Regulation of CFTR stability at the plasma membrane—Mechanisms and therapeutic opportunities in cystic fibrosis

Carlos M Farinha; João Oliveira

doi:10.1002/1873-3468.70407

. 2026 Jul 6;600(14):1966–1991. doi: 10.1002/1873-3468.70407

Regulation of CFTR stability at the plasma membrane—Mechanisms and therapeutic opportunities in cystic fibrosis

Carlos M Farinha ^1,^✉, João Oliveira ¹

PMCID: PMC13404153 PMID: 42403151

Abstract

CFTR stability at the plasma membrane is controlled by a dynamic balance between trafficking, endocytosis, recycling, and degradation. Interactions with scaffold proteins such as NHERF1 and ezrin anchor CFTR to the actin cytoskeleton, reducing mobility and enhancing stability. Cytoskeletal dynamics, regulated by GTPases like RhoA, Rac1, and Rap1, further influence CFTR retention and function. Phosphorylation by kinases including PKA, LMTK2, and SYK modulates channel activity and membrane presence. Whereas rescue of F508del‐CFTR with modulators can rescue its defective folding and premature degradation, reduced membrane stability persists. Overall, understanding the molecular mechanisms governing CFTR regulation provides critical insights for developing more effective treatments targeting its stability and function in cystic fibrosis.

Keywords: CFTR, cystic fibrosis, membrane stability, protein trafficking, therapeutic approaches

CFTR variants cause cystic fibrosis, often by affecting the three‐dimensional structure of the channel and preventing it from reaching the plasma membrane. This review explores the molecular mechanisms regulating CFTR stability at the plasma membrane and shows how they can provide critical insights for developing more effective treatments targeting the protein's stability and function in cystic fibrosis.

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Abbreviations

ABC, ATP‐binding cassette

AC, adenylyl cyclase

AJ, adherent junction

AKAP, A‐kinase anchoring protein

AP2, adaptor protein 2

ARP2/3, actin related protein 2/3 complex

ASL, airway surface liquid

BHK, (baby hamster kidney)

CaAR, calcium‐mediated actin reset

CAL, CFTR‐associated ligand

CAPN1, calpain‐1

CAPZA2, capping acting protein of muscle Z‐line alpha subunit 2

CCV, clathrin‐coated vesicle

CFBE, Cystic Fibrosis bronchial epithelial

CFTR, cystic fibrosis transmembrane conductance regulator

Cif, CFTR inhibitory factor

CKII, casein kinase II

CLASP, clathrin‐associating sorting protein

DAB2, disabled‐2

EB, ezrin binding

EPAC1, Exchange protein directly activated by cAMP

ER, endoplasmic reticulum

ERC, endosomal recycling compartment

ERM, Ezrin‐radixin, Moesin

ERM, ezrin‐radixin‐moesin

ERQC, ER quality control

ESCRT, endosomal sorting complex for transport

F‐actin, filamentous actin

FERM, Four‐point‐one

G‐actin , globular actin

GAP, GTPase‐activating protein

GEF, guanine nucleotide exchange factor

GPCR, G‐protein‐coupled receptor

HBE, human bronchial epithelial

HGF, hepatocyte growth factor

INF2, inverted formin‐2

KD, knockdown

LMTK2, lemur tyrosine kinase 2

LPA ₂, lysophosphatidic acid receptor 2

MRP4, multidrug resistance protein 4

MVB, multivesicular body

MyoVI, myosin VI

NBD, nucleotide‐binding domain

NHERF1, Na⁺/H⁺ Exchanger Regulatory Factor 1

PDE, phosphodiesterase

PDZ, PSD95/DlgA/ZO‐1

PI3Kγ, phosphatidylinositol‐3 kinase gamma

PKA, protein kinase A

PM, plasma membrane

Qdot, quantum dot

R, regulatory

RING, really interests new gene

ROCK, Rho‐associated coiled‐coil protein kinase

STX6, syntaxin 6

SYK, spleen tyrosine kinase

TBB, tetrabromobenzotriazole

TGF‐β1, transforming growth factor beta 1

TGN, trans‐Golgi network

TM, transmembrane

TMD, transmembrane domain

Ub, ubiquitin

USP, ubiquitin specific protease

WASP, Wiskott‐Aldrich Syndrome Protein

WAVE, WASP family Verprolin‐homologous protein

WNK, with‐no‐lysine

β ₂ AR, β₂ adrenergic receptor

Cystic fibrosis (CF) is a multisystem disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial Cl⁻ and HCO₃ ⁻ channel essential for ion and fluid homeostasis. Although CF affects multiple organs in which CFTR possesses a relevant function, its most severe and life‐limiting manifestations arise in the lungs, where CFTR works by regulating intralumenal osmotic pressure through ion transport across the epithelium. Impaired CFTR function disrupts hydration of the airway surface liquid (ASL), leading to mucus stasis, and such CFTR impairment in turn compromises mucociliary clearance and leads to chronic infection and inflammation, culminating in progressive loss of lung function [1]. At the cellular level, disease severity correlates strongly with the amount and activity of CFTR present at the apical plasma membrane (PM) of epithelial cells, underscoring the importance of regulatory mechanisms that control CFTR localization and stability at this site [2].

CFTR is a member of the ATP‐binding cassette (ABC) protein family that functions as a phosphorylation‐ and ATP‐regulated channel. Its structure follows a conserved architecture based on the modular assembly of four ABC‐family canonical domains: two transmembrane domains (TMDs), each containing 6 α‐helical transmembrane segments (TM) that become inserted into the membrane, and two cytosolic nucleotide binding domains (NBDs) (for an extensive review of CFTR structure, see [3]). Additionally, CFTR contains a regulatory (R) region that regulates its gating upon phosphorylation. Such large size and multidomain architecture lead to complex folding requirements, which render CFTR particularly susceptible to perturbations in biosynthesis and regulation. Indeed, many disease‐causing variants compromise CFTR folding, stability, or channel activity, thereby reducing expression of the channel and, consequently, its functional level at the PM [4]. The most prevalent CF‐causing variant, deletion of phenylalanine 508 (F508del), exemplifies this vulnerability by severely limiting CFTR trafficking to the cell surface due to a derived folding defect that leads to early degradation of the channel.

