Limits...
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.

Show MeSH

Related in: MedlinePlus

Simulations of the recovery of isoproterenol-induced signals following a 5-min stimulation and a 5-min washout (the two-pulse protocol). The simulations depict the response of the system to an initial 5-min exposure to 1 μM isoproterenol, followed by a 5-min wash, and then a second exposure to 1 μM isoproterenol. Under control conditions (A), and in the presence of 20 nM PKI (C), only small currents are induced by a second exposure to isoproterenol. (B and D) This was primarily due to a reduction in the number of active receptors. (E and F) Inhibition of GRK activity with 3 μM 59-74E allows a second isoproterenol response that is substantially larger (∼50% of the peak response to the initial pulse) due to an increased number of receptors at the start of the second pulse. (G and H) In the presence of both PKI and 59-74E, the cAMP response was further increased, due to the increased number of receptors at the start of the second pulse, and the loss of PKA-mediated regulation of PDE activity. These simulations are consistent with the data presented in Fig. 4.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2279169&req=5

fig7: Simulations of the recovery of isoproterenol-induced signals following a 5-min stimulation and a 5-min washout (the two-pulse protocol). The simulations depict the response of the system to an initial 5-min exposure to 1 μM isoproterenol, followed by a 5-min wash, and then a second exposure to 1 μM isoproterenol. Under control conditions (A), and in the presence of 20 nM PKI (C), only small currents are induced by a second exposure to isoproterenol. (B and D) This was primarily due to a reduction in the number of active receptors. (E and F) Inhibition of GRK activity with 3 μM 59-74E allows a second isoproterenol response that is substantially larger (∼50% of the peak response to the initial pulse) due to an increased number of receptors at the start of the second pulse. (G and H) In the presence of both PKI and 59-74E, the cAMP response was further increased, due to the increased number of receptors at the start of the second pulse, and the loss of PKA-mediated regulation of PDE activity. These simulations are consistent with the data presented in Fig. 4.

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)

Simulations of the recovery of isoproterenol-induced signals following a 5-min stimulation and a 5-min washout (the two-pulse protocol). The simulations depict the response of the system to an initial 5-min exposure to 1 μM isoproterenol, followed by a 5-min wash, and then a second exposure to 1 μM isoproterenol. Under control conditions (A), and in the presence of 20 nM PKI (C), only small currents are induced by a second exposure to isoproterenol. (B and D) This was primarily due to a reduction in the number of active receptors. (E and F) Inhibition of GRK activity with 3 μM 59-74E allows a second isoproterenol response that is substantially larger (∼50% of the peak response to the initial pulse) due to an increased number of receptors at the start of the second pulse. (G and H) In the presence of both PKI and 59-74E, the cAMP response was further increased, due to the increased number of receptors at the start of the second pulse, and the loss of PKA-mediated regulation of PDE activity. These simulations are consistent with the data presented in Fig. 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279169&req=5

fig7: Simulations of the recovery of isoproterenol-induced signals following a 5-min stimulation and a 5-min washout (the two-pulse protocol). The simulations depict the response of the system to an initial 5-min exposure to 1 μM isoproterenol, followed by a 5-min wash, and then a second exposure to 1 μM isoproterenol. Under control conditions (A), and in the presence of 20 nM PKI (C), only small currents are induced by a second exposure to isoproterenol. (B and D) This was primarily due to a reduction in the number of active receptors. (E and F) Inhibition of GRK activity with 3 μM 59-74E allows a second isoproterenol response that is substantially larger (∼50% of the peak response to the initial pulse) due to an increased number of receptors at the start of the second pulse. (G and H) In the presence of both PKI and 59-74E, the cAMP response was further increased, due to the increased number of receptors at the start of the second pulse, and the loss of PKA-mediated regulation of PDE activity. These simulations are consistent with the data presented in Fig. 4.
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