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.DOI: http://dx.doi.org/10.7554/eLife.03772.001

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. Members of the Hedgehog (Hh)3 family of secreted signaling proteins are present in most metazoans and owe their name to the effects that loss of Hh function has on Drosophila embryos, which lose their normal segmented pattern and develop a uniform coat of bristles reminiscent of the coats of hedgehogs (1). As presaged by this phenotype, Hh proteins mediate essential tissue patterning events during many stages of animal development (2), and abnormal Hh function is associated with birth defects and cancer (3). Hh proteins are also involved in tissue maintenance and wound repair in adult animals (4). Hh proteins achieve their patterning effects by functioning as classical morphogens (5). That is, Hh proteins form gradients of decreasing concentration from sites of secretion and induce concentration-dependent differentiation of distinct cell types (6, 7). As befits a morphogen, Hh expression, release, diffusion, and signal reception are tightly regulated by multiple factors (8).Classical and modern genetic techniques have identified several cell-surface proteins and glycans involved in receiving or modifying Hh signals (9). The core components of this process, conserved in all organisms known to have active Hh signaling, are Patched (Ptc) and Smoothened (Smo) (Fig. 1) (10 -13). Ptc functions upstream of Smo and has been genetically and biochemically defined as a primary component of the Hh receptor (14,15). Ptc is a 12-pass integral membrane protein with distant homology to bacterial resistance-nodulation-cell division (RND) transporters (16,17). Transmembrane helices 2-6 of Ptc are also homologous to sterol-sensing domains, which are found in diverse integral membrane proteins and regulate activity in response to levels of free cellular sterols (18). Smo is a member of the Frizzled family (class F) of G-protein coupled receptors (GPCRs) (19), and contains an N-terminal, ϳ14-kDa extracellular cysteine-rich domain (CRD) connected via a linker to 7 membrane-spanning helices (7TM) and an extended (ϳ200 amino acids, human; ϳ450 amino acids, Drosophila) C-terminal tail.Hh signaling responses are modulated by additional ce...

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.

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 7 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.