Such early degradation is the cause of a stringent quality control during its biogenesis in the secretory pathway, in which a substantial fraction of both mutant and wild‐type protein is eliminated before reaching the plasma membrane [5]. After its co‐translational insertion in the ER membrane, CFTR is glycosylated and navigates through folding‐assessment programs carried out by cytosolic and ER chaperones, which target misfolded channels to proteasomal degradation [6]. At this stage, most F508del‐CFTR is targeted for degradation since it can never achieve a native folding state necessary to overcome such quality control. Later, and if properly folded, CFTR transits to the Golgi apparatus where its sugar moieties undergo modifications, becoming fully glycosylated and following the secretory pathway towards the PM [6]. Even when CFTR successfully exits the ER and Golgi apparatus, delivery to the apical membrane does not represent the endpoint of its regulation. Rather, CFTR enters a highly dynamic lifecycle at the cell surface, where its abundance and activity at the PM are continuously shaped by multiple regulatory interactions [7].

These PM‐associated regulatory processes determine not only the steady‐state abundance of CFTR at the cell surface but also the extent to which the channel can dynamically respond to physiological cues and therapeutic intervention. Interactions with scaffolding proteins, kinases, phosphatases, and components of the endocytic machinery modulate CFTR gating, lateral organization, and stability at the apical membrane, thereby shaping channel activity in response to signals such as cyclic AMP (cAMP) elevation or changes in epithelial demand. Importantly, these same interactions influence the efficacy and durability of CFTR‐targeted therapies for disease‐causing variants (such as the F508del‐CFTR), including potentiators and correctors, by affecting channel residency time, recycling efficiency, and susceptibility to degradation [7].

This review focuses specifically on the molecular interactions that regulate CFTR “stability” at the plasma membrane—here, understood as the balance of all the processes regulating the amount of CFTR that is at the membrane at steady‐state level, and thus including arrival/delivery at the PM, internalization and post‐endocytic fate (either recycling or degradation). We also highlight relevant regulatory nodes that are increasingly recognized as critical determinants of epithelial function, therapeutic responsiveness and disease pathogenesis in CF. Table 1 summarizes the role of the most relevant CFTR interactors in regulating its PM stability.

Table 1.

Summary of the most relevant CFTR interactors that play a role in its plasma membrane stability. ↓, negative regulator (i.e., depletion or inhibition increases CFTR PM levels or residence time); ↑, positive regulator (i.e., depletion or inhibition decreases CFTR PM levels or residence time).

Protein	Effect on plasma membrane stability	Cell type	Ref.
Endocytosis
AP2	↓	Polarized CFBE41o‐ cells	[21, 24]
DAB2	↓	CFBE41o‐ cells	[21, 24]
c‐Cbl	↓	Primary differentiated HBE	[25]
c‐Cbl	↓	CFBE41o‐ cells	[25]
EPAC1	↑	CFBE41o‐ cells	[94, 100]
LMTK2	↓	Polarized primary HBE cells	[108]
LMTK2	↓	Polarized CFBE41o‐ cells	[108]
SYK	↓	CFBE41o‐ cells	[115, 116, 118]
SHC1	↓	CFBE41o‐ cells	[118]
WNK4	↑	CFBE41o‐ cells & HEK cells	[115]
Anchoring/Cytoskeleton remodeling
NHERF1	↑	16HBE14o‐ & CFBE41o‐ cells	[10, 12, 57]
Ezrin	↑	16HBE14o‐ & CFBE41o‐ cells	[10, 12, 57]
RhoA	↑	16HBE14o‐ & CFBE41o‐ cells	[12, 57]
Rac1	↑	16HBE14o‐ & CFBE41o‐ cells	[12]
ARP2/3	↑	16HBE14o‐ cells	[12]
PIP5K	↑	16HBE14o‐ cells	[12]
ROCK	↑	16HBE14o‐ & CFBE41o‐ cells	[57]
N‐WASP	↑	BHK cells	[11]
INF2	↓	CFBE41o‐ cells	[100]
CAPZA2	↑	CFBE41o‐ cells	[100]
Recycling
Rab9	↑	BHK cells	[34]
Rab11	↑	T84 cells	[37, 38]
USP10	↑	CFBE41o‐ cells	[39]
Lysosomal degradation
ESCRT machinery	↓	BHK cells	[36]
Rab7	↓	BHK cells	[34]
CAL	↓	COS7 and 16HBE14o‐ cells	[71, 72]
Syntaxin 6	↓	16HBE14o‐ & CFBE41o‐ cells	[73]
MARCH2	↓	HEK‐ & CFBE41o‐ cells	[74]
Trafficking/maturation
Casein kinase II	↑	16HBE14o‐ & CFBE41o‐ cells	[122]

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CFTR at the plasma membrane: Endocytosis, recycling and the cortical cytoskeleton

Once CFTR's glycosylation state matures in the Golgi, the protein is delivered to the PM where it exerts its function as a chloride channel. In the apical membrane of epithelial cells, CFTR is responsible for regulated ion transport, and its pore acts as a selectivity filter that delimits the nature of the ions that can cross through [6]. At the PM, Cl⁻ flux generated by CFTR depends on the number of channels present at a given moment, as the net ion current generated is the sum of the current of individual CFTR channels—thus, its PM steady‐state levels are intrinsically related to the extent of its overall activity. Such steady‐state levels depend on three major events: (1) its delivery to the PM through biosynthesis and trafficking (following the secretory pathway); (2) its internalization through endocytosis; and (3) the fate of the internalized channel, which can be either recycled back to the PM or sent to lysosomal degradation. Changes in any of these events could alter the amount of CFTR present at the cell surface [8, 9] and the balance of such processes is the main determinant of CFTR PM half‐life which is about 3 h for WT‐CFTR but only 1 h for its F508del counterpart [6]. Beyond that, evidence shows that up to 50% of surface CFTR is present in an immobile pool, anchored to the cortical actin cytoskeleton [10], which characterizes a distinct PM pool of “tethered CFTR” opposed to a “free CFTR” one. The latter is easily recognized by the endocytic machinery for PM withdrawal, whereas the former is stabilized at the cell surface by both molecular partners that tether CFTR to the filamentous (F‐) actin that compose the actin cytoskeleton and on local de novo synthesis of F‐actin, highlighting the role of cytoskeleton regulation on CFTR's PM stability [11–13].

