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Compartmentalized cyclic adenosine 3',5'-monophosphate at the plasma membrane clusters PDE3A and cystic fibrosis transmembrane conductance regulator into microdomains.

Penmatsa H, Zhang W, Yarlagadda S, Li C, Conoley VG, Yue J, Bahouth SW, Buddington RK, Zhang G, Nelson DJ, Sonecha MD, Manganiello V, Wine JJ, Naren AP - Mol. Biol. Cell (2010)

Bottom Line: Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex.Consequently, compartmentalized cAMP signaling is lost.Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

ABSTRACT
Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A-CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

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Actin depolymerization alters PDE3A dynamics and its physical coupling with CFTR. (A) SPT of HA-PDE3A in Calu-3 cells. Pseudocolor images show representative trajectories without or with Lat B (1 μM, 10 min) pretreatment. Histograms represent the diffusion coefficients, and MSD plots show representative mean squared displacement kinetics of HA-PDE3A in untreated and Lat B–pretreated cells (n = 6–9 cells, 150–230 trajectories). (B) Coimmunoprecipitation of HA-PDE3A and Flag-CFTR with or without Lat B pretreatment in HEK293 cells. The total protein from cells cotransfected with HA-PDE3A and Flag-CFTR was immunoprecipitated with α-HA beads and immunoblotted for CFTR. The experiment was repeated for three times.
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Figure 6: Actin depolymerization alters PDE3A dynamics and its physical coupling with CFTR. (A) SPT of HA-PDE3A in Calu-3 cells. Pseudocolor images show representative trajectories without or with Lat B (1 μM, 10 min) pretreatment. Histograms represent the diffusion coefficients, and MSD plots show representative mean squared displacement kinetics of HA-PDE3A in untreated and Lat B–pretreated cells (n = 6–9 cells, 150–230 trajectories). (B) Coimmunoprecipitation of HA-PDE3A and Flag-CFTR with or without Lat B pretreatment in HEK293 cells. The total protein from cells cotransfected with HA-PDE3A and Flag-CFTR was immunoprecipitated with α-HA beads and immunoblotted for CFTR. The experiment was repeated for three times.

Mentions: We first investigated the effects of actin cytoskeleton disruption on HA-PDE3A dynamics in live Calu-3 cells by using the SPT method. SPT is a powerful method to study the dynamics of individual proteins in the plasma membrane of live cells (Dahan et al., 2003; Chen et al., 2006). HA-PDE3A (HA tag on the first outer loop at position 104) was labeled with biotin α-HA antibody and then conjugated to streptavidin-conjugated Qdot-655 for monitoring its lateral mobility on the plasma membrane. The mean diffusion coefficient of PDE3A (0.0025 μm2/s) that we observed in this study is similar to that reported for CFTR (Bates et al., 2006; Jin et al., 2007), indicating the confined diffusion of PDE3A. When the cells were treated with Lat B (1 μM), a significant increase in the MSD and diffusion coefficient of PDE3A was observed (Figure 6A, mean diffusion coefficient: 0.0117 μm2/s; a 4.7-fold increase compared with the untreated cells), which suggests that actin cytoskeleton disruption uncouples PDE3A from the CFTR-containing complex, causes it to move freely (with higher diffusion coefficient), and compromises the integrity of multiprotein complex .


Compartmentalized cyclic adenosine 3',5'-monophosphate at the plasma membrane clusters PDE3A and cystic fibrosis transmembrane conductance regulator into microdomains.

Penmatsa H, Zhang W, Yarlagadda S, Li C, Conoley VG, Yue J, Bahouth SW, Buddington RK, Zhang G, Nelson DJ, Sonecha MD, Manganiello V, Wine JJ, Naren AP - Mol. Biol. Cell (2010)

Actin depolymerization alters PDE3A dynamics and its physical coupling with CFTR. (A) SPT of HA-PDE3A in Calu-3 cells. Pseudocolor images show representative trajectories without or with Lat B (1 μM, 10 min) pretreatment. Histograms represent the diffusion coefficients, and MSD plots show representative mean squared displacement kinetics of HA-PDE3A in untreated and Lat B–pretreated cells (n = 6–9 cells, 150–230 trajectories). (B) Coimmunoprecipitation of HA-PDE3A and Flag-CFTR with or without Lat B pretreatment in HEK293 cells. The total protein from cells cotransfected with HA-PDE3A and Flag-CFTR was immunoprecipitated with α-HA beads and immunoblotted for CFTR. The experiment was repeated for three times.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2836961&req=5

Figure 6: Actin depolymerization alters PDE3A dynamics and its physical coupling with CFTR. (A) SPT of HA-PDE3A in Calu-3 cells. Pseudocolor images show representative trajectories without or with Lat B (1 μM, 10 min) pretreatment. Histograms represent the diffusion coefficients, and MSD plots show representative mean squared displacement kinetics of HA-PDE3A in untreated and Lat B–pretreated cells (n = 6–9 cells, 150–230 trajectories). (B) Coimmunoprecipitation of HA-PDE3A and Flag-CFTR with or without Lat B pretreatment in HEK293 cells. The total protein from cells cotransfected with HA-PDE3A and Flag-CFTR was immunoprecipitated with α-HA beads and immunoblotted for CFTR. The experiment was repeated for three times.
Mentions: We first investigated the effects of actin cytoskeleton disruption on HA-PDE3A dynamics in live Calu-3 cells by using the SPT method. SPT is a powerful method to study the dynamics of individual proteins in the plasma membrane of live cells (Dahan et al., 2003; Chen et al., 2006). HA-PDE3A (HA tag on the first outer loop at position 104) was labeled with biotin α-HA antibody and then conjugated to streptavidin-conjugated Qdot-655 for monitoring its lateral mobility on the plasma membrane. The mean diffusion coefficient of PDE3A (0.0025 μm2/s) that we observed in this study is similar to that reported for CFTR (Bates et al., 2006; Jin et al., 2007), indicating the confined diffusion of PDE3A. When the cells were treated with Lat B (1 μM), a significant increase in the MSD and diffusion coefficient of PDE3A was observed (Figure 6A, mean diffusion coefficient: 0.0117 μm2/s; a 4.7-fold increase compared with the untreated cells), which suggests that actin cytoskeleton disruption uncouples PDE3A from the CFTR-containing complex, causes it to move freely (with higher diffusion coefficient), and compromises the integrity of multiprotein complex .

Bottom Line: Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex.Consequently, compartmentalized cAMP signaling is lost.Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

ABSTRACT
Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A-CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

Show MeSH
Related in: MedlinePlus