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03 03, 2022;12 (1): 3532.

Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand.

Kerrie A Morrison, Laura Wood, Karen J Edler, James Doutch, Gareth J Price, Francoise Koumanov, Paul Whitley
Author Information
  1. Kerrie A Morrison: Department of Biology and Biochemistry, University of Bath, Bath, UK.
  2. Laura Wood: Department of Biology and Biochemistry, University of Bath, Bath, UK.
  3. Karen J Edler: Department of Chemistry, University of Bath, Bath, UK.
  4. James Doutch: ISIS Pulsed Neutron and Muon Source, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, OX11 0QX, UK.
  5. Gareth J Price: Department of Chemistry, University of Bath, Bath, UK.
  6. Francoise Koumanov: Department for Health, Centre for Nutrition, Exercise and Metabolism, University of Bath, Bath, UK. bssfk@bath.ac.uk.
  7. Paul Whitley: Department of Biology and Biochemistry, University of Bath, Bath, UK. bssprw@bath.ac.uk.
PMID: 35241773 DOI: 10.1038/s41598-022-07606-5

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 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.

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Grants

  1. MR/P002927/1/Medical Research Council

MeSH Term

Detergents
Insulin
Ligands
Maleates
Phosphorylation
Polymers
Polystyrenes
Receptor, Insulin

Chemicals

Detergents
Insulin
Ligands
Maleates
Polymers
Polystyrenes
maleic acid
Receptor, Insulin
Journal Article Research Support, Non-U.S. Gov't
Article has an altmetric score of 2

Word Cloud

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