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Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4.

Richter W, Day P, Agrawal R, Bruss MD, Granier S, Wang YL, Rasmussen SG, Horner K, Wang P, Lei T, Patterson AJ, Kobilka B, Conti M - EMBO J. (2008)

Bottom Line: The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex.Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor.We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.

ABSTRACT
Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.

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Selective activation of PDE4D splicing variants after stimulation of β1AR and β2AR. (A, B) Neonatal cardiac myocytes derived from mice deficient in β2AR were stimulated for 3 min with 100 nM ISO (A) and cells deficient in β1AR were treated for 3 min with 10 μM ISO (B). At the end of incubation, cells were lysed, PDE4D5, 8, and 9 were immunoprecipitated with variant-specific antibodies, and the PDE activity recovered in the IP pellet was measured. Data shown are expressed as the means±s.e.m. of at least three experiments performed. (C) Activation of PDE4D splice variants after treatment of neonatal cardiac myocytes with 100 μM Forskolin for 20 min. Shown is the average of five experiments; three experiments performed using myocytes deficient in β2AR and two experiments using cells deficient in β1AR. NS (P⩾0.05); *(P<0.05); **(P<0.005); ***(P<0.0005).
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f5: Selective activation of PDE4D splicing variants after stimulation of β1AR and β2AR. (A, B) Neonatal cardiac myocytes derived from mice deficient in β2AR were stimulated for 3 min with 100 nM ISO (A) and cells deficient in β1AR were treated for 3 min with 10 μM ISO (B). At the end of incubation, cells were lysed, PDE4D5, 8, and 9 were immunoprecipitated with variant-specific antibodies, and the PDE activity recovered in the IP pellet was measured. Data shown are expressed as the means±s.e.m. of at least three experiments performed. (C) Activation of PDE4D splice variants after treatment of neonatal cardiac myocytes with 100 μM Forskolin for 20 min. Shown is the average of five experiments; three experiments performed using myocytes deficient in β2AR and two experiments using cells deficient in β1AR. NS (P⩾0.05); *(P<0.05); **(P<0.005); ***(P<0.0005).

Mentions: All PDE4 long forms are activated by phosphorylation at a conserved PKA consensus site in UCR1 (see Figure 2D); this mechanism provides a ubiquitous negative-feedback loop critical for cAMP signaling (Conti et al, 2003). Accordingly, stimulation of cultured neonatal cardiac myocytes with β-adrenergic agonists leads to a rapid PKA-mediated activation of PDE4D (Supplementary Figure 2A–C). If complexes composed of βARs and PDEs are present in these cells, phosphorylation should be biased toward the PDEs present in the vicinity of the occupied receptors. This is indeed the case when β1AR- and β2AR-stimulated phosphorylation of PDE4D isoforms was monitored. In cardiomyocytes lacking β2AR, PDE4D8 was the PDE4D isoform predominantly activated after stimulation of β1AR with ISO, with a limited activation of PDE4D9, and no significant effect on PDE4D5 (Figure 5A). Conversely, in myocytes lacking β1AR, stimulation of β2AR causes a selective increase in the activity of PDE4D5, with a less pronounced increase in PDE4D9, and no increase in PDE4D8 activity (Figure 5B). Importantly, upon stimulation with the adenylyl cyclase activator, Forskolin, all PDE4D isoforms show the same increase in activity in both cell types (Figure 5C), suggesting that loss of one βAR subtype or the other has not perturbed overall cAMP signaling. It also demonstrates that the spatial dimension of cAMP signaling is lost when generalized adenylyl cyclase activation is induced with Forskolin. The selective activation of PDE4D splicing variants by β1AR and β2AR signaling confirms the selectivity observed in the physical association of β1AR with PDE4D8 (Figure 3A and B) and the preferential sequestration of PDE4D5 to the β2AR by β-arrestin (Baillie et al, 2003). Because these experiments are with endogenous proteins, they strengthen our hypothesis of the presence of PDE4D variants in complex with β1AR and β2AR in vivo.


Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4.