Endocytosis and recycling of CFTR

Endocytic signals and adaptors

Endocytosis is a process in which membrane cargo molecules, such as integral membrane proteins, are internalized and directed towards inner membrane cellular compartments such as early endosomes, lysosomes, and the trans‐Golgi network (TGN). In most cases, protein internalization is mediated by clathrin‐coated vesicles (CCVs) [14]. In this process, adaptor proteins handle cargo selection by binding membrane cargos through short linear sequence motifs, recognizing integral membrane proteins that will be included in endocytic vesicles. Adaptor protein 2 (AP2) is the prototypical adaptor and one of the main components of purified clathrin‐coated vesicles, and it binds a tyrosine‐based motif (YXXΦ) and the dileucine code ([D/E]XXXl[L/I]) in cargo proteins. It is also capable of binding phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P₂), which causes a large conformational change that exposes a clathrin‐binding site, thereby enabling AP2 to directly participate in the clathrin coat recruitment process [15]. Other monomeric adaptors, called clathrin‐associating sorting proteins (CLASPs), also nucleate at endocytic sites at the membrane by binding, beyond their cargos, PI(4,5)P₂, AP2 and the clathrin coat itself, generating a complex endocytic apparatus that can recruit many cargo molecules. During endocytosis, membrane curvature is thought to be driven by proteins such as those from the CALM protein family and epsin. Whether clathrin itself also plays a role in driving membrane curvature is still an open question [16]. Phosphatidylinositols, mainly PI(4,5)P₂, play a major role in regulating endocytosis: most adaptor proteins and dynamin, the protein responsible for pinching off vesicles from the PM, bind PI(4,5)P₂ and depend on this molecule to become active and exert their role [14] [16].

At the membrane, CFTR is endocytosed by CCVs and its ensuing destiny is linked to several Rab small GTPases, as mentioned below (Fig. 1). CFTR possesses three endocytic signals at its C‐terminal: ¹⁴²⁴YDSI, ¹⁴¹³FVLI and ¹⁴³⁰LL; full‐length CFTR binds to the AP2 complex that recognizes the tyrosine‐based motif through its μ2 subunit, turning the channel into a CCV cargo protein [17,18] [19]. Initial reports demonstrate that the μ2 subunit of AP2 binds CFTR C‐terminal and the expression of a dominant negative μ2 subunit mutant inhibits CFTR endocytosis in HeLa cells [20]. Another study showed that CFTR colocalizes and interacts with AP2 and the motor protein myosin VI in rat enterocytes and polarized Caco‐2 cells [19, 21]. Interestingly, the CLASP disabled‐2 (DAB2) influences CFTR levels at the apical membrane without directly binding to the channel, suggesting that AP2 mediates this interaction [19]. Additionally, inhibition of the CLASP protein DAB2 in CFBE41o‐ cells was shown to decrease CFTR's endocytosis and increase the channel's surface steady‐state levels in polarized monolayers [22]. Contrarily, however, depletion of the μ2 subunit of AP2 did not impact CFTR endocytosis or steady‐state surface levels, suggesting that the AP2 adaptor is not as relevant for CFTR endocytosis in bronchial epithelial cells. Interestingly, DAB2 characteristic endocytic motif (consensus NPXY [23]) is absent in CFTR; however, treatment of CFBE cells with a NPXY‐containing peptide completely impaired the channel's internalization and decreased CFTR's presence in CCVs, suggesting that DAB2's action is probably mediated by other molecular partners containing this recognition motif [22]. In contrast, another study performed in CFBE41o‐ cells identified similar effects of AP2 and DAB2 depletion in CFTR endocytosis, in which KD of both proteins increased steady‐state levels of the protein and decreased its internalization, suggesting that they may play a joint role in the process [24]—indeed, DAB2's promotion of CFTR endocytosis has been suggested to depend on other proteins such as AP2 and myosin VI (another CFTR interactor). In this line, studies performed in rat enterocytes showed that DAB2 does not directly bind CFTR but depends on the α subunit of AP2 for CFTR recognition [19]. While AP2 conventionally binds CFTR through its μ2 subunit, the α subunit participates more effectively in CFTR endocytosis with the aid of DAB2, also coupling AP2 to myosin VI (MyoVI) to promote internalization of the channel, given that these proteins were found in molecular complexes with CFTR in Caco‐2 cells. The precise mechanism by which CFTR is recruited to CCVs is, thus, not completely understood, and evidence suggests that it might be variable across tissues, which raises the necessity of specific studies to elucidate how this process takes place and whether it can be modulated to promote rescue of disease‐causing CFTR variants. An adaptor protein and a RING E3 ubiquitin ligase, c‐Cbl, has also been implicated in CFTR endocytosis and endocytic fate: data suggests that it identifies CFTR as an endocytic cargo and promote its endocytosis in a ubiquitin‐independent manner, supposedly functioning as an adaptor protein that binds to either AP2 or directly to clathrin, for instance [25]. Additionally, c‐Cbl has been shown to ubiquitinate CFTR in early endosomes, promoting a shift from the recycling pathway toward lysosomal degradation, decreasing its half‐life [25]—discussed in more detail below.

Small GTPases of the Rab family

After endocytosis, delivery of endocytic vesicles is controlled by small GTPases of the Rab family. Rab small GTPases function as molecular switches by alternating between a GDP‐bound, inactive state and a GTP‐bound, active one whose interconversions are regulated by GTPase‐activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively [26–31].

Endocytosis of CFTR is dependent on Rab small GTPases activities, since CFTR is included as a cargo protein of endocytic vesicles (Fig. 1). Its endocytosis is a rapid process, with a significant proportion of CFTR being internalized. As mentioned, 50% of PM CFTR form a mobile pool at the PM [10], and this detached CFTR is rapidly endocytosed by clathrin‐associated endocytic machinery. Adding to its increased endocytosis, its biosynthesis is a relatively slow process, and together they make the recycling of internalized channels the main process for maintaining a functional level of CFTR at the PM—in fact, CFTR is constantly being recycled from the endosomal recycling compartment (ERC) back to the PM by the action of Rab11 coupled to MyoVb, and its steady‐state levels at the PM heavily depend on the action of this Rab protein [32, 33]. CFTR recycling has also been linked to the small GTPase RhoA, whose activation leads to the formation of actin stress fibers that were suggested to increase recycling of the channel [12]. Other Rab proteins are also associated with CFTR trafficking: after endocytosis, trafficking of CFTR to early endosome is dictated by Rab5, and Rab7 controls movement of CFTR away from the recycling pathway and into late endosomes, which eventually culminates in lysosomal degradation. Rab9, instead, moves CFTR away from lysosomes back to the trans‐Golgi compartment [34].

