cAMP-Related Macromolecular Complexes Regulating CFTR Opening: Comparison
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Cystic fibrosis (CF) is the rare genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). These molecules, known as CFTR modulators, have led to unprecedented improvements in the lung function and quality of life of most CF patients. However, the efficacy of these drugs is still suboptimal, and the clinical response is highly variable even among individuals bearing the same mutation. Furthermore, not all patients carrying rare CFTR mutations are eligible for CFTR modulator therapies, indicating the need for alternative and/or add-on therapeutic approaches. Because the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) represents the primary trigger for CFTR activation and a major regulator of different steps of the life cycle of the channel, there is growing interest in devising ways to fine-tune the cAMP signaling pathway for therapeutic purposes.

  • cystic fibrosis
  • cystic fibrosis transmembrane conductance regulator
  • 3′,5′-cyclic adenosine monophosphate

1. Introduction

Cystic fibrosis (CF) is the most common life-threatening inherited disorder among the Caucasian population, affecting more than 100,000 people worldwide. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane chloride (Cl−) and bicarbonate (HCO3−) channel expressed in epithelial cells, whose dysfunction leads to impaired mucus hydration and clearance [1]. As a consequence, CF impacts the functionality of multiple organs, although the disease primarily affects the respiratory system and digestive tract. If, on the one hand, gastrointestinal symptoms can be limited by pancreatic enzyme replacement therapy, on the other hand, treating lung dysfunction remains the most challenging aspect of disease management. The respiratory pathology of CF is characterized by persistent airway obstruction that progresses from early onset mucus plugging in small airways to chronic airway inflammation and recurrent respiratory infections, ultimately leading to lung destruction and respiratory failure, which is the primary cause of death among CF patients [2].
Despite the cloning of the CFTR gene in 1989, significant progress in the development of CFTR-targeting therapies has only occurred in the past decade, when CFTR modulators, the first molecular treatments directly targeting the underlying molecular defect of CF, were approved. In particular, a game-changing event for CF patients has been the FDA approval in 2019 of Elexacaftor-Tezacaftor-Ivacaftor (ETI), a triple-combination therapy able to substantially improve lung function, especially in patients carrying at least one F508del allele, who represent 80% and 90% of the total European and U.S. CF population, respectively [3,4,5][3][4][5]. However, ETI still has some limitations. Indeed, patients with rare genotypes, who account for about 20% of the European CF population, are currently ineligible for treatment with these modulators, since not all CFTR mutant proteins are responsive to these compounds [6]. Furthermore, ETI can only modulate up to 50–60% of wild-type channel mutant F508del-CFTR activity [7[7][8],8], indicating that alternative and/or add-on therapeutic approaches are still urgently needed for CF.
One possibility to unveil new druggable targets is to pinpoint CFTR interactors and the related signaling pathways involved in regulating channel processing, trafficking, stability, and function, all mechanisms that can be dysregulated in CF patients [9]. A promising candidate is the signaling cascade orchestrated by 3′,5′-cyclic adenosine monophosphate (cAMP), since this second messenger can regulate multiple aspects of CFTR function, from channel open probability to its localization in the plasma membrane [10]. More importantly, cAMP is compartmentalized in space and time thanks to the formation of localized signaling complexes, also known as cAMP signalosomes, anchored to specific subcellular compartments or organelles within the cell, and thus represents an ideal target for fine-tuned therapeutic approaches [11].

