Pathway Map Details

Regulation of CFTR activity (norm and CF)

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Filamin B (TABP), PKC-epsilon, HCO(,3)('-) = HCO(,3)('-), Glutathione extracellular region, L-Adrenaline extracellular region, VIL2 (ezrin), EDG4, Protein kinase G 2, Casein kinase II, alpha chains, ATP, PKA-reg (cAMP-dependent), S100A10, MUNC18, Casein kinase II, beta chain (Phosvitin), Calcineurin A (catalytic), cGMP , AMPK gamma1, PRKAR2A, Adenylate cyclase, SNAP-23, cAMP, COMMD1 (MURR1), PDZK1, chloride ion cytosol, G-protein alpha-s, AMP, G-protein alpha-i family, PKA-cat (cAMP-dependent), PP2A regulatory, Annexin V, chloride ion extracellular, PP2A catalytic, PP2A structural, Glutathione cytoplasm, Tubulin (in microtubules), glutathione = glutathione, RACK1,, HCO(,3)('-) extracellular , CFTR, Adenosine, Lysophosphatidic acid, AMPK alpha 1 subunit, PKA-reg type II (cAMP-dependent), PP2C,, SHANK2, Filamin A, PDE4D, Annexin II, Beta-2 adrenergic receptor, E3KARP (NHERF2), EBP50, Adenosine A2b receptor, Cl(-) = Cl(-), AMPK beta subunit, Syntaxin 1A, HCO(,3)('-)


Regulation of CFTR activity (norm and CF)

The cystic fibrosis transmembrane conductance regulator ( CFTR ) is a member of the ATP-binding cassette (ABC) transporter superfamily. It acts in apical part of the epithelial cells as a plasma-membrane, cyclic AMP-activated chloride anion, bicarbonate anion and glutathione channel [1], [2], [3]. CFTR is required for cell surface water-salt homeostasis and normal function of epithelia lining the airways, intestinal tract, ducts in the pancreas, salivary and sweat glands, liver and others [3], [4].

CFTR is an ATP -dependent membrane transporter which is activated by directly binding to ATP. Opening of the CFTR is initiated by ATP binding at the NBD2 site of this channel [5], [6].

Posttranslational modifications and interactions with several proteins are main regulatory events affecting activity and stabilizing membrane expression of the CFTR channel [4].

Cyclic adenosine monophosphate ( cAMP )/ cAMP-dependent protein kinase A ( PKA ) pathway is a dominant cascade which affects CFTR channel activity [4]. Adenosine is a mediator which activates CFTR channel via cAMP/ PKA. Activation of the Adenosine A2B receptor by physiological ligands such as Adenosine leads to stimulation of Adenylate cyclase by G-protein alpha-s leading to an increase in concentration of highly compartmentalized cAMP, and subsequent activation of the PKA [7], [8].

Phosphorylation of CFTR by PKA-cat is mediated by PRKAR2A which is linked physically and functionally to CFTR by a V illin 2 ( VIL2 (ezrin) ). The latter serves as an anchoring protein for PKA-cat -mediated phosphorylation of CFTR. Anchoring protein VIL2 (ezrin) promotes PKA- to- CFTR interaction [9]. Moreover VIL2 (ezrin) itself exists in a complex with CFTR. This interaction is mediated by Solute carrier family 9 member 3 regulator 2 ( E3KARP (NHERF2) ) - a PDZ-containing binding partner of CFTR [10]. Formation of a VIL2 (ezrin)/ E3KARP (NHERF2)/ CFTR complex enhances the efficacy of cAMP-mediated CFTR activation [10]. In addition, Protein phosphatase 3 catalytic subunit ( Calcineurin A )/ Annexin A2 ( Annexin II )/ S100 calcium binding protein A10 ( S100A10 ) complex participate in PKA -dependent CFTR activation [11].

It is shown, that Annexin A5 ( Annexin V ) is necessary for normal CFTR chloride channel activity as well, but exactly mechanism it acting is unknown [12].

Interestingly, activation of PKA by Adenosine may also increase the activity of Phosphodiesterase 4D ( PDE4D ) leading to attenuation of the cAMP signal. The by-product of cAMP degradation - Adenosine monophosphate ( AMP ) can activate AMP-activated protein kinase ( AMPK ) [13]. AMPK can also phosphorylate CFTR, but unlike PKA, AMPK -dependent phosphorylation has a negative effect on CFTR channel activity [14], [15], [16]. The exact molecular events leading to AMPK-dependent phosphorylation of CFTR are still elusive. Most likely, it starts with activation of PDE4D by PKA-cat followed by conversion of cAMP to AMP [13] . AMP binds to and activates regulatory AMPK gamma 1 (this isoform predominantly and functionally associates with CFTR [14] ) and initiates formation of a complex consisting of regulatory AMPK gamma and beta subunits and catalytic AMPK alpha 1 subunit. In turn, AMPK alpha 1 subunit binds to CFTR. This interaction might be essential for AMPK mediated phosphorylation of CFTR, which reduces chloride anion secretion by inhibiting channel activity without affecting the number of CFTR channels in the plasma membrane [14], [15], [16].

