduction via integrins and G protein-coupled receptors is critical to control cell behavior. These two receptor classes have been traditionally believed to trigger distinct and independent signaling cascades in response to extracellular cues. Here, we report a novel mechanism of integrin signaling that requires activation of the trimeric G protein Gαi by the nonreceptor guanine nucleotide exchange factor (GEF) GIV (also known as Girdin), a metastasis-associated protein. We demonstrate that GIV enhances integrin-dependent cell responses upon extracellular matrix stimulation and makes tumor cells more invasive. These responses include remodeling of the actin cytoskeleton and PI3K-dependent signaling, resulting in enhanced haptotaxis and invasion. We show that both GIV and its substrate Gαi3 are recruited to active integrin complexes and that tumor cells engineered to express GEF-deficient GIV fail to transduce integrin signals into proinvasive responses via a Gßγ-PI3K axis. Our discoveries delineate a novel mechanism by which integrin signaling is rewired during metastasis to result in increased tumor invasiveness.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Integrinas/genética , Células MCF-7 , Camundongos , Proteínas dos Microfilamentos/genética , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Transporte Vesicular/genética
16.
PLoS One ; 10(9): e0139179, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26407179

RESUMO

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-ß and promotes TGF-ß-dependent signaling through interaction with the type II receptor of TGF-ß. Here we explore the implication of ADAM12 in TGF-ß-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-ß-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-ß receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-ß-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.


Assuntos
Proteínas ADAM/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteína ADAM12 , Adulto , Idoso , Idoso de 80 Anos ou mais , Biocatálise/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mesoderma/metabolismo , Pessoa de Meia-Idade , Fenótipo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
17.
Mol Biol Cell ; 23(17): 3461-72, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22767580

RESUMO

Based on its shedding and binding activities, the disintegrin and metalloprotease 12 (ADAM12) has been implicated in cell signaling. Here we investigate the intracellular protein interaction network of the transmembrane ADAM12L variant using an integrative approach. We identify the integrin-linked kinase (ILK) as a new partner for ADAM12L cellular functions. We demonstrate that ADAM12L coimmunoprecipitates with ILK in cells and that its cytoplasmic tail is required for this interaction. In human cultured hepatic stellate cells (HSCs), which express high levels of endogenous ADAM12L and ILK, the two proteins are redistributed to focal adhesions upon stimulation of a ß1 integrin-dependent pathway. We show that down-regulation of ADAM12L in HSCs leads to cytoskeletal disorganization and loss of adhesion. Conversely, up-regulation of ADAM12L induces the Akt Ser-473 phosphorylation-dependent survival pathway via stimulation of ß1 integrins and activation of phosphoinositide 3-kinase (PI3K). Depletion of ILK inhibits this effect, which is independent of ADAM12L proteolytic activity and involves its cytoplasmic domain. We further demonstrate that overexpression of ADAM12L promotes kinase activity from ILK immunoprecipitates. Our data suggest a new role for ADAM12L in mediating the functional association of ILK with ß1 integrin to regulate cell adhesion/survival through a PI3K/Akt signaling pathway.


Assuntos
Proteínas ADAM/metabolismo , Adesão Celular , Sobrevivência Celular , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína ADAM12 , Movimento Celular , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Adesões Focais , Células Estreladas do Fígado/metabolismo , Humanos , Fosforilação , Transdução de Sinais , Regulação para Cima
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