Nobles, K.N., Xiao, K., Ahn, S., Shukla, A.K., Lam, C.M., Rajagopal, S., Strachan, R.T., Huang, T.Y., Bressler, E.A., Hara, M.R., Shenoy, S.K., Gygi, S.P. and Lefkowitz, R.J.
Notes: The authors created a stably transfected HEK293 cell line expressing a luminogenic cAMP-binding protein using GloSensor™ technology to quantify cAMP levels in live cells. The HEK293 cells express a beta2-adrenergic receptor (β2AR ), a Gs-coupled receptor, that when activated with an agonist, stimulates the production of cAMP. The cell line was used to demonstrate that the phosphorylation pattern (“barcode”) of the β2AR created by various G protein-coupled receptor kinases (GPKs) affects the binding and function of beta-arrestin and subsequent internalization of β2AR. GloSensor™ transfection and transcription were confirmed by stimulation of endogenous β2AR with isoproterenol. Endogenous β2ARs in HEK293 cells were prestimulated with either vehicle DMSO or isoproterenol, then washed and rechallenged with serially diluted isoproterenol. In cells transfected with control siRNA, pretreatment with isoproterenol induced a 50% loss of the maximal cAMP signal when rechallenged. Cells transfected with siRNA targeting GRK2, GRK6, or both GRK2 and GRK6 showed impairment of this desensitization. This GRK knockdown effect decreased the change observed in the maximal cAMP response (Emax) after isoproterenol pretreatment. (4138)