POPDC1 scaffolds a complex of adenylyl cyclase 9 and the potassium channel TREK‐1 in heart
2022; Springer Nature; Volume: 23; Issue: 12 Linguagem: Inglês
10.15252/embr.202255208
ISSN1469-3178
AutoresTanya A. Baldwin, Yong Li, Autumn N. Marsden, Susanne Rinné, Anibal Garza Carbajal, Roland F. R. Schindler, Musi Zhang, Mia A. Garcia, Venugopal Reddy Venna, Niels Decher, Thomas Brand, Carmen Dessauer,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoArticle18 October 2022Open Access Source DataTransparent process POPDC1 scaffolds a complex of adenylyl cyclase 9 and the potassium channel TREK-1 in heart Tanya A Baldwin Tanya A Baldwin orcid.org/0000-0001-7142-639X Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Yong Li Yong Li orcid.org/0000-0001-7440-6933 Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Formal analysis, Supervision, Investigation, Methodology, Writing - review & editing Search for more papers by this author Autumn N Marsden Autumn N Marsden Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Susanne Rinné Susanne Rinné orcid.org/0000-0003-2326-7069 Institute for Physiology and Pathophysiology, Vegetative Physiology and Marburg Center for Mind, Brain and Behavior MCMBB, Philipps-University of Marburg, Marburg, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Anibal Garza-Carbajal Anibal Garza-Carbajal orcid.org/0000-0001-9011-0438 Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Roland F R Schindler Roland F R Schindler orcid.org/0000-0001-6823-9622 National Heart and Lung Institute, Imperial College London, London, UK Contribution: Conceptualization, Resources, Methodology Search for more papers by this author Musi Zhang Musi Zhang Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Investigation Search for more papers by this author Mia A Garcia Mia A Garcia Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Investigation Search for more papers by this author Venugopal Reddy Venna Venugopal Reddy Venna orcid.org/0000-0002-7977-4281 Department Neurology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Supervision, Methodology, Writing - review & editing Search for more papers by this author Niels Decher Niels Decher orcid.org/0000-0002-9433-687X Institute for Physiology and Pathophysiology, Vegetative Physiology and Marburg Center for Mind, Brain and Behavior MCMBB, Philipps-University of Marburg, Marburg, Germany Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Thomas Brand Thomas Brand orcid.org/0000-0001-7090-5356 National Heart and Lung Institute, Imperial College London, London, UK Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Carmen W Dessauer Corresponding Author Carmen W Dessauer [email protected] orcid.org/0000-0003-1210-4280 Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration Search for more papers by this author Tanya A Baldwin Tanya A Baldwin orcid.org/0000-0001-7142-639X Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Yong Li Yong Li orcid.org/0000-0001-7440-6933 Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Formal analysis, Supervision, Investigation, Methodology, Writing - review & editing Search for more papers by this author Autumn N Marsden Autumn N Marsden Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Formal analysis, Investigation, Methodology, Writing - review & editing Search for more papers by this author Susanne Rinné Susanne Rinné orcid.org/0000-0003-2326-7069 Institute for Physiology and Pathophysiology, Vegetative Physiology and Marburg Center for Mind, Brain and Behavior MCMBB, Philipps-University of Marburg, Marburg, Germany Contribution: Formal analysis, Investigation, Writing - review & editing Search for more papers by this author Anibal Garza-Carbajal Anibal Garza-Carbajal orcid.org/0000-0001-9011-0438 Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Roland F R Schindler Roland F R Schindler orcid.org/0000-0001-6823-9622 National Heart and Lung Institute, Imperial College London, London, UK Contribution: Conceptualization, Resources, Methodology Search for more papers by this author Musi Zhang Musi Zhang Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Investigation Search