Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand
Morrison, Kerrie A. ; Wood, Laura ; Edler, Karen J. LU
; Doutch, James
; Price, Gareth J.
; Koumanov, Francoise
and Whitley, Paul
(2022)
In Scientific Reports
12.
- Abstract
Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were... (More)
Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.
(Less)
- author
- Morrison, Kerrie A.
; Wood, Laura
; Edler, Karen J.
LU
; Doutch, James
; Price, Gareth J.
; Koumanov, Francoise
and Whitley, Paul
- publishing date
- 2022-12
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Scientific Reports
- volume
- 12
- article number
- 3532
- pages
- 11 pages
- publisher
- Nature Publishing Group
- external identifiers
-
- pmid:35241773
- scopus:85125689515
- ISSN
- 2045-2322
- DOI
- 10.1038/s41598-022-07606-5
- language
- English
- LU publication?
- no
- additional info
- Publisher Copyright: © 2022, The Author(s).
- id
- 5afbb79b-e7b1-4e57-adfa-0e2a30de2b85
- date added to LUP
- 2022-07-12 14:00:05
- date last changed
- 2024-04-16 08:27:53
@article{5afbb79b-e7b1-4e57-adfa-0e2a30de2b85,
abstract = {{<p>Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.</p>}},
author = {{Morrison, Kerrie A. and Wood, Laura and Edler, Karen J. and Doutch, James and Price, Gareth J. and Koumanov, Francoise and Whitley, Paul}},
issn = {{2045-2322}},
language = {{eng}},
publisher = {{Nature Publishing Group}},
series = {{Scientific Reports}},
title = {{Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand}},
url = {{http://dx.doi.org/10.1038/s41598-022-07606-5}},
doi = {{10.1038/s41598-022-07606-5}},
volume = {{12}},
year = {{2022}},
}