Even though vesicle trafficking is largely determined by Rab proteins and their effectors, CFTR's fate in post‐endocytic compartments depends largely on its ubiquitination state. Data support the notion that internalized WT‐CFTR remains mainly in early/recycling endosome compartments and is quickly reinserted into the PM to sustain functional levels of the channel at the cell surface [35]. CFTR ubiquitination, then, works as a signal that diverts its route from recycling endosomes to late endosomes/multivesicular bodies, targeting the ubiquitinated protein to lysosomal degradation—specially in CFTR variants with a folding defect, which are readily recognized by the peripheral quality control machinery [36–38]. Recent results in CFBE cells point to polyubiquitination being necessary for diverting CFTR towards the degradative route, and both K48‐ and K63‐polyubiquitin chains (associated with proteasomal and lysosomal degradation, respectively) collaborate in promoting degradation of the internalized channel [37]. Studies have shown that while ubiquitinated CFTR follows through the lysosomal maturation route, non‐ubiquitinated channels are targeted to the PM through recycling [36, 39]. Such diversion is carried out by the endosomal sorting complex for transport (ESCRT) machinery, which effectively recognizes ubiquitin moieties in cytosolic regions of endocytosed membrane cargos and selects them for inclusion in multivesicular bodies during the endosome maturation process [40]. Consistent with this notion, ubiquitin‐conjugated (Ub‐) WT‐CFTR has been shown to exist in macromolecular complexes with HRS and STAM‐2 from ESCRT 0 machinery, TSG101 from ESCRT I, Vps25 from ESCRT II and Vps32 from ESCRT III, whereas the non‐ubiquitylated protein shows no interaction with these ESCRT machinery subunits [36]. Current models of the fate decision of CFTR between lysosomal degradation and recycling are linked to deubiquitinating enzymes, particularly to USP10, which belongs to the ubiquitin specific protease (USP) family. Internalized, ubiquitinated CFTR can be deubiquitinated by USP10, which colocalizes with it in endosomes, suggesting that this protein is pivotal in regulating the fate of CFTR [39]. Initial reports suggested that ubiquitination of CFTR does not regulate its endocytosis but rather the extent of its lysosomal degradation, and that ubiquitin modification takes place in endocytic compartments rather than at the PM [36, 41], in which case endocytic events appear to be uncoupled from the surface stability of CFTR. Contrarily, other studies suggested that CFTR PM stability was largely linked to the internalization process, which was proposed to become dysregulated in the case of F508del‐CFTR and to account for its decreased PM stability (discussed below) [42]. A recent report, however, demonstrated that both processes influence CFTR PM half‐life in CFBE cells: a CFTR construct with a C‐terminal ubiquitin (CFTR‐Ub) evidenced increased endocytosis than WT‐CFTR, and its internalization was comparable to that of F508del‐CFTR; also, F508del‐CFTR was substantially less recycled to the PM, suggesting that both internalization and recycling are decisive for defining the PM stability of CFTR [37].

CFTR at the cell cortex and the actin cytoskeleton

As stated above, 50% of surface CFTR exists as an immobile pool [10]. Such immobilization takes place due to tethering of CFTR to the cortical actin cytoskeleton, which decreases lateral diffusion of the channel and, therefore, its inclusions in clathrin‐coated pits as an endocytic cargo molecule [10, 17, 43]. The cytoskeleton is a major cellular structure composed of filaments that organize, polarize and structure the cell, allow the movement of organelles across the cytoplasm and cellular movement altogether. There are three major types of cytoskeleton filaments, each associated with different functions: the microtubules, constituted by tubulin, that extend to the cell periphery; the intermediate filaments, constituted by lamins and many other related proteins, which line the inner face of the nuclear envelope; the actin filaments, composed of polymers of actin, that underlie the plasma membrane of epithelial cells to provide strength, making up the cellular cortex, and also found on structures such as the lamellipodia and filopodia, used for cell movement [44].

The actin cytoskeleton

The cell cortex is a thin actin shell that can contract due to the presence of myosin II within the actin filament network. It underlies the inner face of the PM and is several hundreds of nanometers thick [44]. The actin meshwork contains branched and crosslinked actin filaments and is connected to the PM through proteins of the ezrin‐radixin‐moesin (ERM) protein family, discussed in more detail below. This PM association provides contractility that regulates membrane tension and osmotic pressure within the cell [45]. Actin filaments, in turn, are constituted by the polymerization of globular (G)‐actin into its filamentous (F) form. F‐actin polymers are a polarized structure with fast‐growing barbed ends and slow‐growing pointed ends, and such polarity is the result of both G‐actin monomer asymmetry, which delimits both ends of the polymer and its capacity to bind and hydrolyse ATP. G‐actin is recruited to F‐actin polymers and binds the barbed end, extending the growing filament [46].

Polymerization is regulated by many actin‐cytoskeleton‐associated proteins in vivo, allowing a tight temporal and spatial control of F‐actin elongation and disassembly: in the cytoplasm, G‐actin is usually bound to the inhibitory protein profilin, which binds opposite to the G‐actin ATP‐binding site [44]. In the cell cortex, two protein complexes mainly mediate polymerization: formins, proteins that bind the profilin‐actin dimer and include them in the barbed end of F‐actin, extending it linearly; and the ARP2/3 complex, which acts as a nucleating factor to promote branched actin networks [45]. The former are homodimers that recruit profilin‐inhibited G‐actin through its proline‐rich FH1 domain and add them to the barbed end of F‐actin, to which they are usually bound through their FH2 domain [46, 47]. ARP2/3, on the other hand, is an actin‐like protein that associates with and nucleates actin monomers to begin elongation [45]. ARP2/3‐F‐actin complexes [48] are regulated by the WASP/WAVE family of proteins [49] that promote ARP2/3 complex nucleation and elongation of F‐actin [50]. Capping proteins (CP) are also relevant to the ARP2/3 complex polymerization role: they bind to the barbed ends of F‐actin and prevent further polymerization (and depolymerization) of grown filaments. In a model of how they impact polymerization dynamics, CPs allow new ARP2/3 complex‐derived branches to be formed by inhibiting elongation at the barbed end and promoting a new round of nucleation, which implies an important role in the formation of branched F‐actin architectures as opposed to linear bundles [51].

ERM proteins in CFTR anchoring

PM CFTR tethering at the cell cortex is mediated by adaptor proteins responsible for effectively anchoring the channel in place (Fig. 2). The best‐characterized adaptor responsible for this process is the PDZ protein NHERF1, which directly binds CFTR and mediates its connection to the cell cortex. NHERF1 contains two tandem PDZ (PSD95/DlgA/ZO‐1) domains that recognize a C‐terminal PDZ‐binding motif (consensus: C‐terminal X‐[S/T]‐X‐[V/I/L]) [43], which is present at the C terminus of CFTR (residues ¹⁴⁷⁷DTRL). In addition, NHERF1 also contains a C‐terminal EB (ERM‐binding) domain that binds members of the ERM family. ERM family proteins, in turn, act as physical linkers between the plasma membrane and cortical actin [52, 53]. Thus, ERM proteins effectively connect the cortical cytoskeleton to the PM and are essential in transmitting to the latter the cell cortex contractility, participating in the regulation of cellular shape. Ezrin is initially at an autoinhibitory state in which the interaction of its N and C terminii precludes the protein from binding actin. PI(4,5)P₂ relieves this autoinhibition, and further stabilization of its open, active conformation is achieved through phosphorylation at a tyrosine residue, T567, by the Rho‐associated coiled‐coil protein kinase (ROCK) (among other kinases), which is a RhoA effector [54]. Active‐ezrin not only tethers CFTR in an immobile pool, but it also functions as a versatile scaffold that organizes signaling complexes. One of its best‐described roles is in binding protein kinase A (PKA), a major cAMP effector that is crucial for CFTR channel function—discussed in detail below. Ezrin can also recruit RhoGDI to counteract its inhibitory activity over Rho small GTPases such as RhoA and Rac1, allowing them to become activated by their respective GEF [55, 56].

