Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs

Thomas E. Cleveland, Jacqueline M. McCabe, Daniel J. Leahy

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)92-102
Number of pages11
JournalProtein Expression and Purification
Volume104
DOIs
StatePublished - 2014

Fingerprint

Sf9 Cells
Hedgehogs
Detergents
Hedgehog Proteins
Drosophila
Proteins
Cell Membrane

Keywords

  • Hedgehog signaling
  • Patched
  • Protein purification

ASJC Scopus subject areas

  • Biotechnology

Cite this

Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs. / Cleveland, Thomas E.; McCabe, Jacqueline M.; Leahy, Daniel J.

In: Protein Expression and Purification, Vol. 104, 2014, p. 92-102.

Research output: Contribution to journalArticle

@article{a43cb5e1546041f9b65346c3f62280c9,
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.",
keywords = "Hedgehog signaling, Patched, Protein purification",
author = "Cleveland, {Thomas E.} and McCabe, {Jacqueline M.} and Leahy, {Daniel J.}",
year = "2014",
doi = "10.1016/j.pep.2014.09.012",
language = "English (US)",
volume = "104",
pages = "92--102",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs

AU - Cleveland, Thomas E.

AU - McCabe, Jacqueline M.

AU - Leahy, Daniel J.

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Hedgehog signaling

KW - Patched

KW - Protein purification

UR - http://www.scopus.com/inward/record.url?scp=84908204135&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84908204135&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2014.09.012

DO - 10.1016/j.pep.2014.09.012

M3 - Article

C2 - 25261717

AN - SCOPUS:84908204135

VL - 104

SP - 92

EP - 102

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

ER -