In addition, enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin) activate intracellular cAMP/ PKA and cGMP/ Protein kinase G 2 and signal CFTR on the apical plasma membrane [17].

CFTR membrane expression is also regulated by the Tubulin/ Solute carrier family 9 member 3 regulator 1 ( EBP50 )/ Guanine nucleotide binding protein beta polypeptide 2-like 1 ( RACK1 )/ Protein kinase C epsilon ( PKC-epsilon ) pathway. PKC-epsilon phosphorylates CFTR and thus stabilizes expression of CFTR in the apical plasma membrane of epithelial cells. [18], [19]. Apparently, constitutive phosphorylation by PKC-epsilon is essential for the acute activation of CFTR by PKA-cat, since phosphorylation by PKA-cat alone is not a sufficient stimulus to open the CFTR [20].

SH3/ankyrin domain gene 2 ( SHANK2 ) inhibits CFTR activity by breaching the CFTR - EBP50 association and by bringing PDE4D, which precludes cAMP/ PKA signaling [21].

Dephosphorylation also affects activity of CFTR channel. For instance, Protein phosphatase 2 ( PP2A ) and PP2C domain containing protein phosphatases ( PP2C ) inhibit CFTR activity [22], [23], [24].

In addition to posttranscriptional modifications, the binding partners (especially PDZ-domain containing proteins) can also modulate CFTR activity [4]. These are EBP50, E3KARP (NHERF2), PDZ domain containing ( PDZK1 ) 1 and others.

E3KARP (NHERF2) functions as a scaffold (see above [10] ). PDZK1 is capable of linking CFTR molecules to form dimers. In this dimeric form, CFTR channel activity is enhanced. Disrupting PDZK1/ CFTR complex abrogates the functional coupling of cAMP transporter activity to CFTR function [25], [26]. EBP50, which exists in a complex with CFTR at the apical surface of epithelial cells, is a main PDZ-domain containing binding partner which positively regulates CFTR channel activity [4].

EBP50 may stimulate CFTR expression on apical membrane in receptor-dependent fashion - mainly with Beta-2 adrenergic receptor. Beta-2 adrenergic receptor and CFTR are physically and functionally coupled into a macromolecular signaling complex via interactions with EBP50 [27]. Importantly, this process is independent of the agonist-mediated cAMP/ PKA pathway [28]. On the other hand, PKA-cat -mediated phosphorylation of CFTR strongly inhibits formation of the macromolecular complex consisting of Beta-2 adrenergic receptor/ EBP50/ CFTR [27]. Functional consequences of the disruption of this complex are elusive.

Copper metabolism domain containing 1 ( COMMD1 ) (Drevillion, L et al., The 21st annual north American cystic fibrosis conference, California, 2007), Filamin A and Filamin B [29] stabilize expression of CFTR in the apical plasma membrane.

In addiction to positive regulation of CFTR by PDZ-containing scaffold proteins other binding partners such as Synaptosomal-associated protein 23kDa ( SNAP-23 ) - S yntaxin 1A complex can sterically interfere with CFTR. This results in a decrease of channel activity, although inhibitory influences of Syntaxin binding protein 1 ( MUNC18 ) can be diminished by its binding to S yntaxin 1A [30], [31].

In addition , Endothelial differentiation lysophosphatidic acid G-protein-coupled receptor 4 ( EDG4 ) activated by a Lysophosphatidic acid rapidly inhibits CFTR channel activity through G-protein alpha-i family by suppressing PKA-cat -mediated activation of CFTR. EDG4 is most typical for gut but EDG4/ E3KARP (NHERF2)/ CFTR macromolecular complex may be form in different cell as HT29-CL19A (colonic epithelial cells) as Calu-3 (airway serous gland epithelial cells). And so it is possibly, that EDG4 participates in CFTR regulation in airway cells too [32].

The most common CFTR mutation is loss of a Phe residue at position 508 ( deltaF508-CFTR ). Majority of regulators - to- CFTR are equal interactions for wt CFTR and deltaF508- CFTR. One of the exclusion is a Casein kinase II. Casein kinase II associates with and phosphorylates wt CFTR but not deltaF508- CFTR. This interaction activates CFTR -dependent chloride transport [33].


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