RESUMO
Caveolae are lipid raft microdomains that regulate endocytosis and signal transduction. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates caveolin 1, supporting a role for these subcellular regions in the compartmentalization of IGF-I signaling. Src homology 2/alpha-collagen related protein (Shc) is the main mediator of IGF-I mitogenic action, coupling IGF-IR phosphorylation to Ras-MAPK activation. Here we show that IGF-I induces Shc tyrosine phosphorylation in the caveolae with a time course significantly different from that observed in the nonraft cellular fractions. In the same time, IGF-I recruits growth factor receptor bound protein 2 (Grb2) to caveolae and activates p42/p44 MAPKs in these microdomains. Src family kinases regulate IGF-I action through an Shc-dependent mechanism. In R-IGF-IRWT cells, IGF-I causes Fyn enrichment in the caveolae with a time course consistent with Shc phosphorylation and Grb2 recruitment in these regions. Finally, we have observed that after IGF-I stimulation, IGF-IR and Fyn colocalize in lipid raft caveolin 1-enriched microdomains. As insulin and IGF-I share common substrates, the effect of insulin on these cellular processes was measured. Here we show that insulin also induces Shc phosphorylation and Grb2 recruitment to caveolae, but with a significantly different time course compared with IGF-I. Our results suggest that 1) IGF-I causes the colocalization of signaling proteins in caveolae through a phosphorylation-regulated mechanism; and 2) the time course of phosphorylation and recruitment of substrates in caveolae by insulin receptor and IGF-IR could determine the specific actions of these receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Cavéolas/metabolismo , Hipoglicemiantes/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Insulina/farmacologia , Proteínas/metabolismo , Domínios de Homologia de src/fisiologia , Animais , Cavéolas/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteína Adaptadora GRB2 , Camundongos , Fosforilação , Agregação de Receptores/efeitos dos fármacos , Agregação de Receptores/fisiologia , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
We report two new mutations in the myophosphorylase gene (PYGM) in two unrelated Italian patients with myophosphorylase deficiency (McArdle's disease). In one, we identified a missense C-to-T mutation at codon 269 in exon 7, changing CGA (arginine) to TGA (stop codon) (R269X). The second patient carried a G-to-C mutation, changing GCT (alanine) to CCT (proline) at codon 686 (A686P) in exon 17. Both were compound heterozygous, with the common mutation at codon 49 (R49X) on the other allele. Our data further expand the genetic heterogeneity in patients with McArdle's disease, suggesting that the possibility of novel mutations has to be taken into account when performing genetic analysis in distinct ethnic groups.
Assuntos
Glicogênio Fosforilase Muscular/genética , Doença de Depósito de Glicogênio Tipo V/genética , Mutação/genética , Códon sem Sentido/genética , Feminino , Heterozigoto , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genéticaRESUMO
We report a 16-month-old boy with psychomotor regression, muscle hypotonia, peripheral neuropathy, and lactic acidosis. Brain magnetic resonance imaging showed a bilateral abnormal signal in the substantia nigra and in the subthalamic nucleus, suggestive of Leigh disease. Histochemical analysis of skeletal muscle showed decreased cytochrome-c oxidase activity. Biochemical analysis of respiratory chain enzymes in muscle homogenate and in cultured fibroblasts showed isolated cytochrome-c oxidase deficiency. Western blot analysis in fibroblasts showed the absence of Surf1 protein. Genetic analysis of the SURF1 gene revealed that the patient was compound heterozygous for a previously reported mutation at the splice-junction site of intron 3 (240 + 1G > T), and for a novel 4-bp deletion in exon 6 (531_534delAAAT). Our data further enlarge the spectrum of mutations in SURF1 gene in patients with Leigh disease and cytochrome-c oxidase deficiency, contributing to better characterization of the clinical and neuroradiologic features of this group of patients for genotype-phenotype correlations.
Assuntos
Deficiência de Citocromo-c Oxidase/complicações , Doença de Leigh/complicações , Doença de Leigh/genética , Proteínas de Membrana/genética , Doenças do Sistema Nervoso Periférico/complicações , Mutação Puntual/genética , Biópsia , Southern Blotting , Encéfalo/patologia , Deficiência de Citocromo-c Oxidase/enzimologia , Análise Mutacional de DNA , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologiaRESUMO
Caveolae are hot spots in IGF-I signalling as suggested by the facts that IGF-I receptors localize in caveolae, directly interact with and tyrosine phosphorylate caveolin 1, the major caveolar protein. Also a number of IGF-IR substrates reside in caveolae, supporting a role of these organelles in the regulation of IGF-I action. Recently, we have demonstrated that IGF-I could specifically regulate Shc phosphorylation in caveolae. Here we show that also IRS1 localizes in this region where it is tyrosine phosphorylated in the presence of IGF-I. Moreover, IRS1 co-immunoprecipitates with caveolin 1 and the specific phosphocaveolin 1-IRS1 interaction is increased by IGF-I.
Assuntos
Cavéolas/metabolismo , Caveolinas/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fosfoproteínas/metabolismo , Animais , Cavéolas/efeitos dos fármacos , Caveolina 1 , Linhagem Celular , Proteínas Substratos do Receptor de Insulina , Fosfoproteínas/química , Fosforilação , Testes de Precipitina , Tirosina/metabolismoRESUMO
Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.