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

• Published in: Protein expression and purification Vol. 168; p. 105547
• Main Authors: Beyett, Tyler S., Chen, Qiuyan, Labudde, Emily J., Krampen, Joseph, Sharma, Prateek V., Tesmer, John J.G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2020
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Autophosphorylation; Bacterial expression; Binding Sites; Cloning, Molecular; Escherichia coli - genetics; Escherichia coli - metabolism; G protein-coupled receptor kinase 5; G-Protein-Coupled Receptor Kinase 5 - chemistry; G-Protein-Coupled Receptor Kinase 5 - genetics; G-Protein-Coupled Receptor Kinase 5 - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Humans; Kinetics; Mass spectrometry; Models, Molecular; Mutation; Phosphorylation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Processing, Post-Translational; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; G protein-coupled receptor kinase 5; Autophosphorylation; Phosphorylation; Mass spectrometry; Bacterial expression
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2019.105547

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. •Expression system for G protein-coupled receptor (GPCR) kinase 5 (GRK5) in E. coli.•E. coli expressed GRK5 is hyperphosphorylated, especially at C-terminus.•Hyperphosphorylation seems required for efficient expression in E. coli•Some autophosphorylation sites inhibit activity towards GPCR substrates.•Represents convenient system for producing pure GRK5 for biophysical studies.