2. cAMP Is Recognized as the Master Regulator of CFTR Activity

cAMP elevation in the subcortical compartment leads to the activation of PKA and PKA-mediated phosphorylation of the CFTR is required to increase the channel open probability and to allow efflux of Cl– anions [10]. In epithelial cells, the cAMP pool responsible for CFTR function is primarily produced upon the activation of a specific GPCR, namely the β2AR channel (Figure 1). This protein co-localizes with the CFTR at the apical membrane of polarized lung epithelial cells (Calu-3), and it has also been found to co-immunoprecipitate with the channel itself [26,27][12][13]. The physical protein-protein interaction (PPI) between the anion channel and the β2AR is coupled with CFTR activation since βAR agonists, such as isoproterenol (β1AR and β2AR agonist) and albuterol (β2AR-selective agonist), can induce a concentration-dependent increase in short-circuit current (ISC), which is sensitive to CFTR-selective inhibition in polarized epithelial cells [26][12]. It is also notable that the β2AR-dependent CFTR opening can be stimulated only upon the interaction between the two proteins, since the removal of the PDZ-binding motif of CFTR, which abolishes the physical interaction between the channel and the receptor, specifically reduces Cl− efflux after β2AR stimulation in vitro [26][12]. The relevance of β2ARs to CFTR activation is corroborated by the observation that β2AR agonists induce the swelling of intestinal organoids, a gold-standard model in CF research, indicating that these molecules can potently activate the wild-type (wt) CFTR [28][14]. Again, in intestinal epithelial cells, it has been shown that AC6 plays a central role in regulating CFTR function upon GPCR activation (Figure 1). AC6, the most abundant AC isoform in the gut, can be detected as a CFTR interactor in the intestinal mucosa of mice and in colon epithelial cells [29][15]. In further support of the key role of AC6 in producing the cAMP pool responsible for CFTR activity in the gut, Thomas and colleagues showed that the AC activator forskolin failed to induce the swelling of intestinal organoids derived from AC6 knockout mice [29][15]. Another well-characterized interactor with the CFTR protein is PKA, one of the main effectors of the second messenger cAMP. PKA phosphorylates the CFTR protein on several serines mainly located within the regulatory R domain of the channel (Figure 1), an unstructured polypeptide sequence with a predominant inhibitory function. In its unphosphorylated state, the R domain intercalates between the two nucleotide-binding domains (NBDs), preventing their dimerization and CFTR opening [30][16], whereas phosphorylation of residues S422, S660, S795, and S813, can release the channel from its closed conformation, increasing its open probability up to 100-fold [31][17]. Unlike the aforementioned serines, whose post-translational modification is considered as being activating, some residues such as S737 and S768 have been proposed to be either activating or inhibitory, depending on contextual modifications of other phosphorylation sites [32][18]. In this regard, the phosphorylation of different serine residues can be interdependent, as seen in S795 and S813. The latter can be phosphorylated only when S795 has already been post-translationally modified, showcasing S813 phosphorylation as a limiting step in CFTR activation [33][19].
Figure 1. Schematic representation of CFTR interactors of the cAMP signalosomes that positively (A) and negatively (B) regulate CFTR-mediated anion secretion. (A) Endogenous stimulation of β2ARs leads to Gs-mediated activation of AC6, which is responsible for cAMP production. cAMP in turn activates PKA that, by phosphorylating the R domain of the CFTR channel, leads to anion secretion. PKA localizes in the vicinity of the CFTR via ezrin, an AKAP that interacts with the C terminus of the channel via the adaptor protein NHERF1. (B) PDE4, which interacts with the CFTR protein via Shank2, is anchored to PKA by the AKAP PI3Kγ, which favors PKA-mediated phosphorylation and consequent activation of the cAMP-hydrolyzing enzyme, switching off β2AR-mediated activation of the CFTR channel. The signal can also be terminated by the cAMP exporter MRP4, which interacts with the CFTR via the scaffolding protein PDZK1. PM: plasma membrane.
A recent restudyearch suggested that, in addition to the phosphorylation of key serine residues, the simple binding of PKA to the CFTR contributes to the activation of the channel [31][17]. Mihályi and colleagues demonstrated that, while the post-translational modification of the unstructured R domain is required for sustained channel activation, the binding of the kinase to the CFTR is by itself able to release the unphosphorylated R domain from its inhibitory position, thus leading to Cl− secretion [31][17]. PKA was found to interact with the CFTR channel and in particular to co-immunoprecipitate with both the catalytic and type II regulatory subunits of the kinase in the early 2000s [34][20]. The early finding that this interaction is abrogated upon treatment with Ht31, a peptide disrupting the interaction between AKAPs and the