RESUMO
Receptor Tyrosine Kinases (RTKs) comprise a diverse group of cell-surface receptors that mediate key signaling events during animal development and are frequently activated in cancer. We show here that deletion of the extracellular regions of 10 RTKs representing 7 RTK classes or their substitution with the dimeric immunoglobulin Fc region results in constitutive receptor phosphorylation but fails to result in phosphorylation of downstream signaling effectors Erk or Akt. Conversely, substitution of RTK extracellular regions with the extracellular region of the Epidermal Growth Factor Receptor (EGFR) results in increases in effector phosphorylation in response to EGF. These results indicate that the activation signal generated by the EGFR extracellular region is capable of activating at least seven different RTK classes. Failure of phosphorylated Fc-RTK chimeras or RTKs with deleted extracellular regions to stimulate phosphorylation of downstream effectors indicates that either dimerization and receptor phosphorylation per se are insufficient to activate signaling or constitutive dimerization leads to pathway inhibition.
Assuntos
Receptores ErbB/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Receptores ErbB/genética , Humanos , Fosforilação/genética , Fosforilação/fisiologia , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
A general aim of studies of signal transduction is to identify mediators of specific signals, order them into pathways, and understand the nature of interactions between individual components and how these interactions alter pathway behavior. Despite years of intensive study and its central importance to animal development and human health, our understanding of the Hedgehog (Hh) signaling pathway remains riddled with gaps, question marks, assumptions, and poorly understood connections. In particular, understanding how interactions between Hh and Patched (Ptc), a 12-pass integral membrane protein, lead to modulation of the function of Smoothened (Smo), a 7-pass integral membrane protein, has defied standard biochemical characterization. Recent structural and biochemical characterizations of Smoothened domains have begun to unlock this riddle, however, and lay the groundwork for improved cancer therapies.
Assuntos
Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Hedgehog/genética , Humanos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/químicaRESUMO
Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.
Assuntos
Detergentes/química , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Hedgehog/química , Glicoproteínas de Membrana/química , Receptor Patched-1/genética , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Animais , Baculoviridae/genética , Proteínas de Drosophila/isolamento & purificação , Camundongos , Receptor Patched-1/química , Receptor Patched-1/isolamento & purificação , Ligação Proteica , Conformação Proteica , Receptores de Superfície Celular/isolamento & purificação , Células Sf9 , SolubilidadeRESUMO
The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation.