Phosphorylation of the β2-Adrenergic Receptor in Plasma Membranes by Intrinsic GRK5

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Abstract
Characterization of the GRKs participating in the phosphorylation of the β2-adrenergic receptor (β2AR) have in part been limited by the lack of a simple cell-free assay with membrane-bound β2AR and GRKs. We describe here a cell-free assay for GRK phosphorylation of the β2AR in a postnuclear 600g fraction and washed membranes by intrinsic GRK activity using the GRK phosphosite-specific antibody that recognizes pS(355,356). Treatment of these cell-free preparations with 1.0 μM isoproterenol (ISO) caused a rapid maximal 10−15-fold increase in GRK site phosphorylation of the β2AR (t1/2 = 1 min) with an EC50 for ISO stimulation of ∼80 nM. Extensively washed plasma membrane fractions retained the 10−15-fold ISO stimulation of GRK site phosphorylation and GRK5 levels while being depleted of GRK2 and GRK6. Stimulation of GRK site phosphorylation by a range of partial agonists correlated well with their intrinsic efficacy for stimulation of adenylyl cyclase. GRK phosphorylation of the β2AR in the washed membrane fraction caused minimal desensitization of ISO stimulation of adenylyl cyclase activity. Association of GRK5 with the β2AR in intact cells was demonstrated by a high level of basal BRET2 using β2AR-Rluc and GRK5-GFP2 that was not diminished by agonist stimulation. BRET2 between the β2AR-Rluc and GFP2-βarrestin 2 was increased by agonist, whereas BRET2 between the β2AR and GRK2-GFP2 was not significant. On the basis of the level of GRK5-mediated phosphorylation we observe in isolated membrane fractions and co-localization of the β2AR and GRK5, we conclude that GRK5 plays a distinctive role in the phosphorylation of the β2AR.