Chapter 13
Identifying the Heterotrimeric Complex Stoichiometry
of AMPK in Skeletal Muscle by Immunoprecipitation
Jesper B. Birk and Jørgen F. P. Wojtaszewski
Abstract
The 5^0 -AMP-activated protein kinase is a complicated enzyme consisting of three different subunits, each of
which is expressed as two or three isoforms. This gives the possibility of 12 different heterotrimeric
complexes, which could have diverse functions within the cell. To map out which of these complexes are
present and to what extent in skeletal muscle, we have used the immunoprecipitation technique and
analyzed both the precipitates and the remaining supernatants for coprecipitation of complex partners.
We have fine-tuned this method to give the best results on lysates from the skeletal muscle, liver, and heart
muscle from mouse to man.
Key wordsAMPK, Homogenization, Protein interaction, Immunoprecipitation, Western blot, In
vitro setting, Antibody specificity1 Introduction
The 5^0 -AMP-activated protein kinase (AMPK) is a trimeric protein
complex consisting of a catalyticαsubunit and two regulatoryβand
γsubunits. Not including splice variants, each of these subunits is
expressed as two or three isoforms (α1,α2,β1,β2,γ1,γ2, andγ3)
giving the possibility of 12 different heterotrimeric complexes. It is
well established that the expression profile of each of these isoforms
is more or less tissue specific; α1, γ1, and γ2 being broadly
expressed in most tissues throughout the body. The α2 and
β2 being more restricted to the skeletal muscle, heart muscle, and
liver, while theγ3 subunit isoform is highly restricted to skeletal
muscle and especially fast-twitch/white fiber types [1–4]. Although
this tissue specificity narrows down the number of possible hetero-
trimeric complexes in a certain tissue, all seven subunit isoforms
have been identified in skeletal muscle leaving the stoichiometry
and regulation of AMPK in this tissue highly complex. Adding to
this complexity is the heterogeneity of the skeletal muscle tissue
containing not only myofibers but also endothelial and adiposeDietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_13,©Springer Science+Business Media, LLC 2018
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