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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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Schematic presentation of the feedback mechanism primarily responsible for the regulation of β2 adrenergic signaling in HEK-293 cells. Ligand binding to β2ARs leads to activation of adenylyl cyclase and cAMP production. cAMP activates PKA, which in turn phosphorylates and activates PDE4. GRK-mediated phosphorylation of the receptor, and subsequent arrestin binding, is also sufficient to trigger a decay in the near-membrane cAMP signal.
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fig5: Schematic presentation of the feedback mechanism primarily responsible for the regulation of β2 adrenergic signaling in HEK-293 cells. Ligand binding to β2ARs leads to activation of adenylyl cyclase and cAMP production. cAMP activates PKA, which in turn phosphorylates and activates PDE4. GRK-mediated phosphorylation of the receptor, and subsequent arrestin binding, is also sufficient to trigger a decay in the near-membrane cAMP signal.

Mentions: To gain further insight into the molecular mechanisms underlying the cAMP signals described above, we developed a mathematical model of the system. The model is summarized in the schematic depicted in Fig. 5. Aspects of the model (e.g., PKA-mediated regulation of PDE4 activity) have been published previously (Rich et al., 2007). According to this model, β2ARs couple to Gs which in turn leads to activation of adenylyl cyclase (AC). Conversely, endogenous β2ARs do not couple to Gi in HEK-293 cells, or, if they do, activation of Gi has no significant effect on cAMP signals (Fig. 3). Isoproterenol-induced increases in cAMP concentration subsequently lead to activation of PKA, which exerts its effects on cAMP signals through phosphorylation and stimulation of PDE4 activity. We have excluded other potential mechanisms of PKA-mediated feedback, such as phosphorylation of adenylyl cyclase or β2ARs, because we observed little or no PKA-mediated inhibition of forskolin-induced cAMP synthesis in HEK-293 cells (Rich et al., 2007). This indicates that PKA-dependent inhibition of adenylyl cyclase activity does not play a significant role in shutting down cAMP signals in HEK-293 cells. In addition, we have assumed that at saturating isoproterenol concentrations there is little PKA-mediated desensitization of β2AR because PKA inhibitors (10 μM H89) have no additional effects on isoproterenol-induced cAMP accumulation in the presence of rolipram (Fig. 2 B). This assumption is only valid at high agonist concentration where GRK-mediated desensitization of β2ARs is predominant (Krasel et al., 2004, 2005; Tran et al., 2004; Violin et al., 2006b). For a considered discussion of PKA-mediated desensitization of β2ARs, see Whaley et al. (1994). The equations are detailed in the Materials and methods.


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)

Schematic presentation of the feedback mechanism primarily responsible for the regulation of β2 adrenergic signaling in HEK-293 cells. Ligand binding to β2ARs leads to activation of adenylyl cyclase and cAMP production. cAMP activates PKA, which in turn phosphorylates and activates PDE4. GRK-mediated phosphorylation of the receptor, and subsequent arrestin binding, is also sufficient to trigger a decay in the near-membrane cAMP signal.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Schematic presentation of the feedback mechanism primarily responsible for the regulation of β2 adrenergic signaling in HEK-293 cells. Ligand binding to β2ARs leads to activation of adenylyl cyclase and cAMP production. cAMP activates PKA, which in turn phosphorylates and activates PDE4. GRK-mediated phosphorylation of the receptor, and subsequent arrestin binding, is also sufficient to trigger a decay in the near-membrane cAMP signal.
Mentions: To gain further insight into the molecular mechanisms underlying the cAMP signals described above, we developed a mathematical model of the system. The model is summarized in the schematic depicted in Fig. 5. Aspects of the model (e.g., PKA-mediated regulation of PDE4 activity) have been published previously (Rich et al., 2007). According to this model, β2ARs couple to Gs which in turn leads to activation of adenylyl cyclase (AC). Conversely, endogenous β2ARs do not couple to Gi in HEK-293 cells, or, if they do, activation of Gi has no significant effect on cAMP signals (Fig. 3). Isoproterenol-induced increases in cAMP concentration subsequently lead to activation of PKA, which exerts its effects on cAMP signals through phosphorylation and stimulation of PDE4 activity. We have excluded other potential mechanisms of PKA-mediated feedback, such as phosphorylation of adenylyl cyclase or β2ARs, because we observed little or no PKA-mediated inhibition of forskolin-induced cAMP synthesis in HEK-293 cells (Rich et al., 2007). This indicates that PKA-dependent inhibition of adenylyl cyclase activity does not play a significant role in shutting down cAMP signals in HEK-293 cells. In addition, we have assumed that at saturating isoproterenol concentrations there is little PKA-mediated desensitization of β2AR because PKA inhibitors (10 μM H89) have no additional effects on isoproterenol-induced cAMP accumulation in the presence of rolipram (Fig. 2 B). This assumption is only valid at high agonist concentration where GRK-mediated desensitization of β2ARs is predominant (Krasel et al., 2004, 2005; Tran et al., 2004; Violin et al., 2006b). For a considered discussion of PKA-mediated desensitization of β2ARs, see Whaley et al. (1994). The equations are detailed in the Materials and methods.

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