Signaling from β1- and β2-adrenergic receptors is defined by differential interactions with PDE4
2008; Springer Nature; Volume: 27; Issue: 2 Linguagem: Inglês
10.1038/sj.emboj.7601968
ISSN1460-2075
AutoresWito Richter, Peter Day, Rani Agrawal, Matthew D. Bruss, Sébastien Granier, Yvonne L. Wang, Søren G. F. Rasmussen, Kathleen Horner, Ping Wang, Lei Tao, Andrew J. Patterson, Brian K. Kobilka, Marco Conti,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoArticle10 January 2008Open Access Signaling from β1- and β2-adrenergic receptors is defined by differential interactions with PDE4 Wito Richter Wito Richter Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Peter Day Peter Day Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Rani Agrawal Rani Agrawal Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Matthew D Bruss Matthew D Bruss Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Sébastien Granier Sébastien Granier Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Yvonne L Wang Yvonne L Wang Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Søren G F Rasmussen Søren G F Rasmussen Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Kathleen Horner Kathleen Horner Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Ping Wang Ping Wang Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Tao Lei Tao Lei Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Andrew J Patterson Andrew J Patterson Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Brian Kobilka Brian Kobilka Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Marco Conti Corresponding Author Marco Conti Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Wito Richter Wito Richter Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Peter Day Peter Day Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Rani Agrawal Rani Agrawal Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Matthew D Bruss Matthew D Bruss Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Sébastien Granier Sébastien Granier Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Yvonne L Wang Yvonne L Wang Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Søren G F Rasmussen Søren G F Rasmussen Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Kathleen Horner Kathleen Horner Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Ping Wang Ping Wang Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Tao Lei Tao Lei Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Andrew J Patterson Andrew J Patterson Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Brian Kobilka Brian Kobilka Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Marco Conti Corresponding Author Marco Conti Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Author Information Wito Richter1,‡, Peter Day2,‡, Rani Agrawal3, Matthew D Bruss1, Sébastien Granier2, Yvonne L Wang1, Søren G F Rasmussen2, Kathleen Horner1, Ping Wang1, Tao Lei1, Andrew J Patterson3, Brian Kobilka2 and Marco Conti 1 1Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA 3Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA ‡These authors contributed equally to this work *Corresponding author. Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, Grant Building, Room S301, Stanford, CA 94305-5317, USA. Tel.: +1 650 725 2452; Fax: +1 650 725 7102; E-mail: [email protected] The EMBO Journal (2008)27:384-393https://doi.org/10.1038/sj.emboj.7601968 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info β1- and β2-adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR 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 β1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β-arrestin/PDE complex reported previously. The β1AR 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 β2AR is a prerequisite for the recruitment of a complex consisting of β-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 β1- and β2-adrenoceptor signaling. Introduction To meet the increased metabolic demands of stress or exercise, the sympathetic nervous system stimulates cardiac function through activation of the closely related β1- and β2-adrenergic receptors (β1AR and β2AR). Even though these highly homologous receptors both activate the G protein stimulatory for adenylyl cyclase (Gs), signaling through β1AR and β2AR produces clearly distinguishable biological effects (Xiang and Kobilka, 2003; Xiao et al, 2004). The β1AR plays the dominant role in stimulating heart rate and strength of myocyte contraction, whereas β2AR produces only modest chronotropic effects. Chronic stimulation of β1AR produces myocyte hypertrophy and apoptosis, whereas β2AR signaling