The functional importance of the NHERF1‐ezrin interaction for CFTR membrane organization is supported by studies examining CFTR diffusion at the plasma membrane. Full‐length CFTR is less mobile at the cell surface than a C terminus truncated CFTR construct in which the PDZ motif is absent [43], indicating this site is responsible for lower mobility of the channel at the PM. Moreover, the PDZ motif does not affect the channel's diffusion at the ER, indicating that it immobilizes CFTR specifically at the cell surface. Similar results were obtained with quantum dot (Qdot) labelling of CFTR molecules, which allowed single molecule PM diffusion analysis, revealing that the expression of a C terminus truncated NHERF1 lacking its EB domain promotes increased CFTR PM mobility, showcasing that the CFTR‐NHERF1‐ezrin axis is crucial to restrict the channel movement at the cell surface [10]. Interestingly, NHERF1 overexpression in CFBE41o‐ cells (a CF cell model which was shown to have lower NHERF1 expression than 16HBE14o‐ cells) increased phosphorylated (p‐) ezrin levels and restored its localization to the apical membrane, strongly suggesting that the presence of apical NHERF1 is necessary for the proper activation and function of ezrin [57, 58]. Data available suggest that NHERF1 not only binds to ERM proteins to promote anchoring of PM integral proteins but is intrinsically responsible for ezrin's regulation and function, participating in the adhesion of the cytoskeleton to the PM [58]. On this basis, NHERF1 itself is subject to conformational regulation that modulates these interactions: an autoinhibitory domain‐domain interaction between these C terminus EB and PDZ2 is relieved upon ezrin binding [59, 60]. Thus, NHERF1 and ezrin come together to mutually promote their functions, and their presence at the cellular cortex implicates in many regulatory processes discussed below.

PDZ proteins, the cytoskeleton and CFTR

Cytoskeleton remodeling is also important for the PM stability of CFTR (Fig. 2). For instance, it has been shown that N‐WASP (a WASP family protein) inhibition decreases PM CFTR levels while increasing the intracellular pool of the channel [11]. As mentioned, WASP proteins regulate ARP2/3‐mediated actin polymerization; beyond that, they are regulated by Rho GTPases such as Rho, Cdc42, and Rac [50]. Thus, N‐WASP inhibition possibly leads to actin polymerization deficiencies that influence CFTR's internalization and recycling, even though its precise mechanism is currently unknown. Interestingly, N‐WASP inhibition leads to an increased endocytic rate of CFTR, suggesting that its actin polymerization role is responsible for increased CFTR PM residency. In line with that, actin disruption through latrunculin B reduces steady‐state levels of CFTR at the PM, leading to decreased CFTR‐mediated Cl⁻ currents [11].

In polarized 16HBE41o‐ cells expressing WT‐CFTR, the NHERF1‐ezrin interaction was deemed crucial for both cytoskeletal structure and CFTR apical localization [57]. The abolishment of this interaction, either by modulating NHERF1 or ezrin binding capacity, decreased CFTR‐dependent Cl⁻ currents and surface levels, as well as F‐actin content, leading to major disorganization of the actin cytoskeleton [57]. Such changes in cytoskeleton dynamics were comparable to treatment with cytochalasin D, which affects CFTR function to a similar degree as destabilizing the NHERF1‐ezrin interaction, suggesting that the latter has a role in the organization of actin cytoskeleton, which, in turn, regulates CFTR PM residency and function [57]. In line with this, a study in mice showed that NHERF1^−/− intestines presented a disorganized microvillus and a disordered and thick actin terminal web, as well as lower p‐ezrin in the brush border membrane (BBM) of the epithelium, suggesting that NHERF1 is necessary for proper ezrin function and F‐actin organization [61]. Also, ezrin has been shown to colocalize with N‐WASP and ARP2/3 complex, but not with mDia1 (a formin), in the membrane of phagosomes in RAW264.7 macrophages, and the active, open form conformation of ezrin readily interacts with N‐WASP, suggesting that it may elicit de novo actin polymerization [62, 63]. Altogether, these results point to a regulatory role of the NHERF1‐ezrin axis in cytoskeleton dynamics, plausibly by allowing the actin polymerization machinery recruitment to promote cytoskeleton remodeling.

Such regulation might be linked to N‐WASP activity, which in turn seems to be crucial for proper PM CFTR function, but a specific mechanism elucidating this regulatory process is still absent (Fig. 2). In this sense, N‐WASP might be responsible for driving the formation of the cytoskeleton frame onto which the CFTR‐NHERF‐ezrin axis resides, and both NHERF1 and ezrin themselves seem to participate in the recruitment of such cytoskeleton remodeling machinery. Interestingly, CFTR itself seems to participate in this process: CFTR silencing in polarized cells leads to a disruption of the cortical actin cytoskeleton, and CFTR overexpression in CFBE cells expressing F508del‐CFTR, whose cortical cytoskeleton is intrinsically disordered, restores the cortical F‐actin structure [64]. Accordingly, studies in CF airway epithelial cells showed that the absence or mislocalization of CFTR destabilizes the cortical actin network by disrupting its scaffolding interactions with NHERF1 and ezrin, leading to the collapse of tight‐junction organization, the accumulation of cytosolic stress fibers, and a marked increase in epithelial leakiness—defects that can be reversed by restoring CFTR or NHERF1 expression. Also, F508del‐CFTR correction through chemical modulators, which leads to increased surface levels of the mutant channel, restores the actin cytoskeleton organization and increases p‐ezrin levels [65]. Altogether, this suggests that CFTR is not a passive user of the cortical actin cytoskeleton: it actively participates in its remodeling, which is expected to have larger consequences in the cell (this topic is specifically reviewed in [66]) (Fig. 2).

Small GTPases as CFTR regulators

Given that small GTPases are key regulators of the cytoskeleton, they have also been implicated in the PM residency and channel function of CFTR (Fig. 1). Rho GTPases are found in all eukaryotic organisms and are divided into three subfamilies, grouped according to their functional and structural similarity to their three founding members, RhoA, Rac1, and Cdc42. Rho proteins are key regulators of actin cytoskeleton dynamics [67, 68], and have also been implicated in the regulation of cell polarity and membrane trafficking through the modulation of F‐actin remodeling [69, 70]. In CFBE cells, NHERF1 overexpression not only increased p‐ezrin levels but also the activity of RhoA small GTPase. This increased activity was linked to full‐length NHERF1, since NHERF1‐ΔERM, a construct lacking the ezrin‐binding C‐terminal region of NHERF1, could not elicit the same effect [57]. Interestingly, the dominant‐active RhoA‐V14 was also able to increase p‐ezrin levels and the assembly and organization of F‐actin in the apical side of polarized CFBE cells, which was accompanied by increased Cl⁻ channel function and apical localization of F508del‐CFTR; however, this dominant‐active mutant elicited no effect in CFBE cells overexpressing NHERF1, suggesting that NHERF1 promoted RhoA activation and signaling at the apical side of polarized epithelial cells, which is then speculated to drive cytoskeleton reorganization that sustains CFTR PM residency and function. In agreement with that, the effects of NHERF1 overexpression were abrogated by treating transfected cells with the ROCK inhibitor Y‐27632.

Another study showed that both RhoA and Rac1 increase PM levels of CFTR without affecting the overall CFTR level within cells [12]: dominant‐active RhoA and Rac1 both increased cell surface of WT‐CFTR levels and CFTR‐derived Cl⁻ currents, and their dominant‐negative variants elicited the opposite changes. NHERF1 overexpression produced comparable results, suggesting that these small GTPases act downstream of NHERF1 to promote cytoskeleton remodeling and CFTR function. Moreover, both RhoA and Rac1 were shown to be necessary for the NHERF1‐overexpression‐induced increased PM CFTR levels [12]. Interestingly, the consequences of both Rho small GTPases were quite different in regulating PM CFTR: Rac1 was shown to delay internalization of CFTR, changing its endocytosis rate, and to promote CFTR accumulation in F‐actin‐rich membrane ruffles in 16HBE14o‐ cells expressing WT‐CFTR. Active Rac1 also increased the interaction of NHERF1 with ezrin, actin and CFTR, indicating that it promotes the strengthening of the CFTR‐NHERF1‐ezrin axis which increases CFTR retention at the cell surface. Rac1 effect was dependent on PIP5K and the ARP2/3 complex, in which case a kinase‐dead PIP5K and knockdown (KD) of ARP3 reverted the increased CFTR PM levels promoted by dominant‐active Rac1 [12]. This suggests that Rac1 promotes local production of PIP2 through PIP5K, which allows both the actin polymerization machinery (such as N‐WASP through the ARP2/3 complex) to remodel the cortical cytoskeleton and the opening of ezrin at the PM, which effectively anchors CFTR to the underlying F‐actin.

RhoA, on the other hand, had no effect in the initial internalization step of the channel but increased its recycling in the same cellular model. On the same line, it promoted the formation of stress fibers and CFTR was shown to be distributed along its length, accumulating at the stress fibers PM anchoring point, consistent with a role in CFTR recycling for RhoA [12]. Finally, RhoA failed to strengthen the CFTR‐NHERF1‐ezrin axis despite promoting increased p‐Ezrin levels, which suggests its role in PM CFTR dynamics is not to simply restrict it to the PM, even though it must lead to the sustained activation of ezrin through phosphorylation of its C terminus. How both small GTPases jointly regulate CFTR PM levels is yet unknown, and further studies dissecting how each of them participates in this process could reveal a major crosstalk between the actin cytoskeleton regulation and CFTR.

Additional regulators of CFTR membrane anchoring

Even though the CFTR‐NHERF1‐ezrin axis is one of the best‐characterized CFTR‐containing complexes at the cell surface, many other PDZ domain proteins interact with CFTR and regulate its activity and PM residency. For instance, the CFTR‐associated ligand (CAL) is a PDZ protein that binds to CFTR C terminus at the Golgi and the PM. CAL decreases the steady‐state levels of CFTR at the PM by both reducing its anterograde trafficking from the Golgi and decreasing its PM half‐life, but also by competing with NHERF1 to bind CFTR's PDZ motif—whereas NHERF1 stabilizes CFTR at the apical membrane and promotes its recycling, CAL favors intracellular retention and turnover of the protein [71]. CAL has been shown to act exclusively on fully glycosylated CFTR, and its effects are blocked by the lysosomal proton‐pump inhibitor bafilomycin 1, suggesting it targets CFTR to lysosomal degradation both from the Golgi and the PM [72]. More recently it has been shown that syntaxin 6 (STX6) (a Q‐SNARE important for vesicle trafficking and fusion between endosomes, the TGN and lysosomes) and MARCH2, a RING E3 ubiquitin ligase, mediate the CAL‐dependent fate decision of CFTR [73, 74]. STX6 interacts with CAL and post‐ER CFTR, and its inhibition increases CFTR surface expression and Cl⁻ currents; also, inhibition of CAL abolishes STX6 overexpression effects, indicating that they cooperate in targeting CFTR to lysosomal degradation [73]. MARCH2, in turn, seems to mediate CAL's targeting by ubiquitinating CFTR. Its overexpression increases ubiquitinated CFTR and it interacts and colocalizes with CAL and STX6 [74]. Importantly, the effect of increased MARCH2 overexpression is inhibited when lysosome degradation is blocked, and MARCH2 and CAL are mutually necessary for decreasing CFTR half‐life, yet MARCH2's role on cortical CFTR is still unclear, since it has been shown to colocalize with CFTR mainly in the Golgi.

Other PDZ domain‐containing proteins that interact with CFTR include NHERF2, PDZK1, PDZK2, and SHANK2 (Fig. 3). NHERF2, similarly to NHERF1, has two tandem PDZ domains and has been implicated in macromolecular complexes that exert an inhibitory effect on CFTR. It tethers the channel to the lysophosphatidic acid receptor 2 (LPA₂), which, in turn, decreases CFTR‐dependent iodide efflux and single‐channel activity in polarized Calu‐3 cells and HT29‐CL19A cells [75]. Such LPA₂‐NHERF2 inhibitory effect depends on compartmentalization of cAMP (Fig. 3). Given that LPA₂ signals through the G_i pathway (see below), the authors showed that specifically blocking this signaling pathway abrogates LPA₂ inhibition of CFTR, possibly by local inhibition of the adenylyl cyclase enzyme [75, 76]. Further data from mouse intestines suggest that this effect is strictly NHERF2‐dependent, given that the loss of NHERF1 or PDZK1 did not affect LPA₂‐mediated CFTR inhibition [77]. Thus, NHERF2 couples CFTR to an inhibitor signal transducer that limits the extent of its channel function activation by cAMP.

