title: Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs.
abstract: 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.
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Cleveland
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: McCabe
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA. Electronic address: dleahy@jhmi.edu.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Leahy
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003902:Detergents / Q000737:chemistry*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D053823:Hedgehog Proteins / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D051379:Mice; D000072081:Patched-1 Receptor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D061987:Sf9 Cells; D012995:Solubility
chemical_list: D003902:Detergents; D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D008562:Membrane Glycoproteins; D000072081:Patched-1 Receptor; D011956:Receptors, Cell Surface; C510880:iHog protein, Drosophila; C070844:ptc protein, Drosophila
keywords: Hedgehog signaling; Patched; Protein purification