Characterization of a hyperphosphorylated variant of G protein-coupled receptor kinase 5 expressed in E. coli

Research output: Contribution to journalArticle

Authors

  • Tyler S. Beyett
  • Qiuyan Chen
  • Emily J. Labudde
  • Joseph Krampen
  • Prateek V. Sharma
  • John J.G. Tesmer

External Institution(s)

  • University of Michigan, Ann Arbor
  • Purdue University

Details

Original languageEnglish (US)
Article number105547
JournalProtein Expression and Purification
Volume168
StatusPublished - Apr 2020
Peer-reviewedYes

Abstract

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK subfamilies that does not need post-translational lipidation or other binding partners to exhibit full activity against GPCRs, rendering it a useful tool for biophysical studies directed at characterizing GRK function. However, recombinant expression of GRK5 has thus far been limited to insect and mammalian systems. Here, we describe the expression of functional GRK5 in E. coli and its purification and biochemical characterization. Bacterially expressed GRK5 is hyperphosphorylated, primarily in regions known to be flexible from prior crystal structures, which slightly decreases its catalytic activity toward receptor substrates. Mutation of a single phosphorylation site, Thr10, restores kinetic parameters to those of GRK5 purified from insect cells. Consequently, bacterial expression will allow for production of GRK5 at a reduced cost and faster pace and would facilitate production of isotopically labeled kinase for NMR studies or for the incorporation of unnatural amino acids.

    Research areas

  • Autophosphorylation, Bacterial expression, G protein-coupled receptor kinase 5, Mass spectrometry, Phosphorylation

Citation formats

APA

Beyett, T. S., Chen, Q., Labudde, E. J., Krampen, J., Sharma, P. V., & Tesmer, J. J. G. (2020). Characterization of a hyperphosphorylated variant of G protein-coupled receptor kinase 5 expressed in E. coli. Protein Expression and Purification, 168, [105547]. https://doi.org/10.1016/j.pep.2019.105547

Harvard

Beyett, TS, Chen, Q, Labudde, EJ, Krampen, J, Sharma, PV & Tesmer, JJG 2020, 'Characterization of a hyperphosphorylated variant of G protein-coupled receptor kinase 5 expressed in E. coli', Protein Expression and Purification, vol. 168, 105547. https://doi.org/10.1016/j.pep.2019.105547