SHANK2, in turn, contains a PDZ domain along with a SH3 domain, a prolin‐rich region and a SAM domain that enables other protein–protein interactions [78]. Its interaction with CFTR was assessed in yeast two‐hybrid systems and its overexpression suppresses cAMP‐induced phosphorylation and activation of CFTR without changing the channel's PM half‐life in NIH‐3 T3 cells, indicating it intervenes in the cAMP regulatory axis of CFTR [79]. Also, SHANK2 knockdown (KD) increased CFTR‐dependent currents in T84 cells. Later it was shown that SHANK2 competes with NHERF1 for binding to CFTR C terminus, with both proteins having similar binding affinities to the channel's PDZ motif [80]. Importantly, SHANK2 was shown to colocalize and associate with phosphodiesterase 4 (PDE4) isoforms, which degrade cAMP, both in overexpression systems and in mice colonic crypts (Fig. 3). This indicates that SHANK2 provides a scaffold onto which cAMP degradative machinery limits the extension of this second messenger production and regulates CFTR activity, plausibly influencing the compartmentalization of this molecule in its physiological role [80].

PDZK1 has been reported to associate with CFTR at the apical membrane of polarized colonic HT29‐CL19A and T84 cells and gather the protein in a macromolecular complex with the multidrug resistance protein 4 (MRP4) channel, which can transport cAMP out of the cell [81]. This protein, in turn, inhibits the extent of CFTR activation, decreasing local cAMP levels through its efflux out of the cell, and PDZK1 is necessary for this MRP4‐derived inhibition effect. PDZK1 effectively couples both proteins at the cell surface—presumably, it may also participate in the compartmentalization by inhibiting diffusion of the second messenger. Interestingly, all PDZ proteins described to interact with CFTR participate in the cAMP regulatory axis of the channel; how they are all spatiotemporally coupled, however, is not well defined. Furthermore, their role in different tissues and the macromolecular complexes in which they participate have not been completely determined, leaving a gap of how and whether all these PDZ interactors are relevant in multiple locations across the body (reviewed in [76]).

cAMP regulation of CFTR: Function and stabilization at the PM

cAMP is a second messenger molecule in cells that transduce external signals from a plethora of signaling cues. It is synthesized by integral membrane proteins named adenylyl cyclases (ACs), which in turn are regulated by the activation of G‐protein‐coupled receptors (GPCRs). These receptors bind to external ligands and function as a GEF for trimeric GTP‐binding proteins (more commonly called G proteins), which are composed of α, β and γ gamma subunits—the α subunit can either activate AC (in which case it is called stimulatory, Gα_s) or inhibit it (Gα_i). The signaling cascade that emerges from activation of GPCRs is contingent and depends on the cell type or even on a particular cellular state [82]. The axis GPCR‐G protein‐AC is highly variable since a given GPCR can be coupled with different G proteins and different ACs, which generates a diversity of signaling avenues. Even though the overall consequence of every stimulatory cascade is cAMP increase, the effect also depends on signal compartmentalization, which plays a fundamental functional role. Indeed, a similar increase in cAMP can lead to drastically different outcomes depending on the spatial confinement of the generated cAMP pool, since it causes only a limited subset of effectors to become activated [83]. Surprisingly, such confinement, or cAMP domains, can be as small as a few tens of nanometers [84], and it is mainly dependent on two classes of proteins: cAMP phosphodiesterases (PDEs), that degrade cAMP into 5′‐AMP, and A‐kinase anchoring proteins (AKAPs). While PDEs are known for regulating compartmentalization possibly by limiting the diffusion of cAMP from its nanodomain, AKAPs are responsible for joining the pieces of cAMP signaling cascade together, giving rise to a specific cAMP signalosome. Even though the name is given after the most well‐known cAMP effector, protein kinase A (PKA), AKAPs are responsible for assembling member‐specific sets of cAMP effectors, ACs, kinases, PDEs, phosphatases, etc., in order to effectively convey the external stimuli recognized by the cell—making it so that local cAMP signaling is not dependent on random molecular collisions.

In epithelial cells, the GPCR β₂ adrenergic receptor (β₂AR) is responsible for regulating the cAMP pool responsible for CFTR function: this protein colocalizes with CFTR at the apical membrane (Fig. 3) and is co‐immunoprecipitated with the channel [85]. AC6, acting downstream of the G_s protein, was detected as a CFTR interactor and knockout of this adenyl cyclase leads to failure in forskolin‐induced swelling of intestinal organoids pointing to a specific role of AC6 in producing CFTR‐regulating cAMP pool [86]. Importantly, cAMP compartmentalization is also dependent on the actin cytoskeleton, since F‐actin depolymerization through latrunculin B was shown to increase the cytosolic cAMP pool at the expense of the cortical one even when PDEs were inhibited in lung epithelial cellular models [64]. Such cytoskeleton integrity also promotes localized subcortical PKA function: PKA activity and localization become distributed through the cytosol when cytoskeleton organization is disrupted, whereas intact cortical architecture supports the enrichment of PKA activity in subcortical regions. This demonstrates that the integrity of the actin cytoskeleton itself is essential for establishing and maintaining cortical cAMP microdomains, thereby enabling spatially restricted cAMP signaling at the plasma membrane. As mentioned before, the CFTR‐NHERF1‐ezrin axis regulates the cortical cytoskeleton, and this regulation has direct influence on the cAMP compartmentalization. NHERF1 overexpression and ezrin activation restore cAMP compartmentalization and cortical PKA activity in CFBE cells, and this effect is also observed upon expression of WT‐CFTR and by modulator rescue of F508del‐CFTR in this cellular model [64, 65]. Thus, the CFTR‐NHERF1‐ezrin axis promotes the necessary subcortical architecture for cAMP compartmentalization, and PM CFTR not only needs local cAMP pool to promote its function but also enables the very cellular structure that supports such compartmentalization in the first place (Fig. 1).

