Abstract
We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.
Highlights
The b2 adrenergic receptor (b2AR) is intimately involved in the control of smooth muscle relaxation in airways and the vasculature, in stimulation of the heart, and numerous other physiologically important actions
At high concentrations of agonist we have shown that the G protein coupled receptor kinase (GRK) pathway accounts for almost all of the desensitization of the b2AR [8]
As expected from the contribution of the protein kinase A (PKA) desensitization, we found that our GRK-mediated b2AR regulation model underestimated the experimentally determined desensitization over the first two minutes of agonist treatment for the WTb2AR, but there was good agreement with longer treatment times
Summary
The b2 adrenergic receptor (b2AR) is intimately involved in the control of smooth muscle relaxation in airways and the vasculature, in stimulation of the heart, and numerous other physiologically important actions. Agonist stimulation of the b2AR causes activation of the Gs/cAMP/ protein kinase A (PKA) pathway. Agonist triggers b2AR desensitization that involves two pathways, G protein-dependent highly amplified PKA phosphorylation, and G protein-independent G protein coupled receptor kinase (GRK) phosphorylation that in turn triggers arrestin binding, internalization, recycling and resensitization [1,2,3,4]. Our study of the b2AR desensitization was based on characterization of membrane localized cAMP in single cells with genetically encoded cyclic nucleotide-gated (CNG) channels in the presence of GRK and/or
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