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Developing Reagents and Methodologies to Measure Post‐Translational Modification of G Protein‐coupled Receptor Kinase 2
Author(s) -
Keyoskey Joshua,
Shareef Afzaal,
Grogan Alyssa,
Tagliafierro Teresa,
SterneMarr Rachel
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.728.30
Subject(s) - posttranslational modification , measure (data warehouse) , chemistry , receptor , kinase , computational biology , microbiology and biotechnology , biochemistry , biology , computer science , data mining , enzyme
G protein‐coupled receptors (GPCRs) are transmembrane proteins that are activated by sensory and hormonal signals and regulate many cellular functions. Upon binding agonist, the GPCR undergoes a conformational change that activates the heterotrimeric G protein. GPCR kinases (GRKs) are recruited to the membrane and initiate the desensitization of GPCRs via phosphorylation of the receptor. GRKs belong to the AGC kinase family and are themselves targets of phosphorylation. GRK2 is phosphorylated by protein kinase A, protein kinase C, ERK and Src in addition to undergoing nitrosylation. Our lab has recently developed an assay that measures the phosphorylation of the β 2 ‐adrenergic receptor by GRK2 in intact cells. The long‐term goal of this work is to determine the kinetics and significance of these reported GRK2 post‐translational events. Thus we have prepared a plasmid construct that allows expression of hexahistidine‐tagged GRK2 (GRK2‐H 6 ) in COS‐7 cells. First we will determine whether this tagged GRK2 is functional in intact cells. We then hope to purify enough GRK2‐H 6 to carry out isoelectric focusing and mass spectrometry studies. This work was supported by a grant from the National Science Foundation to RSM.