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1.
J Cell Physiol ; 236(12): 8160-8170, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34170016

RESUMEN

Epidermal growth factor (EGF) receptors (ErbB1-ErbB4) promote cardiac development and growth, although the specific EGF ligands and receptor isoforms involved in growth/repair versus pathology remain undefined. We challenged ventricular cardiomyocytes with EGF-like ligands and observed that selective activation of ErbB4 (the receptor for neuregulin 1 [NRG1]), but not ErbB1 (the receptor for EGF, EGFR), stimulated hypertrophy. This lack of direct ErbB1-mediated hypertrophy occurred despite robust activation of extracellular-regulated kinase 1/2 (ERK) and protein kinase B. Hypertrophic responses to NRG1 were unaffected by the tyrosine kinase inhibitor (AG1478) at concentrations that are selective for ErbB1 over ErbB4. NRG1-induced cardiomyocyte enlargement was suppressed by small interfering RNA (siRNA) knockdown of ErbB4 and ErbB2, whereas ERK phosphorylation was only suppressed by ErbB4 siRNA. Four ErbB4 isoforms exist (JM-a/JM-b and CYT-1/CYT-2), generated by alternative splicing, and their expression declines postnatally and following cardiac hypertrophy. Silencing of all four isoforms in cardiomyocytes, using an ErbB4 siRNA, abrogated NRG1-induced hypertrophic promoter/reporter activity, which was rescued by coexpression of knockdown-resistant versions of the ErbB4 isoforms. Thus, ErbB4 confers cardiomyocyte hypertrophy to NRG1, and all four ErbB4 isoforms possess the capacity to mediate this effect.


Asunto(s)
Hipertrofia/metabolismo , Miocitos Cardíacos/metabolismo , Isoformas de Proteínas/metabolismo , Receptor ErbB-4/metabolismo , Empalme Alternativo/genética , Animales , Proliferación Celular/fisiología , Humanos , Fosforilación/fisiología , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Receptor ErbB-4/genética , Transducción de Señal/fisiología
2.
J Cell Sci ; 126(Pt 23): 5377-90, 2013 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-24046455

RESUMEN

The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer.


Asunto(s)
Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Glándulas Mamarias Humanas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Activación Transcripcional , Angiotensina II/metabolismo , Angiotensina II/farmacología , Línea Celular Transformada , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/genética , Colina Quinasa/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Receptores ErbB/genética , Femenino , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor de Angiotensina Tipo 1/genética , Transducción de Señal , Trombina/metabolismo , Trombina/farmacología
3.
Biochem Pharmacol ; 158: 232-242, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30347205

RESUMEN

The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of ß-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Activación Transcripcional/fisiología , Animales , Células CHO , Cricetulus , Receptores ErbB/análisis , Receptores ErbB/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/análisis , Células HEK293 , Humanos , Masculino , Ratones , Células 3T3 NIH , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/análisis
5.
J Biol Chem ; 279(8): 6720-9, 2004 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-14623894

RESUMEN

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.


Asunto(s)
Fenilalanina/análogos & derivados , Receptores de Calcitonina/química , Secuencia de Aminoácidos , Animales , Células COS , Calcitonina/química , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Bromuro de Cianógeno/farmacología , AMP Cíclico/química , Cisteína/química , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Endopeptidasas/farmacología , Ligandos , Lisina/química , Metaloendopeptidasas/farmacología , Metionina/química , Datos de Secuencia Molecular , Mutagénesis , Mutación , Oxígeno/metabolismo , Péptidos/química , Fenilalanina/química , Etiquetas de Fotoafinidad/farmacología , Mutación Puntual , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Salmón/metabolismo , Transfección
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