for more papers by this author Mia A Garcia Mia A Garcia Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Investigation Search for more papers by this author Venugopal Reddy Venna Venugopal Reddy Venna orcid.org/0000-0002-7977-4281 Department Neurology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Supervision, Methodology, Writing - review & editing Search for more papers by this author Niels Decher Niels Decher orcid.org/0000-0002-9433-687X Institute for Physiology and Pathophysiology, Vegetative Physiology and Marburg Center for Mind, Brain and Behavior MCMBB, Philipps-University of Marburg, Marburg, Germany Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Thomas Brand Thomas Brand orcid.org/0000-0001-7090-5356 National Heart and Lung Institute, Imperial College London, London, UK Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Carmen W Dessauer Corresponding Author Carmen W Dessauer [email protected] orcid.org/0000-0003-1210-4280 Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration Search for more papers by this author Author Information Tanya A Baldwin1,†, Yong Li1,†, Autumn N Marsden1, Susanne Rinné2, Anibal Garza-Carbajal1, Roland F R Schindler3, Musi Zhang1, Mia A Garcia1, Venugopal Reddy Venna4, Niels Decher2, Thomas Brand3 and Carmen W Dessauer *,1 1Department Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA 2Institute for Physiology and Pathophysiology, Vegetative Physiology and Marburg Center for Mind, Brain and Behavior MCMBB, Philipps-University of Marburg, Marburg, Germany 3National Heart and Lung Institute, Imperial College London, London, UK 4Department Neurology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA † These authors contributed equally to this work *Corresponding author. Tel: +1 713-500-6308; E-mail: [email protected] EMBO Reports (2022)23:e55208https://doi.org/10.15252/embr.202255208 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the β-adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control. Synopsis Adenylyl cyclase type 9 (AC9) regulates resting heart rate and binds the novel scaffolding protein POPDC1. The two-pore potassium channel TREK-1 is released in a cAMP-dependent manner from a POPDC1:AC9 complex to regulate TREK-1 potassium currents. The Popeye domain-containing 1 (POPDC1) protein serves as a novel scaffold for adenylyl cyclase type 9 (AC9) that recruits the two-pore potassium channel TREK-1 in the absence of cAMP. Deletion of Adcy9 gives rise to a bradycardia at rest, heart rate variability during recovery from stress, and reduces the TREK-1-associated adenylyl cyclase activity in heart. Upon beta-adrenergic stimulation of AC9, TREK-1 association with AC9 and POPDC1 is reduced while interactions between AC9 and POPDC1 remain unchanged. AC9 activity within the POPDC1 complex controls the enhancement of TREK-1 currents by POPDC1, representing a novel form of regulation for heart rate control. Introduction Cyclic AMP mediates the sympathetic regulation of many physiological functions in the heart, including contractility, relaxation, heart rate (HR), conduction velocity, and stress responses (Baldwin & Dessauer, 2018). The robust and specific control of numerous activities by cAMP within a single chamber and/or nodal cardiomyocyte is due not only to the diversity of adenylyl cyclase (AC) isoforms and cAMP effector molecules but also to the spatial control of cAMP signaling by the generation of macromolecular complexes (Baldwin & Dessauer, 2018; Musheshe et al, 2018; Marsden & Dessauer, 2019). These complexes are organized by a family of A-kinase anchoring proteins (AKAPs) that place protein kinase A (PKA) in close proximity to its targets for regulation by phosphorylation (Omar & Scott, 2020). Many of these complexes also contain AC and phosphodiesterase, providing a framework for integration and modulation of local cAMP signaling within distinct AC-PKA protein complexes or nanodomains (Scott et al, 2013; Ahmad et al, 2015). There are 9 transmembrane AC isoforms that display diverse patterns of regulation, yet only a subset of AC isoforms binds to an individual AKAP (Dessauer, 2009; Johnstone et al, 