RI or RII subunits of PKA, indicated that a PKA-anchoring protein is responsible for the association between the kinase and the CFTR [34][20]. Subsequent studies identified ezrin as the scaffold protein bringing PKA into close proximity with the Cl− channel (Figure 1). Ezrin can co-immunoprecipitate with both the RII subunit of the kinase and the CFTR, and the inhibition of its AKAP function with the Ht31 peptide reduces the cAMP-activated membrane conductance of Calu-3 cells by 83%, indicating that the association between ezrin and PKA is key to the kinase-dependent regulation of CFTR gating [34][20]. The binding of ezrin to the channel is not direct, rather it involves an adaptor known as the Na+/H+ exchanger regulatory factor 1 (NHERF1), which can interact with the C terminus of the CFTR via its PDZ domains, serving as a bridge between ezrin and the Cl− channel (Figure 1) [35][21]. In particular, in immortalized bronchial epithelial cell (16HBE14o-) monolayers, the deletion of NHERF1 PDZ domains, as well as of its FERM-binding domain responsible for the association with ezrin, abrogates CFTR-dependent Cl− efflux, pointing to a crucial role of NHERF1 in the formation of the CFTR multi-protein complex which is key to the regulation of the activity of the channel [36][22]. In support of this data, the absence of NHERF1 completely abolishes duodenal bicarbonate secretion in response to clenbuterol-mediated β2AR stimulation in vivo [37][23]. Another level of NHERF1-dependent CFTR regulation resides in the fact that this adaptor protein competes with the PDZ domain-containing protein Shank2 for CFTR binding [38][24]. Different from NHERF1, Shank2 is a negative regulator of cAMP-mediated channel function (Figure 1). Patch clamp experiments in the whole cell configuration revealed that the overexpression of Shank2 in CFTR-expressing mouse embryonic fibroblasts (NIH 3T3) decreased CFTR activity by 50% upon forskolin-mediated AC activation, as a consequence of a significant reduction in the cAMP-induced phosphorylation of the channel [39][25]. This negative effect of Shank2 overexpression on CFTR activity is instead lost upon treatment with rolipram, a PDE4-specific inhibitor, suggesting a functional association between the cAMP-hydrolyzing enzyme and Shank2, which was confirmed by an increase in Shank2/PDE4 physical association upon forskolin treatment [38][24]. As further validation of the presence of PDE4 in the cAMP signalosome of the CFTR, this cAMP-hydrolyzing enzyme was found to interact with the channel in airway epithelial cells. The use of chamber measurements revealed that PDE4 inhibition increases CFTR activity after βAR stimulation and significantly reduces the time required for the channel to return to baseline activity levels [40][26]. In addition, the expression of a catalytically inactive PDE4, which acts by displacing the endogenous protein from the cAMP signalosome, significantly augments PKA-mediated phosphorylation of the CFTR, pointing out the key role of PDE4 in controlling CFTR via regulation of local cAMP/PKA levels [40][26]. Another key regulator of this CFTR-anchored pool of PDE4 is the AKAP phosphoinositide 3-kinase γ (PI3Kγ). By anchoring PKA to PDE4, PI3Kγ favors its PKA-mediated phosphorylation and consequent activation (Figure 1) [41][27]. Displacement of the PI3Kγ-anchored pool of PKA inhibits the activity of subcortical PDE4, leading to a localized cAMP elevation responsible for PKA-mediated CFTR phosphorylation and gating, which unveils PI3Kγ as the regulator of a cAMP microdomain central to epithelial fluid secretion in the airways [41][27]. Another PDE isotype, namely PDE8, was found to regulate CFTR function. Selective pharmacological PDE8 inhibition in primary bronchial epithelial cells stimulates CFTR-dependent ion transport, both under basal conditions and after pre-treatment with cAMP-elevating agents, suggesting that this negative regulator of the channel may also be a component of the cAMP signalosome regulating the CFTR [42][28]. In addition to PDEs, MRP channels are also responsible for limiting the pool of cAMP that regulates CFTR function, with MRP4 being the isoform found to functionally and physically associate with the CFTR protein [43,44][29][30]. In particular, MRP4 inhibition leads to a restricted increase in cAMP levels in the subcortical compartment where the transporter interacts with the CFTR channel via the PDZ Domain Containing 1 protein (PDZK1, also known as CAP70), suggesting that MRP4 modulates the pool of cAMP responsible for CFTR opening (Figure 1) [44][30]. This model is strengthened by the observation that MRP4 inhibition increases CFTR-mediated ISC in response to an endogenous stimulus triggering a compartmentalized cAMP increase, while it does not affect the current elicited by the global cAMP elevation induced by forskolin [44][30]. Finally, disruption of MRP4/PDZK1 protein-protein interaction significantly attenuates CFTR-mediated currents activated by MRP4 inhibition in gut epithelial cells, confirming the role of MRP4 as one of the crucial CFTR interactors regulating the function of the channel [44][30].

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