Richter W, Day P, Agrawal R, Bruss MD, Granier S, Wang YL, Rasmussen SG, Horner K, Wang P, Lei T, Patterson AJ, Kobilka B, Conti M - EMBO J. (2008)

Selective activation of PDE4D splicing variants after stimulation of β1AR and β2AR. (A, B) Neonatal cardiac myocytes derived from mice deficient in β2AR were stimulated for 3 min with 100 nM ISO (A) and cells deficient in β1AR were treated for 3 min with 10 μM ISO (B). At the end of incubation, cells were lysed, PDE4D5, 8, and 9 were immunoprecipitated with variant-specific antibodies, and the PDE activity recovered in the IP pellet was measured. Data shown are expressed as the means±s.e.m. of at least three experiments performed. (C) Activation of PDE4D splice variants after treatment of neonatal cardiac myocytes with 100 μM Forskolin for 20 min. Shown is the average of five experiments; three experiments performed using myocytes deficient in β2AR and two experiments using cells deficient in β1AR. NS (P⩾0.05); *(P<0.05); **(P<0.005); ***(P<0.0005).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196435&req=5

f5: Selective activation of PDE4D splicing variants after stimulation of β1AR and β2AR. (A, B) Neonatal cardiac myocytes derived from mice deficient in β2AR were stimulated for 3 min with 100 nM ISO (A) and cells deficient in β1AR were treated for 3 min with 10 μM ISO (B). At the end of incubation, cells were lysed, PDE4D5, 8, and 9 were immunoprecipitated with variant-specific antibodies, and the PDE activity recovered in the IP pellet was measured. Data shown are expressed as the means±s.e.m. of at least three experiments performed. (C) Activation of PDE4D splice variants after treatment of neonatal cardiac myocytes with 100 μM Forskolin for 20 min. Shown is the average of five experiments; three experiments performed using myocytes deficient in β2AR and two experiments using cells deficient in β1AR. NS (P⩾0.05); *(P<0.05); **(P<0.005); ***(P<0.0005).
Mentions: All PDE4 long forms are activated by phosphorylation at a conserved PKA consensus site in UCR1 (see Figure 2D); this mechanism provides a ubiquitous negative-feedback loop critical for cAMP signaling (Conti et al, 2003). Accordingly, stimulation of cultured neonatal cardiac myocytes with β-adrenergic agonists leads to a rapid PKA-mediated activation of PDE4D (Supplementary Figure 2A–C). If complexes composed of βARs and PDEs are present in these cells, phosphorylation should be biased toward the PDEs present in the vicinity of the occupied receptors. This is indeed the case when β1AR- and β2AR-stimulated phosphorylation of PDE4D isoforms was monitored. In cardiomyocytes lacking β2AR, PDE4D8 was the PDE4D isoform predominantly activated after stimulation of β1AR with ISO, with a limited activation of PDE4D9, and no significant effect on PDE4D5 (Figure 5A). Conversely, in myocytes lacking β1AR, stimulation of β2AR causes a selective increase in the activity of PDE4D5, with a less pronounced increase in PDE4D9, and no increase in PDE4D8 activity (Figure 5B). Importantly, upon stimulation with the adenylyl cyclase activator, Forskolin, all PDE4D isoforms show the same increase in activity in both cell types (Figure 5C), suggesting that loss of one βAR subtype or the other has not perturbed overall cAMP signaling. It also demonstrates that the spatial dimension of cAMP signaling is lost when generalized adenylyl cyclase activation is induced with Forskolin. The selective activation of PDE4D splicing variants by β1AR and β2AR signaling confirms the selectivity observed in the physical association of β1AR with PDE4D8 (Figure 3A and B) and the preferential sequestration of PDE4D5 to the β2AR by β-arrestin (Baillie et al, 2003). Because these experiments are with endogenous proteins, they strengthen our hypothesis of the presence of PDE4D variants in complex with β1AR and β2AR in vivo.

Bottom Line: The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex.Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor.We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.

ABSTRACT
Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.

Show MeSH
Related in: MedlinePlus