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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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Measurement of isoproterenol-induced cAMP signals near the plasma membrane of HEK-293 cells. HEK-293 cells were infected with CNG-channels that transduce cAMP signals to electrical current. Responses were elicited by exposure of each cell to the indicated concentrations of isoproterenol. Shown are cAMP signals in response to 20 nM (A), 50 nM (B), and 1 μM (C) isoproterenol. All traces are representative of the range of responses from at least five experiments.
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fig1: Measurement of isoproterenol-induced cAMP signals near the plasma membrane of HEK-293 cells. HEK-293 cells were infected with CNG-channels that transduce cAMP signals to electrical current. Responses were elicited by exposure of each cell to the indicated concentrations of isoproterenol. Shown are cAMP signals in response to 20 nM (A), 50 nM (B), and 1 μM (C) isoproterenol. All traces are representative of the range of responses from at least five experiments.

Mentions: We and others recently reported that PKA-mediated activation of PDE4 is an important mechanism for shaping GPCR-mediated signals (Terrin et al., 2006; Willoughby et al., 2006; Rich et al., 2007). To better understand the interplay between PDE activation and GPCR desensitization in regulating cAMP signals we examined signaling via the endogenous β2ARs in HEK-293 cells. Cyclic AMP signals were monitored in single cells using CNG channels as real-time biosensors. CNG channels with the C460W and E583M mutations (C460W/E583M channels) were expressed in HEK-293 cells via adenovirus infection. These mutations increase both the specificity and the sensitivity of the channels to cAMP (Rich et al., 2001b). CNG channel activity was measured using the whole cell voltage clamp technique, as described in the Materials and methods. The activation of C460W/E583M channels in response to 20, 50, and 1000 nM isoproterenol is shown in Fig. 1. CNG channel activation in response to 20 nM isoproterenol reached a steady plateau over a 7-min stimulation, indicating a sustained increase in cAMP levels. Conversely, channel activation in response to 50 and 1000 nM isoproterenol was transient, indicating an initial increase and subsequent decrease in cAMP levels near the plasma membrane. We next sought to determine the cellular mechanisms responsible for the transient cAMP signals observed in response to high concentrations of isoproterenol.


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)

Measurement of isoproterenol-induced cAMP signals near the plasma membrane of HEK-293 cells. HEK-293 cells were infected with CNG-channels that transduce cAMP signals to electrical current. Responses were elicited by exposure of each cell to the indicated concentrations of isoproterenol. Shown are cAMP signals in response to 20 nM (A), 50 nM (B), and 1 μM (C) isoproterenol. All traces are representative of the range of responses from at least five experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Measurement of isoproterenol-induced cAMP signals near the plasma membrane of HEK-293 cells. HEK-293 cells were infected with CNG-channels that transduce cAMP signals to electrical current. Responses were elicited by exposure of each cell to the indicated concentrations of isoproterenol. Shown are cAMP signals in response to 20 nM (A), 50 nM (B), and 1 μM (C) isoproterenol. All traces are representative of the range of responses from at least five experiments.
Mentions: We and others recently reported that PKA-mediated activation of PDE4 is an important mechanism for shaping GPCR-mediated signals (Terrin et al., 2006; Willoughby et al., 2006; Rich et al., 2007). To better understand the interplay between PDE activation and GPCR desensitization in regulating cAMP signals we examined signaling via the endogenous β2ARs in HEK-293 cells. Cyclic AMP signals were monitored in single cells using CNG channels as real-time biosensors. CNG channels with the C460W and E583M mutations (C460W/E583M channels) were expressed in HEK-293 cells via adenovirus infection. These mutations increase both the specificity and the sensitivity of the channels to cAMP (Rich et al., 2001b). CNG channel activity was measured using the whole cell voltage clamp technique, as described in the Materials and methods. The activation of C460W/E583M channels in response to 20, 50, and 1000 nM isoproterenol is shown in Fig. 1. CNG channel activation in response to 20 nM isoproterenol reached a steady plateau over a 7-min stimulation, indicating a sustained increase in cAMP levels. Conversely, channel activation in response to 50 and 1000 nM isoproterenol was transient, indicating an initial increase and subsequent decrease in cAMP levels near the plasma membrane. We next sought to determine the cellular mechanisms responsible for the transient cAMP signals observed in response to high concentrations of isoproterenol.

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