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

doi:10.1016/j.pep.2014.09.012

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

School of Medicine

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

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 language	English (US)
Pages (from-to)	92-102
Number of pages	11
Journal	Protein Expression and Purification
Volume	104
DOIs	https://doi.org/10.1016/j.pep.2014.09.012
State	Published - Dec 2014

Keywords

Hedgehog signaling
Patched
Protein purification

ASJC Scopus subject areas

Biotechnology

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10.1016/j.pep.2014.09.012

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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",

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doi = "10.1016/j.pep.2014.09.012",

language = "English (US)",

volume = "104",

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journal = "Protein Expression and Purification",

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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 - 2014/12

Y1 - 2014/12

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

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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

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SN - 1046-5928

VL - 104

SP - 92

EP - 102

JO - Protein Expression and Purification

JF - Protein Expression and Purification

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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