promotes cell survival. The assembly of distinct macromolecular signaling complexes with transducer, scaffold, and effector proteins, which determine signaling properties and subcellular localization of the βARs, is thought to be at the core of the divergent properties of these receptors (Xiang and Kobilka, 2003; Xiao et al, 2004). Thus, understanding the differences of these receptor complexes has important pharmacological and clinical implications. One of the emerging mechanisms that safeguard the specificity of G-protein-coupled receptor/cAMP signaling is the control of cAMP transients via degradation by cyclic nucleotide phosphodiesterases (PDEs) (Conti and Beavo, 2007). Biochemical, electrophysiological, and in vivo imaging studies are consolidating the idea that occupancy of different receptors generates a nonuniform pattern of activation of cAMP effector proteins such as PKA (cAMP-dependent protein kinase). PDEs play a critical role for the specificity in cAMP-signaling by preventing the free diffusion of cAMP, thus, effectively creating cyclic nucleotide microdomains and/or cAMP gradients that can be sensed by the cell (Zaccolo and Pozzan, 2002; Xiang et al, 2005; Fischmeister et al, 2006). PDEs comprise a large group of over 20 genes that are divided into 11 PDE families based on their amino-acid sequence homology, substrate specificities, and pharmacological properties (Conti and Beavo, 2007). Each of the 11 PDE families encompasses one to four distinct genes. In addition, most PDE genes encode for multiple splicing variants through the use of multiple promoters and alternative splicing. Previous studies indicated that occupancy of the β2AR initiates the recruitment of a preformed complex consisting of β-arrestin and the cyclic AMP-specific PDE, PDE4D5 (Perry et al, 2002; Baillie et al, 2003). Conversely, no data are available on complexes between PDEs and the β1AR even though it has been shown that PDE4 inhibitors potentiate cAMP accumulation induced by either β1AR or β2AR (Xiang et al, 2005). Here, we show that β1AR forms a signaling complex with a PDE4D splicing variant in a manner inherently different from the β2AR/β-arrestin/PDE complex reported previously. Thus, this study challenges the assumption that the regulation of receptor signaling by PDEs described for the β2AR also applies to β1AR. We propose that the distinct modes of interaction with PDEs provide an additional layer of complexity to enforce the specificity of β1- and β2-adrenoceptor signaling. Results Detection of a β1AR/PDE4D signaling complex in mouse neonatal cardiac myocytes To probe for a possible signaling complex including the β1AR and a PDE, mouse neonatal cardiomyocytes were infected with an adenovirus encoding a Flag-tagged β1AR, and the receptor was subsequently immunoprecipitated using an antibody against the tag. A significant amount of endogenous PDE activity was recovered in the β1AR immunoprecipitation (IP) pellet (Figure 1A). The PDE activity associated with the β1AR was inhibited by the PDE4-selective inhibitor, Rolipram, identifying this activity as PDE4. Three PDE4 subtypes, PDE4A, PDE4B, and PDE4D, are expressed in neonatal cardiomyocytes at comparable levels (Figure 2B). The fourth gene, PDE4C, is not expressed in the heart and was not investigated. To assess which of the PDE4 subtypes contribute to the activity recovered in the β1AR IP, cardiomyocytes deficient in PDE4A, PDE4B, and PDE4D were subjected to pull-down experiments. Whereas ablation of PDE4A or PDE4B had no effect, inactivation of the PDE4D gene prevented co-IP of PDE activity with the β1AR (Figure 1B). Thus, PDE4D is the endogenous PDE recovered in complex with the receptor. This conclusion is further supported by western blot analysis of the immunoprecipitated PDE. A band immunoreactive with PDE4D-selective antibodies was consistently detected in the IP pellet (Figure 1C), and its mobility is consistent with that of a subset of PDE4D splicing variants that include PDE4D3, PDE4D8, and PDE4D9 (Richter et al, 2005). Together, these data suggest the presence of a signaling complex containing the β1AR, a PDE4D isoform, and perhaps other components of the cAMP signaling pathway in cardiac myocytes. Figure 1.A β1AR/PDE4D signaling complex in mouse neonatal cardiomyocytes. Shown are IPs of a Flag-tagged β1AR from detergent extracts of mouse neonatal cardiomyocytes. (A) Total, non-PDE4, and PDE4 activity in the IP pellet. (B) Co-IP of β1AR and PDE activity from cardiomyocytes deficient in PDE4A, PDE4B, or PDE4D, and wild-type controls. (C) Western blot with the