2018). For example, coupling AC5 with a downstream effector of PKA (i.e., TRPV1) on AKAP79 can sensitize this anchored effector to local cAMP production, reducing the IC50 for agonists by 100-fold (Li et al, 2012, 2019; Efendiev et al, 2013). Scaffolding of AC9 to AKAP9 (a.k.a. Yotiao) promotes the PKA-dependent phosphorylation of KCNQ1 and is required for regulation of the IKs current in cardiomyocytes (Li et al, 2012, 2019). We show herein that the Popeye domain-containing (POPDC) protein represents a novel scaffolding protein for AC isoforms. POPDC isoforms 1–3 were named for their conserved and abundant expression in skeletal and cardiac muscle and are important for cardiac pacemaking and conduction (Schindler et al, 2016a). POPDC proteins are heavily glycosylated, containing a short amino-terminal extracellular domain, three transmembrane domains, and a cytosolic Popeye domain that displays structural similarity to the regulatory subunit of PKA and binds cAMP with high (~ 120 nM) affinity (Froese et al, 2012). Loss of either Popdc1 (a.k.a. Bves) or Popdc2 in mice or zebrafish results in arrhythmic and bradycardic phenotypes with high HR variability (Froese et al, 2012; Kirchmaier et al, 2012; Schindler et al, 2016b), while human mutations of POPDC family members cause limb-girdle muscular dystrophy (LGMD), cardiac arrhythmia, familial atrioventricular (AV) block, and are implicated in long-QT syndrome and heart failure (Gingold-Belfer et al, 2011; Tan et al, 2013; Wang et al, 2016; Schindler et al, 2016b; Brand, 2019; Han et al, 2019; Vissing et al, 2019; Indrawati et al, 2020; Rinné et al, 2020). POPDC1 and POPDC2 bind the two-pore-domain potassium channel TREK-1 (KCNK2 or K2P2.1) to enhance channel density at the plasma membrane and increase K+ currents (Froese et al, 2012; Schindler et al, 2016b; Rinné et al, 2020). Upon cAMP binding to POPDC proteins, the POPDC:TREK-1 complex is dissociated (Froese et al, 2012; Rinné et al, 2020); loss of this regulation is proposed in part to give rise to HR variability upon deletion of Popdc1 or -2. AC9 regulates HR as well (Li et al, 2017). We show herein that deletion of Adcy9 gives rise to a bradycardia at rest and HR variability during recovery from stress, albeit with a milder phenotype than the loss of Popdc1 or Popdc2 (Froese et al, 2012). AC9 binds all three POPDC isoforms, interacting with both the transmembrane regions and the cytosolic Popeye domain of POPDC1. TREK-1 co-localizes and associates with the AC9:POPDC complex, while the deletion of Adcy9 or Popdc1 reduces TREK-1-associated Gαs-stimulated AC activity in heart. TREK-1 also interacts with the calcium calmodulin-stimulated AC isoforms (AC1 and AC8); however, this interaction does not require POPDC1. Binding of AC9 and POPDC is independent of cAMP production, while AC9 association with TREK-1 is reduced in an isoproterenol (ISO)-dependent manner, requiring an intact Popeye domain and local production of cAMP within the complex. Moreover, AC9 regulates POPDC1 effects on TREK-1 currents. POPDC1 therefore represents a novel adaptor protein for AC9 to regulate downstream effectors for HR control. Results Deletion of Adcy9 decreases HR at rest and increases HR variability after ISO injection Previous studies using Doppler imaging of isoflurane-anesthetized mice revealed a mild bradycardia in Adcy9−/− male and female mice from 1 to 7 months of age (Li et al, 2017). To confirm the effects of Adcy9 deletion on HR in conscious animals, we measured electrocardiograms (ECGs) by telemetry. HR was measured over a 24-h period in which the animals were given free access to a running wheel. Adcy9−/− mice displayed a daytime bradycardia during periods of rest (defined as no movement of the running wheel for > 10 min), but not while actively moving (> 5 min on running wheel; Fig 1A). Similar trends were also observed at night. Figure 1. Deletion of Adcy9 results in bradycardia at rest and HR variability during the recovery period after ISO injection A. Heart rate (HR) of WT and Adcy9−/− mice averaged over 5 min of nonactive versus sustained activity on home cage running wheels. The Student's