RESUMO
Abstract A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 159(3.53) to glutamic acid and isoleucine160(3.54) to arginine (A159E/I160R) in TM-III of the human ß(1)-adrenergic receptor (ß(1)-AR) to disrupt the function of the hydrophobic cluster. Structurally, the combined mutations of A159E/I160R caused an almost 90° tilt in the rotation of Arg156(3.50) in the E/DRY motif of TM-III and displaced Tyr166(3.60) in intracellular loop 2. The A159E/I160R ß(1)-AR was uncoupled from G(s) as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the ß(1)-AR and G(s)α. Isoproterenol induced ß-arrestin trafficking in cells expressing both the wild-type ß(1)-AR and the A159E/I160R ß(1)-AR. Isoproterenol markedly increased the phosphorylation of ERK1/2 in cells expressing the WT ß(1)-AR and this effect was dependent on the activation of the G(s)-cyclic AMP-dependent protein kinase â Rap â B-raf axis. However, in cells bearing the A159E/I160R ß(1)-AR, isoproterenol failed to increase the phosphorylation of ERK(1/2). These results indicate that mutations in the G(s)α-binding pocket of the GPCR interfered with receptor coupling to G(s) and with its downstream signaling cascades.
Assuntos
Aminoácidos/química , Citoplasma/metabolismo , Proteínas de Ligação ao GTP/química , Receptores Adrenérgicos beta 1/química , Aminoácidos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Isoproterenol/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Fosforilação/efeitos dos fármacos , Conformação Proteica , Receptores Adrenérgicos beta 1/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
PURPOSE: To determine whether ß-adrenergic receptors require insulin receptor substrate (IRS)-1 activity to regulate apoptosis in retinal Müller cells. METHODS: Müller cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium grown in normal (5 mm) or high glucose (25 mM) conditions. The medium was supplemented with 10% fetal bovine serum and antibiotics. Cells were allowed to reach 80%-90% confluence. After becoming appropriately confluent, cells were placed in medium with reduced serum (2%) for 18-24 h to eliminate any effects of fetal bovine serum. Cells were then transfected with 10 ug of IRS-1 small hairpin RNA (shRNA). Forty-eight hours following transfection, cells were lysed and harvested for protein analysis using western blotting. In additional experiments, some cells were treated with 10 uM salmeterol for 24 h following transfection with IRS-1 shRNA. To determine whether IRS-1 directly regulates apoptotic events in the insulin-signaling pathway in retinal Müller cells, a cell death assay kit was used. In tumor necrosis factor (TNF)α inhibitory studies, cells were treated with 5 ng/ml of TNFα alone for 30 min or 30 min pretreatment with TNFα followed by salmeterol for 4 h. RESULTS: Müller cells treated with 5 ng/ml TNFα in 25 mM glucose significantly increased phosphorylation of IRS-1(Ser307). Treatment with the selective beta-2-adrenergic receptor agonist, salmeterol, significantly decreased phosphorylation of IRS-1(Ser307). Following IRS-1 shRNA transfection+salmeterol treatment, Bcl-2-associated X protein (Bax) and cytochrome c levels were significantly decreased. Salmeterol+IRS-1 shRNA also decreased cell death and increased protein levels of B-cell lymphoma-extra large (Bcl-xL), an anti-apoptotic factor. CONCLUSIONS: In these studies, we show for the first time that salmeterol, a beta-2-adrenergic receptor agonist, can reduce retinal Müller cell death through IRS-1 actions. These findings also suggest the importance of IRS-1 in beta-adrenergic receptor signaling in the prevention of cell death in retinal Müller cells.
Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Neuroglia/metabolismo , Retina/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Albuterol/análogos & derivados , Albuterol/farmacologia , Animais , Western Blotting , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Citocromos c/antagonistas & inibidores , Citocromos c/genética , Citocromos c/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Glucose/farmacologia , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno/genética , Ratos , Receptores Adrenérgicos beta 2/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Xinafoato de Salmeterol , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/agonistas , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
PURPOSE: The goal of this study was to determine the relationship of TNF-α and the downregulation of insulin receptor signaling in retinal Müller cells cultured under hyperglycemic conditions and the role of ß-adrenergic receptors in regulating these responses. METHODS: Retinal Müller cells were cultured in normal (5 mM) or high (25 mM) glucose until 80% confluent and then were reduced to 2% serum for 18 to 24 hours. The cells were then treated with 10 µM salmeterol followed by Western blot analysis or ELISA. For TNF-α inhibitory studies, the cells were treated with 5 ng/mL of TNF-α for 30 minutes or by a 30-minute pretreatment with TNF-α followed by salmeterol for 6 hours. In the TNF-α short hairpin (sh)RNA experiments, the cells were cultured until 90% confluent, followed by transfection with TNF-α shRNA for 18 hours. RESULTS: TNF-α-only treatments of Müller cells resulted in significant decreases of tyrosine phosphorylation of the insulin receptor and Akt in high-glucose conditions. Salmeterol (10 µM), a ß-2-adrenergic receptor agonist, significantly increased phosphorylation of both insulin receptor and Akt. TNF-α shRNA significantly decreased phosphorylation of IRS-1(Ser307), which was further decreased after salmeterol+TNF-α shRNA. Both TNF-α shRNA and salmeterol significantly reduced death of the retinal Müller cells. CONCLUSIONS: These studies demonstrate that ß-adrenergic receptor agonists in vitro can restore the loss of insulin receptor activity noted in diabetes. By decreasing the levels of TNF-α and decreasing the phosphorylation of IRS-1(Ser307) while increasing tyrosine phosphorylation of insulin receptor, these results suggest a possible mechanism by which restoration of ß-adrenergic receptor signaling may protect the retina against diabetes-induced damage.