title: Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.
abstract: In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K.
authors.forename: D A
authors.initials: DA
authors.lastname: Schiffmann
authors.forename: J H
authors.initials: JH
authors.lastname: White
authors.forename: A
authors.initials: A
authors.lastname: Cooper
authors.forename: M A
authors.initials: MA
authors.lastname: Nutley
authors.forename: S E
authors.initials: SE
authors.lastname: Harding
authors.forename: K
authors.initials: K
authors.lastname: Jumel
authors.forename: R
authors.initials: R
authors.lastname: Solari
authors.forename: K P
authors.initials: KP
authors.lastname: Ray
authors.forename: N J
authors.initials: NJ
authors.lastname: Gay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002151:Calorimetry; D017931:DNA Primers; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins; D006868:Hydrolysis; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D051193:Toll-Like Receptors; D014461:Ultracentrifugation
chemical_list: D017931:DNA Primers; D029721:Drosophila Proteins; D019476:Insect Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C068721:Tl protein, Drosophila; D051193:Toll-Like Receptors