t-test P-values for indicated comparisons are shown. Circles represent data from individual animals. The boxplot central band is the median while whiskers represent the 10th and 90th percentiles. B, C. HR and R-R interval of WT (black bars) and Adcy9−/− mice (red bars) before and after ISO (1 μg/g) injection; mean of 5 min intervals ± SE are shown. D. Representative ECG recordings of 7 months WT and Adcy9−/− mice, ~ 55 min post-ISO injection. E, F. HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences (RMSSD; panel (E)) and percentage of sequential R-R intervals differing by >6 ms (pNN6; panel (F)) methods. Data information: Two-way ANOVA repeated-measures analysis (time and genotype) with the Holm–Sidak method used for pairwise comparisons for HR, RR, and HR variability with ISO; n = 9 WT and n = 10 Adcy9−/− mice; *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [embr202255208-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Mice were subjected to behavioral tests postimplantation of telemetry devices to rule out effects on general locomotor activity levels and exploratory drive (running wheels and open field activity box) or anxiety (elevated plus maze and forced swim test) that may affect overall HR. Overall, no significant differences were found in the maximal velocities or total distance traveled on the running wheel or during the 25 min open field test (Table 1). Adcy9−/− also showed no exploratory differences in the open field test and displayed similar levels of anxiety and depression-like behavior as demonstrated in the elevated plus maze and forced swim tests (Table 1). Table 1. Behavioral assays for WT and Adcy9−/− mice. Testsa WT Adcy9 −/− P-value* Running wheel Distance, 7 pm – 7 am (m) 1,700 ± 1,080 1,070 ± 790 0.20 Distance, 1st h (m) 180 ± 120 140 ± 90 0.48 Max velocity (m/min) 25 ± 15 24 ± 22 0.92 Open field box Zone 1 (cm) 301 ± 126 431 ± 199 0.32 Zone 1 entries 66 ± 39 62 ± 27 0.50 Total distance 1,701 ± 350 2,250 ± 1,170 0.48 Avg velocity (cm/min) 43.3 ± 11.1 40.7 ± 12.0 0.65 EPM Time, open arm (s) 24 ± 13 18 ± 24 0.25 Forced swim Immobility (s) 147 ± 36 158 ± 41 0.58 a Mean ± SD is given for combined male and female animals. * Student's t-tests were performed, except for EPM, which was analyzed by the Mann–Whitney rank-sum test, n = 9 WT mice (4 females, 5 males), n = 10 Adcy9−/− mice (4 females, 6 males). Source data are available online for this table. To induce an increase in HR in a controlled and timed manner, we administered ISO (1 μg/g) by a single intraperitoneal injection and recorded ECGs. Both genotypes and sexes showed a substantial increase in HR upon ISO administration (39 ± 10% and 48 ± 23% for WT and Adcy9−/−, respectively; Fig 1B), reaching a similar maximal HR. However, Adcy9−/− displayed a more rapid recovery to baseline with significantly longer R-R intervals (the time between two successive R-waves of the ECG) and slower HR (the reciprocal of R-R) at 55 and 60 min postinjection (Fig 1B and C). A further examination of ECG traces revealed a variability in beat-to-beat timing in Adcy9−/− mice at these time points (Fig 1D). HR variability was quantified by two distinct methods, which calculate the beat-to-beat consistency: RMSSD (Root mean square of successive RR interval differences) and pNN (Percentage of successive RR intervals that differ by more than 6 ms). Both methods showed statistically significant HR variability for Adcy9−/− mice in the recovery phase after ISO injection (Fig 1E and F). POPDC proteins bind AC9 in HEK293 cells and cardiomyocytes Despite the effects on HR and HR variability during recovery from beta-adrenergic stimulation, it was not immediately clear why deletion of Adcy9 would give rise to this phenotype. AC9 is present in the sinoatrial (SA) node (Li et al, 2017), but the Ca2+/calmodulin-stimulated ACs are generally thought to be responsible for pacemaker activity within the SA node (Mattick et al, 2007; Kryukova et al, 2012; Moen et al, 2019; Robinson et al, 2020). Thus, we searched the literature for proteins that were regulated by cAMP and when deleted, displayed HR variability. Two proteins of interest