β1AR IP from wild-type myocytes. The migration of the PDE4D-immunoreactive band in the IP pellet corresponds to that of PDE4D splicing variants PDE4D3, PDE4D8, and PDE4D9. Data shown represent the means±s.e.m. (A, B) or are representative (C) of at least three experiments performed. Download figure Download PowerPoint Figure 2.PDE4 subtypes and splice variants expressed in mouse neonatal cardiac myocytes. (A) Total, PDE4, and non-PDE4 activity in detergent extracts of cultured neonatal cardiac myocytes. PDE3 is the major non-PDE4 subtype expressed in these cells contributing 24±5 pmol/min/mg to the total PDE activity. (B, C) Detergent extracts from neonatal cardiac myocytes were immunoprecipitated with PAN-selective antibodies for the PDE4 subtypes, PDE4A, PDE4B, and PDE4D (B), or with splice variant-selective anti-PDE4D antibodies (C). PDEs recovered in the IP pellet were detected by PDE activity assay (B, C) or western blotting (C). All results are expressed as the means±s.e.m. of at least three experiments performed. (D) Schematic representation of the domain organization of PDE4D splice variants. Domains are depicted as barrels connected by wires (putative linker regions). The variants are distinguished into long forms (PDE4D3, 4, 5, 7, 8, and 9) and short forms (PDE4D1, 2, and 6) by the complete or partial presence of the UCR1/2 (upstream conserved regions 1 and 2) module (green barrels), respectively. The PKA phosphorylation site conserved among long splice variants is indicated with red circles. Download figure Download PowerPoint Distinct PDE4D splice variants co-IP with the β1AR Through alternate splicing and the use of multiple promoters, nine different proteins, PDE4D1–9, originate from the PDE4D gene (Richter et al, 2005; Figure 2D). These proteins are identical in the catalytic domain and C-terminus but diverge at the N-terminus. Long forms contain a conserved UCR1/UCR2 (upstream conserved regions 1 and 2) motif, whereas short forms lack UCR1 and part of UCR2 (Conti et al, 2003; Houslay and Adams, 2003). Using antibodies raised against the unique N-terminus of each variant, we determined that PDE4D5, PDE4D8, and PDE4D9 are the splicing variants most abundant in cardiomyocytes, with trace amounts of PDE4D3 (Figure 2C). The co-IP of these PDE4D splice variants expressed exogenously in HEK293 cells identified PDE4D8 as the variant that most efficiently interacts with β1AR. Other long PDE4D splice variants were also recovered in β1AR IP pellets with the following rank order: PDE4D8>PDE4D9>PDE4D3>PDE4D5 (Figure 3A and B). Conversely, the short PDE4D form, PDE4D2, did not co-IP with the β1AR, indicating that the UCR domains unique to long PDE4 splice variants may contribute to the formation of the β1AR/PDE4D complex. Figure 3.Interaction of exogenous β1AR and PDE4D. (A, B) Co-IP of exogenous β1AR and Myc-tagged PDE4D splice variants expressed in HEK293 cells. The efficiency with which β1AR pulls down the different PDE4D splice variants is quantified in (B). (C) Shown is the co-IP of exogenous β1AR and PDE4D8-Myc from extracts of MEFs derived from mice deficient in β-arrestin 1 and 2 (βarr1/2KO) or from wild-type controls (WT-MEF). (D, E) PDE4D3, and Flag-tagged receptors, β1AR and β2AR, were affinity purified after baculovirus expression (see Supplementary Figure 1). Purified PDE and (βARs) were then combined and the βARs immunoprecipitated. Quantification of PDE4D recovered in the βAR IP pellet is shown in (E). All data shown are representative of (A, C, D) or are expressed as the means±s.e.m. (B, E) of at least three experiments performed. *(P<0.05); **(P<0.005); ***(P 90% purity (see Supplementary Figure 1 for the characterization of the purified proteins). In this paradigm, PDE4D shows robust association with β1AR but not with β2AR (Figure 3D and E). This confirms that β-arrestins are not required for the β1AR/PDE4D complex. More importantly, this approach clearly indicates that PDE4D binds directly to the β1AR but has no significant, or a much reduced affinity for β2AR. Binding of β-adrenergic agonists induces dissociation of the β1AR/PDE4D complex To determine whether receptor occupancy affects the β1AR/PDE4D complex, HEK293 cells expressing exogenous β1AR and PDE4D8 were incubated with different ligands. Treatment with the physiological β1AR agonist, (−)-Norepinephrine (NorEpi), caused dissociation of the β1AR/PDE4D complex (Figure 4A and B), whereas the stereoisomer, (+)-norepinephrine, which is a poor β1AR ligand, had no effect. Dissociation of the β1AR/PDE4D complex was observed also in cardiac myocytes