Figure 1.
Reaction diagram of the GRK-mediated β2AR regulation.
L is ligand; R* is active state of β2AR; Rs and Ri are surface/plasma membrane and internalized β2AR; Rg is GRK-phosphorylated β2AR; Arr is arrestin. This reaction diagram describes the default model for simulations using the rate constants as described in Table 1.
Table 1.
Model parameters.
Figure 2.
Comparisons of four experimental results with simulations of the model.
Panels A–D: Comparisons of simulations (continuous lines) of the model shown in Figure 1, with experimental data obtained in HEK 293 cells stably overexpressing the FLAG WT β2AR (discrete data points). (A) Time course of GRK phosphorylation of the receptor on treatment with different concentrations of epinephrine [19]. (B) Dephosphorylation of the GRK phosphorylated site on the receptor after 5 min treatment with either 1.0 µM or 10 nM ISO (red bar) followed by addition of propranolol (blue bar) and measure of loss of GRK site phosphorylation. Phosphorylated receptor is expressed as a percent of phosphorylation achieved at the end of 5 min treatment with either agonist concentration [16]. (C) Recycling of the receptor after 20 min treatment with 1µM ISO followed by rapid washout of agonist [33]. (D) Internalization of the β2AR on treatment with various concentrations ISO as indicated. Surface receptor is measured by the loss of [3H]CGP-12177.
Figure 3.
Validation of the model with two sets of experimental results.
Validation of the simulations (continuous lines) of the model shown in Figure 1, with experimental data obtained in HEK 293 cells stably overexpressing the FLAG WT β2AR (discrete data points). (A) Desensitization of β2AR stimulation of adenylyl cyclase after treatment with 10 µM ISO. Inset shows desensitization obtained on 30 nM ISO stimulation. Red – simulated results; Black – WT cells; Blue - cells stably overexpressing β2AR lacking PKA phosphorylation sites (PKA-). At lower concentrations the model matches PKA- desensitization more closely since it does not include PKA-mediated β2AR desensitization. (B) Resensitization of the β2AR stimulation of adenylyl cyclase. WT β2AR were stimulated with 1µM ISO for 15 min, followed by addition of metoprolol as described in methods. Dotted line shows simulated % activity of the β2AR when ligand dissociation from arrestin-bound receptor complex is reduced by ten-fold.
Figure 4.
Simulated effects of varying rates of GRK phosphorylation and arrestin binding.
A–C: Simulated effects of ten-fold variation in GRK phosphorylation rates on (A) phosphorylation, (B) internalization and (C) desensitization. D–E: Simulated effects of ten-fold variation in arrestin binding rates on (D) phosphorylation, (E) internalization and (F) desensitization. Experimental data as given in Figure 2A, C, and Figure 3B.
Figure 5.
Simulated effects of phosphatase location and recycling of phosphorylated β2AR on receptor dephosphorylation.
HEK 293 cells stably overexpressing WT β2AR were treated for 5 min with 1 µM ISO (red bar) followed by washout and addition of 1 µM propranolol (blue bar). Experimental data [16] are shown as discrete points with standard errors and the simulations are shown as continuous lines. The black lines are the total phosphorylated receptor, red indicates the phosphorylated receptor on the plasma membrane and blue indicate internalized levels of phosphorylated β2AR. (A) Model A allows for dephosphorylation of both the internalized and plasma membrane bound receptor along with recycling of phosphorylated and dephosphorylated receptor. (B) Model B disallows plasma membrane dephosphorylation but allows both dephosphorylation of the internalized receptor and recycling of phosphorylated receptor. (C) Model C allows for dephosphorylation of both the internalized and plasma membrane bound receptor but limits recycling to only dephosphorylated receptor. (D) Model D allows for dephosphorylation only at the plasma membrane and also allows recycling of phosphorylated receptor. (E) Model E allows for dephosphorylation only at the plasma membrane and disallows recycling of phosphorylated receptor. (F) Model F allows for dephosphorylation only after internalization and prevents recycling of phosphorylated receptor.
Figure 6.
Simulations of the effects of frequency modulation.
In these panels we describe the effect of varying the frequency of stimulation on surface (black), phosphorylated (red) and active (green) receptor species. (A) Rapid stimulation with a train of 1 µM ISO pulses for 0.5 min followed by a 0.5 min immediate washout. Note that this achieves more than 80% desensitization with only 20% internalization. (B) This panels shows the results of a 5 min stimulation with 1.0 µM ISO and a 5 min washout. (C) Simulation of a prolonged 30 min stimulation with 50 nM ISO followed by washout of 30 min. This panel shows that even with low β2AR occupancy (15%) the prolonged stimulation time gives substantial desensitization.
Figure 7.
Basis for “Cellular Memory” in the β2AR signaling machinery.
(A) Simulation of activation of β2AR by paired pulses of 1 µM ISO. Higher desensitization is obtained for the second and third pulse. Colors indicate simulated receptor species as indicated in the figure. (B) Decay in memory of prior stimuli on increase in inter-pulse period from 1–120 min. (C) Effect of up to 50 fold increase in surface dephosphorylation rates on memory of prior stimuli. Default dephosphorylation rate is 0.036/min. (D) Effect of arrestin-β2AR complex stability on desensitization time courses simulated by varying arrestin dissociation rates from the ligand-free complex on the surface. Default arrestin surface dissociation rate from the ligand-free complex is 10/min.