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Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling.

Xin W, Tran TM, Richter W, Clark RB, Rich TC - J. Gen. Physiol. (2008)

Bottom Line: We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels.This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane.Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine and Center for Lung Biology, University of South Alabama, Mobile, AL 36688, USA.

ABSTRACT
An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous beta(2) adrenergic receptors (beta2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of beta2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of beta2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

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Mathematical simulations of the system described by the schematic in Fig. 5. Panels on left depict the signal being measured, panels on right depict the underlying cAMP signals. (A and B) Simulations of normalized current through CNG channels (I/Imax) due to isoproterenol-induced cAMP signals under control conditions. (C and D) Simulations of isoproterenol-induced currents and cAMP signals in the presence of 20 nM PKI (included in the pipette solution). Including PKI in the pipette solution inhibits PKA-mediated stimulation of PDE activity, allowing increased cAMP levels, but the signal is still transient due to basal PDE4 activity and GRK-mediated receptor desensitization. (E and F) Simulations describing the effect of GRK inhibition (3 μM 59-74E) on isoproterenol-induced signals. The signals are still transient due to PKA-mediated stimulation of PDE4 activity. (G and H) Simulations of currents and cAMP signals when both PKA and GRK activity are inhibited (with PKI and 59-74E). Inhibiting receptor desensitization and the stimulation of PDE4 activity prevents the decay of isoproterenol-induced signals.
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fig6: Mathematical simulations of the system described by the schematic in Fig. 5. Panels on left depict the signal being measured, panels on right depict the underlying cAMP signals. (A and B) Simulations of normalized current through CNG channels (I/Imax) due to isoproterenol-induced cAMP signals under control conditions. (C and D) Simulations of isoproterenol-induced currents and cAMP signals in the presence of 20 nM PKI (included in the pipette solution). Including PKI in the pipette solution inhibits PKA-mediated stimulation of PDE activity, allowing increased cAMP levels, but the signal is still transient due to basal PDE4 activity and GRK-mediated receptor desensitization. (E and F) Simulations describing the effect of GRK inhibition (3 μM 59-74E) on isoproterenol-induced signals. The signals are still transient due to PKA-mediated stimulation of PDE4 activity. (G and H) Simulations of currents and cAMP signals when both PKA and GRK activity are inhibited (with PKI and 59-74E). Inhibiting receptor desensitization and the stimulation of PDE4 activity prevents the decay of isoproterenol-induced signals.

Mentions: The model reproduces the experimental data presented (compare Figs. 3 and 6). In the presence of either PKI or 59-74E, the transient nature of the signal is maintained; however, in the presence of both PKI and 59-74E the decline in the signal is blunted. Fig. 7 depicts simulations of the two pulse protocol. Panels A, C, E, and G depict currents through CNG channels. Panels B, D, F, and H depict active receptor levels. In these simulations, active receptors are receptors in the surface membrane that have not been phosphorylated. While it is likely that the binding of arrestin, and not receptor phosphorylation, is responsible for desensitizing the receptor (by blocking receptor–G protein interactions), the binding of arrestin to phosphorylated receptors is fast compared with the rate of GRK-mediated phosphorylation (Krasel et al., 2004, 2005), and therefore is not considered in the model.


Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling.

Xin W, Tran TM, Richter W, Clark RB, Rich TC - J. Gen. Physiol. (2008)

Mathematical simulations of the system described by the schematic in Fig. 5. Panels on left depict the signal being measured, panels on right depict the underlying cAMP signals. (A and B) Simulations of normalized current through CNG channels (I/Imax) due to isoproterenol-induced cAMP signals under control conditions. (C and D) Simulations of isoproterenol-induced currents and cAMP signals in the presence of 20 nM PKI (included in the pipette solution). Including PKI in the pipette solution inhibits PKA-mediated stimulation of PDE activity, allowing increased cAMP levels, but the signal is still transient due to basal PDE4 activity and GRK-mediated receptor desensitization. (E and F) Simulations describing the effect of GRK inhibition (3 μM 59-74E) on isoproterenol-induced signals. The signals are still transient due to PKA-mediated stimulation of PDE4 activity. (G and H) Simulations of currents and cAMP signals when both PKA and GRK activity are inhibited (with PKI and 59-74E). Inhibiting receptor desensitization and the stimulation of PDE4 activity prevents the decay of isoproterenol-induced signals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279169&req=5

fig6: Mathematical simulations of the system described by the schematic in Fig. 5. Panels on left depict the signal being measured, panels on right depict the underlying cAMP signals. (A and B) Simulations of normalized current through CNG channels (I/Imax) due to isoproterenol-induced cAMP signals under control conditions. (C and D) Simulations of isoproterenol-induced currents and cAMP signals in the presence of 20 nM PKI (included in the pipette solution). Including PKI in the pipette solution inhibits PKA-mediated stimulation of PDE activity, allowing increased cAMP levels, but the signal is still transient due to basal PDE4 activity and GRK-mediated receptor desensitization. (E and F) Simulations describing the effect of GRK inhibition (3 μM 59-74E) on isoproterenol-induced signals. The signals are still transient due to PKA-mediated stimulation of PDE4 activity. (G and H) Simulations of currents and cAMP signals when both PKA and GRK activity are inhibited (with PKI and 59-74E). Inhibiting receptor desensitization and the stimulation of PDE4 activity prevents the decay of isoproterenol-induced signals.
Mentions: The model reproduces the experimental data presented (compare Figs. 3 and 6). In the presence of either PKI or 59-74E, the transient nature of the signal is maintained; however, in the presence of both PKI and 59-74E the decline in the signal is blunted. Fig. 7 depicts simulations of the two pulse protocol. Panels A, C, E, and G depict currents through CNG channels. Panels B, D, F, and H depict active receptor levels. In these simulations, active receptors are receptors in the surface membrane that have not been phosphorylated. While it is likely that the binding of arrestin, and not receptor phosphorylation, is responsible for desensitizing the receptor (by blocking receptor–G protein interactions), the binding of arrestin to phosphorylated receptors is fast compared with the rate of GRK-mediated phosphorylation (Krasel et al., 2004, 2005), and therefore is not considered in the model.

Bottom Line: We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels.This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane.Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine and Center for Lung Biology, University of South Alabama, Mobile, AL 36688, USA.

ABSTRACT
An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous beta(2) adrenergic receptors (beta2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of beta2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of beta2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

Show MeSH
Related in: MedlinePlus