and β-arrestin-deficient MEFs (Supplementary Figures 3 and 4) and occurred whether the overexpressed PDE4D was catalytically active or inactive (Supplementary Figure 5). Dissociation of the β1AR/PDE4D complex by NorEpi binding is rapid (T1/2<1 min; Figure 4C and D) and dose-dependent (Figure 4E and F), reaching maximum at approximately 100 μM NorEpi. Thus, the concentration-dependence of dissociation of the β1AR/PDE4D complex is comparable to that of receptor occupancy by NorEpi rather than that of receptor-induced cAMP production, which is in the nanomolar range. In addition, washout of the agonist results in β1AR/PDE4D reassociation (data not shown). This dynamic, receptor occupancy-dependent regulation of β1AR/PDE4D complex formation may explain why β1AR/PDE4D dissociation is not complete and some portion of receptor/PDE complexes (∼30%) remain at any given time point. Treatment with the β-adrenergic agonists, isoproterenol (ISO; 10 μM; see Supplementary Figure 5) or Epinephrine (100 μM; data not shown), also promoted dissociation of the β1AR/PDE4D complex. Figure 4.Binding of β-adrenergic agonists dissociates the β1AR/PDE4D complex. HEK293 cells expressing exogenous β1AR and PDE4D8-Myc were treated with β-adrenergic agonists before cell lysis and IP of the β1AR. (A) Cells were treated for 10 min with 100 μM of the physiological β1AR agonist (−)-Norepinephrine or the stereoisomer (+)-Norepinephrine, which is not an efficient ligand for the β1AR. The amount of PDE4D recovered in the IP pellet is quantified in (B). (C–F) Time course and dose-dependency of the ligand-induced dissociation of the β1AR/PDE4D complex. Cells were treated for various times with 100 μM NorEpi (C, D) or for 15 min with increasing concentrations of NorEpi (E, F) before cell lysis and β1AR IP. The amount of PDE4D recovered in the IP pellet is quantified in (D, F). Data shown are representative of (A, C, E) or represent the means±s.e.m. (B, D, F) of at least three experiments performed. Download figure Download PowerPoint Selective activation of PDE4D splice variants upon stimulation of β1AR and β2AR 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. Figure 5.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). Download figure Download PowerPoint PDE4D controls the activity of PKA in the vicinity of the β1AR The presence of a PDE4D in the vicinity of the β1AR should affect the activity of PKA localized with the receptor as well as the PKA-phosphorylation state of the receptor itself. This possibility was tested by blocking PDE activity with selective PDE4 inhibitors in cardiomyocytes (Figure 6A and B), by using MEFs deficient in PDE4D (Figure 6C and D), or by overexpressing a catalytically inactive PDE4D in cardiomyocytes, which acts as a dominant-negative construct (Perry et al, 2002; Baillie et al, 2003) by displacing endogenous PDE4D from the β1AR complex (Figure 6E and F; Supplementary Figure 5). In all instances, blockage of PDE4 activity or, more specifically, ablation or displacement of PDE4D caused a significant increase in the phosphorylation of the transfected β1AR in the absence of β-adrenergic agonists. It should be noted that inhibition of PDE3 activity or an overexpression of a dominant-negative PDE3A construct has no effect on β1AR phosphorylation, confirming the specificity of the interactions. These findings indicate that PDE4D controls the access of cAMP to PKA localized with the β1AR, effectively creating a domain with low basal cAMP/PKA activity. PDE4D also limits PKA-phosphorylation of β1AR in response to low concentrations of β-adrenergic agonists that do not disrupt a large number of β1AR/PDE4D complexes. This is likely due to the control of cAMP levels and PKA-activity in the vicinity of unoccupied, and thus, PDE4D-associated receptors in response to elevated cellular cAMP levels. These findings suggest a function of PDE4D in complex with the β1AR in the intact cell. Figure 6.PDE4D in the β1AR complex controls local PKA activity. (A, B) Neonatal cardiac myocytes expressing a Flag-tagged β1AR were treated for 3 min with 100 nM Norepinephrine before cell lysis and IP with M1 (α-Flag) resin. The effect of a 5 min pre-treatment with 10 μM of the PDE4-specific inhibitor, Rolipram, or the PDE3-selective inhibitor, Cilostamide, on PKA-phosphorylation of the β1AR is detected in IBs using a PKA-site-specific antibody. (C, D) MEFs derived from mice deficient in PDE4D or wild-type controls were infected with adenovirus to express a Flag-tagged β1AR construct. At 40 h post-infection, cells were treated for 3 min with 100 nM Norepinephrine (NorEpi) before cell lysis and IP with M1 (α-Flag) resin. PKA-phosphorylation of the β1AR is detected in IB using a PKA-site-specific antibody. (E, F) Neonatal cardiac myocytes coexpressing a Flag-tagged β1AR and either GFP, a catalytically inactive PDE4D8 construct (PDE4D-DN; see also Supplementary Figure 5), or a catalytically inactive PDE3A1 (PDE3A1-DN) were subjected to α-Flag(M1)-IP, and the phosphorylation of the β1AR was subsequently detected in IB using a PKA-substrate-specific antibody. Quantification of all results (B, D, F) is expressed as the means±s.e.m. of three experiments performed. NS (P⩾0.05; *(P<0.05); **(P<0.005). Download figure Download PowerPoint PDE4D ablation promotes β1AR desensitization in vivo To assess the role of PDE4D in β1AR function in a more physiological context, changes in the heart rate of mice in response to β-adrenergic stimulation were measured, as it is established that in vivo contraction rate is primarily controlled by β1AR (Rohrer et al, 1996, 1999; Devic et al, 2001). Wild-type and PDE4DKO mice, matched by age, sex, and genetic background, were sedated using isoflurane. While their heart rate was continuously measured using a mouse pulse oximeter sensor, the mice were then injected with a submaximal concentration of ISO. An additional group of mice was first injected with glucagon-like peptide 1 (GLP1) to enhance the heterologous desensitization of β1AR before the ISO injection. Wild-type and PDE4DKO mice showed no significant differences in basal heart rate (WT=410±52 and 4DKO=386±46 beats/min, means±s.e.m.), the maximal heart rate after ISO injection (WT=544±34 and 4DKO=515±32 beats/min), the maximal heart rate after GLP1 injection (WT=487±14 and 4DKO=448±19 beats/min), or the maximal heart rate after sequential injection of GLP1 and ISO (WT=539±24 and 4DKO=581±17 beats/min). The rate of return to basal heart rate after the initial response to ISO was slightly faster in PDE4DKO mice compared with wild-type controls (Figure 7A); however, this effect was greatly magnified by pretreatment of mice with GLP1 (Figure 7B; P<0.0001). The faster decrease in heart rate is in agreement with our stated hypothesis that elevated levels of cAMP/PKA activity in the vicinity of the β1AR, due to absence of PDE4D in this compartment, causes an increased phosphorylation and heterologous desensitization of β1AR (see Figure 6). Figure 7.PDE4D ablation promotes desensitization of β1AR signaling in vivo. Anesthetized mice were sequentially injected with GLP1 followed by a submaximal dose of ISO as described in Materials and methods and the heart rate of the animals was continuously recorded using pulse oximeter sensor (B). Control mice received ISO only (A). The decline in heart rate after ISO injection in PDE4DKO and wild-type control mice is reported. Data are expressed as percent of the initial, maximal heart rate in response to ISO injection. Number of mice used for each measurement is reported among brackets. Download figure Download PowerPoint Discussion With the above findings, we have identified a novel signaling complex that distinguishes β1AR from β2AR. Although both receptors are in complexes with PDEs, their interactions differ in terms of the PDE4D splice variant recruited to the receptor, the mode of interaction with the PDE4D variant, and the effect of receptor agonists on the complex (see the illustration in Figure 8). β1AR preferentially associates with PDE4D8 in cardiomyocytes as shown by co-IP of endogenous PDE with the β1AR (Figure 1C), as well as the selective activation of PDE4D8 in intact cells (Figure 5A). This preference of β1AR for PDE4D8 was confirmed by co-IP experiments with exogenous proteins (Figure 3A and B). Conversely, PDE4D5 is the variant tethered to the β2AR/β-arrestin complex (Baillie et al, 2003) concurring with the preferential activation of PDE4D5 upon β2AR signaling (Figure 5B). In pull-down experiments using purified proteins (Figure 3D and E), β1AR efficiently interacts with PDE4D, whereas β2AR has negligible affinity for PDE4D, underscoring the direct mode of PDE4D–β1AR interaction versus the indirect, β-arrestin-dependent mode of PDE4D–β2AR interaction. The most important difference regarding the function of βARs is the effect of receptor occupancy on the PDE4D complexes. The β1AR/PDE4D complex is present in the absence of agonist and dissociates after receptor occupancy, whereas agonist binding to the β2AR is a prerequisite for the recruitment of the β-arrestin/PDE4D complex to the receptor. T
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