included POPDC1 and TREK-1; upon deletion in mice, both mutants show a sinoatrial pacemaking phenotype and HR variability (Froese et al, 2012; Unudurthi et al, 2016). POPDC proteins represent a family of cAMP effectors that bind to the two-pore potassium channel TREK-1 (Brand, 2019; Swan et al, 2019). Additionally, a high-throughput affinity purification-mass spectrometry screen identified AC9 and AC3 with POPDC2 as bait; however, these potential interactions were never biochemically verified (Huttlin et al, 2015). Unfortunately, AC9 and POPDC1 suffer from a lack of antibodies sufficient for immunoprecipitation, with poor detection of endogenous proteins by western blotting. Therefore, we first tested for potential interactions between AC9 and POPDC using the expression of tagged proteins in cellular assays of HEK293 cells and COS-7 cells. These tagged proteins are fully functional, as demonstrated by activity assays and AKAP association for AC9 (Li et al, 2012, 2017; Baldwin et al, 2019; Lazar et al, 2020) and cAMP binding and TREK-1 interactions for POPDC1 (Froese et al, 2012; Schindler et al, 2016b). Proximity ligation assay (PLA) can amplify the detection of protein–protein interactions that occur within a range of < 60 nm (Fredriksson et al, 2002). YFP-tagged AC9 and POPDC-MYC-tagged proteins were expressed in HEK293 cells; interactions between AC9 and Gβγ (as detected with antibodies against Gβ) were used as a positive control (Li et al, 2017). PLA puncta (red) are readily detected with AC9 and POPDC isoforms 1 and 2, suggesting close proximity and possible complex formation (Fig EV1A and B). As a control, overexpression of nontagged POPDC1 or AC9 was used to compete with the PLA signal between tagged POPDC1 and AC9 (Fig EV1C). These nontagged proteins reduced the PLA signal compared with the noninteracting transmembrane protein EGFR. Figure 2. Cellular interaction of POPDC isoforms with AC9 A, B. PLA assay performed in neonatal cardiomyocytes expressing GFP-Flag or AC9-Flag using antibodies against Flag and endogenous POPDC1, POPDC2, or Gβγ. Images (A); scale bar is 20 μm; nuclear staining by DAPI shown in blue) and quantitation (B) of PLA signal is shown. The unpaired two-sided Student's t-test (n = 4 independent experiments, **P = 0.0086) was performed for each antibody pair. Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range. C. Cartoon of AC9-POPDC BiFC complex. D. Quantification of COS-7 cells expressing BiFC constructs for AC9, POPDC 1–3. The expressed proteins tagged with VN (top line) and VC (bottom line) are shown. The Kruskal–Wallis one-way ANOVA analysis on ranks was performed (n = 4 experiments) with multiple comparisons with control by the Dunnett's method (#P < 0.05). Individual comparisons were performed with a two-tailed Student's t-test (**P = 0.003, *P = 0.018) or Mann–Whitney rank-sum test (^P = 0.004 versus control) Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range. E. Representative live-cell images of indicated BiFC combinations in HEK293 cells (n > 20 cells; scale bar: 10 μm). Quantification of POPDC1-VN:AC9-VC is shown in Figs 3 and 7. Source data are available online for this figure. Source Data for Figure 2 [embr202255208-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. PLA signal between GFP-tagged AC9 and POPDC1-Myc A, B. Images (A) of PLA signal (red) and DAPI (blue) performed in HEK293 cells using antibodies against GFP and MYC tags. Scale bars are 20 μm. Mean cellular fluorescence intensity of PLA signal (B) was quantified by high content microscopy. The Kruskal–Wallis one-way ANOVA analysis was performed (n = 7 experiments, P = 0.003 between groups) with multiple comparisons by the Bonferroni t-test (*P < 0.05). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the 10th and 90th percentiles are represented by the whiskers. C. Competition of PLA signal between GFP-tagged AC9 and POPDC1-Myc with overexpression of nontagged EGFR (control), Flag-AC9, or POPDC1-Flag in HEK293 cells. Mean cellular fluorescence intensity was quantified by high content microscopy and normalized to GFP control. The Kruskal–Wallis one-way ANOVA analysis was performed (n = 3 experiments, >1,000 cells per experiment) with multiple comparisons by the Student–Newman–Keuls method (*P < 0.05). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range. Download figure Download PowerPoint AC9 also interacts with endogenous POPDC1 in neonatal cardiomyocytes (CMs). Using antibodies that were validated for immunohistochemistry against endogenous POPDC1 or POPDC2 (see methods), PLA signals were detected in CMs between POPDC1 and adenovirally expressed Flag-tagged AC9 but not Flag-tagged GFP (Fig 2A and B). Interaction of AC9 with endogenous Gβγ served as a positive control, while PLA signals were not significant for endogenous POPDC2 and AC9. A complementary cellular interaction technique is Bimolecular Fluorescence Complementation (BiFC). Here, potential interacting pairs were tagged with either the N-terminal half of Venus (VN) or C-terminal half of Venus (VC; Fig 2C). Since HEK293 cells express endogenous POPDC1 and POPDC2, we performed BiFC experiments in COS-7 cells that lack detectable endogenous POPDC proteins (Appendix Table S1). A strong BiFC signal was detected between AC9 and POPDC 1 and 3, and to a lesser extent with POPDC2 (Fig 2D), with AC9:POPDC1 BiFC signal localizing to PM in HEK293 cells (Fig 2E). To test whether AC and POPDC interactions could also be detected by co-immunoprecipitation (co-IP), Flag-tagged AC9 was co-expressed with or without Myc-tagged POPDC1 or POPDC2 in HEK293 cells. Complexes were isolated with anti-MYC antibodies and associated Gαs-stimulated AC activity was measured (IP-AC assay; Fig 3A). From the IP-AC assay, it appeared that AC9 interacted with POPDC1 but not POPDC2. However, when the co-IPs were evaluated via western blot, AC9 co-precipitates with both POPDC1 and POPDC2 (Fig 3B). Thus, POPDC1 pulls down AC9 and maintains associated AC activity, while AC9 complexed with POPDC2 does not respond to Gαs stimulation. POPDC2:AC9 may represent a different conformation than POPDC1:AC9, consistent with the decreased BiFC and PLA signals and AC9-associated activity. POPDC interactions with AC9 were confirmed by performing co-IP in reverse by pulling down the complex with the associated Flag-tag on AC9 (Fig 3C). POPDC1 and POPDC2 were both detected in pull-downs of Flag-AC9. Figure 3. AC9 interacts with the POPDC1 transmembrane and Popeye domains HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or − 2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. The Kruskal–Wallis one-way ANOVA analysis on ranks was performed (n = 6 experiments, P = 0.003 between all groups) with multiple comparisons with AC9 control by the Dunn's method and Mann–Whitney rank-sum test (**P = 0.002). Data are plotted as mean ± SEM. A portion of the lysates and Co-IP from (A) were subjected to western blot (WB) analysis. Sf9 cells expressing Flag-AC9 served as a positive WB control. Molecular weight markers are denoted as M. Quantitation of Flag-AC9 WB by one-way ANOVA, n = 3–4 experiments, with comparisons by the Tukey test, **P = 0.003, ***P = 0.001. Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range. HEK293 cells expressing Flag-AC9 +/− POPDC1-Myc or POPDC2-Myc were subjected to Co-IP with anti-FLAG and subjected to WB analysis with anti-MYC and anti-FLAG. Quantitation of anti-MYC WB by one-way ANOVA, n = 4 experiments, with comparisons by the Tukey test, **P = 0.003, ***P < 0.001). Boxplots show the median as the central band, the box size as the lower and upper quartiles, while the whiskers are the range. HEK293 cells expressing POPDC1-Myc +/− GFP-tagged AC9 and control TM proteins EGFR, or LAMP1 were subjected to Co-IP with anti-MYC. Western blotting of lysates and Co-IPs for GFP (top) and MYC (bottom) are shown (n = 3 experiments). Schematic of POPDC1 truncations. BiFC of AC9 and indicated POPDC1 truncations in COS-7 cells. The Kruskal–Wallis one-way ANOVA analysis was performed (n = 4 experiments) with multiple comparisons by the Tukey test, ***P < 0.001 compared with VN control, **P = 0.008. Boxplots show the median as the central band, the box size as the
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