Expression, purification, and analysis of G-protein-coupled receptor kinases.
G-protein-coupled receptor (GPCR) kinases (GRKs) were first identified based on their ability to specifically phosphorylate activated GPCRs. Although many soluble substrates have since been identified, the chief physiological role of GRKs still remains the uncoupling of GPCRs from heterotrimeric G-proteins by promoting β-arrestin binding through the phosphorylation of the receptor. It is expected that GRKs recognize activated GPCRs through a docking site that not only recognizes the active conformation of the transmembrane domain of the receptor but also stabilizes a more catalytically competent state of the kinase domain. Many of the recent gains in understanding GRK-receptor interactions have been gleaned through biochemical and structural analysis of recombinantly expressed GRKs. Described herein are current techniques and procedures being used to express, purify, and assay GRKs in both in vitro and living cells.
Biology Department, Siena College, Morrell Science Center, Loudonville, New York, USA.
Rachel
R
Sterne-Marr
Alison I
AI
Baillargeon
Kevin R
KR
Michalski
John J G
JJ
Tesmer
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002847:Chromatography, Agarose / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization / Q000379:methods; D054768:G-Protein-Coupled Receptor Kinases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection / Q000379:methods
D011994:Recombinant Proteins; D054768:G-Protein-Coupled Receptor Kinases