PKA and its anchoring in the vicinity of CFTR

After cAMP production, PKA acts as a cAMP effector in CFTR's function: it phosphorylates CFTR in serine residues mainly located in the R domain, allowing dimerization of the NBDs and ATP binding, which in turn drives the channel's gating by increasing open pore probability (Fig. 3). PKA binds directly to CFTR, and its binding has also been postulated to activate its function as a Cl⁻ channel [87, 88]. This interaction is mainly established by the AKAP function of ezrin, which binds PKA and places it in close proximity to CFTR [89]. Also, PKA's phosphorylation of CFTR is responsible for the intracellular binding of 14–3‐3 proteins, which stabilize CFTR's steady‐state levels when overexpressed and reduce the channel's PM stability upon knockdown [90]. PKA activation is also included in a negative feedback loop to decrease β₂AR‐derived cAMP production: PKA phosphorylates and activates PDEs (in particular, PDE4B and PDE4D), which in turn counterbalance cAMP production [91]. PKA and PDEs are coupled together through PI3Kγ, a PIP2 kinase that acts as an AKAP for the docking of PKA and its substrates in a kinase‐independent manner. Interestingly, PKA also phosphorylates PI3Kγ at a threonine residue, T1024, which inhibits its phospholipid kinase activity and leads to increased levels of β₂AR density in mice myocardium, showing that this phospholipase works as a negative regulator of β₂AR signaling [91]. The use of a PI3Kγ mimetic peptide (MP) that blocks the PKA‐PI3Kγ interaction resulted in a β₂AR‐induced rise in cAMP levels in 16HBE14o‐ cells, which specifically blocks PDE activation by PKA [92]. This cAMP rise was PM‐confined and increased both the amount of PKA‐phosphorylated CFTR and CFTR‐dependent Cl⁻ currents in polarized CFBE cells expressing WT‐CFTR, an effect similarly obtained with PDE4 inhibitors, confirming the negative feedback effect of PKA through PDE4 in this cell model. The effect of PI3Kγ MP occurs through the activation of PKD1, a known regulator of PM trafficking [93].

EPAC1 regulation of CFTR PM levels

Even though PKA action on CFTR regulates its function and trafficking, other cAMP signalosomes are also important for PM stabilization of CFTR. Low concentrations of cAMP elicit PKA‐mediated phosphorylation of the channel, while higher concentrations lead to EPAC1 signaling. EPAC1 is a GEF for the small GTPases Rap1 and Rap2 [94] which is activated upon binding of cAMP. Its activation culminates in increased CFTR PM stability through attenuation of its internalization. EPAC1 interacts with both CFTR and NHERF1 and this interaction is abolished by KD of the PDZ protein, suggesting that NHERF1 bridges EPAC1 and CFTR's interaction [94]. Interestingly, its effects on PM CFTR are ezrin‐independent, and EPAC1 still promoted increased steady‐state CFTR PM levels even upon ezrin KD. This suggests that EPAC1 acts independently of the cytoskeleton machinery elicited by the CFTR‐NHERF1‐ezrin axis and might work as a bypass to promote cortical cytoskeleton remodeling and CFTR stabilization. On this basis, Rap1 has been linked to increased integrin adhesion by promoting talin, a subcortical protein containing a N‐terminal FERM domain and a C terminus actin‐binding region, to move to the cell cortex and promote integrin activation [95]. This process is dependent on the Rap1 scaffold RIAM1, which seems to mediate the Rap1‐induced talin activation. Interestingly, and in parallel to the CFTR‐NHERF1‐ezrin axis, RIAM1‐dependent talin activation promotes F‐actin remodeling [96], suggesting that this cortical signaling axis might be involved in cytoskeleton maintenance and integral membrane protein regulation. EPAC1‐Rap1 has also been shown to regulate adherent junctions (AJ) in endothelial cells, and such regulation is cytoskeleton dependent [97]. Another study showed that EPAC1 activation leads to Rac activity, promoting increased cortical actin cytoskeleton and improving barrier function of endothelial cells, and Rac then inhibits RhoA and its downstream promotion of cell contractility through stress fiber formation [98]. Indeed, Rap effectors form a particular combination: numerous Rac GEFs (Tiam1, Vav2) and RhoA GAPs (Arap3, RA‐RhoGAP) have been identified to act downstream of Rap proteins and transduce signaling (specifically reviewed in [99]). It is curious how these processes parallel the Rac1 and RhoA activity in 16HBE41o‐ cells, in which RhoA activation leads to stress fibers while Rac1 improves cortical actin cytoskeleton and is directly linked to increased CFTR retention [12]. Thus, it is likely that EPAC1 activation effectively leads to major cytoskeleton remodeling that promote cortical F‐actin assembly, but further studies dissecting the role of EPAC1 and Rap1 signaling in epithelial cells should clarify this matter, as data on epithelial tissues is still scarce.

Cytoskeleton regulators involved in cAMP regulation of CFTR

EPAC1 activation recruits several cytoskeleton regulators. Among these, capping acting protein of muscle Z‐line alpha subunit 2 (CAPZA2) and inverted formin‐2 (INF2) might be involved in the cytoskeleton remodeling responsible for the EPAC1‐dependent increased CFTR PM stabilization (Fig. 4) [94, 100]. Indeed, it was found that knockdown of both these proteins culminates in opposite effects: INF2 knockdown increases CFTR PM levels while CAPZA2 knockdown decreases it, being the former a negative regulator of CFTR while the latter, a positive one (Fig. 4A) [100]. Also, INF2 interacts with CFTR in CFBE cells, and this interaction increases upon EPAC1 activation using its specific activator 007‐AM; CAPZA2, on the other hand, interacts with CFTR only upon EPAC1 activation—suggesting a cAMP‐driven role in the regulation of PM CFTR. The precise mechanism through which these cytoskeleton regulators promote different CFTR PM level outcomes and the link between these two proteins and cAMP signaling is not fully understood. CAPZA2 is a subunit of the CAPZ protein, and its main role is to limit actin polymerization by binding to the barbed end of actin filaments in a way that prevents new actin monomer from elongating filaments [101]. The fact that CAPZA2 is recruited to CFTR‐containing complexes upon EPAC1 activation suggests that it aids in promoting a proper actin cytoskeleton architecture elicited by the GEF's signaling [51]. Thus, CAPZA2 presence plausibly supports the underlying cytoskeleton remodeling, which agrees with CAPZA2 being a positive regulator of PM CFTR levels.

Fig. 4 — EPAC1 activation‐induced recruitment of cytoskeleton regulators INF2 and CAPZA2. (A) CFTR interaction axis with NHERF1 and ezrin anchors the channel to the cortical cytoskeleton, decreasing endocytosis. When EPAC1 becomes activated by increased cAMP levels, it induces increased CFTR retention at the PM and recruits the cytoskeleton regulators CAPZA2 (a subunit of a capping protein) and INF2 into a molecular complex with CFTR, which might be responsible for the increased EPAC1‐mediated stabilization of CFTR at the PM. Such effect might be associated with the CAPZA2 ability to stabilize F‐actin by preventing G‐actin loss and, thus, depolymerization, and with the INF2 ability to either polymerize or sever actin. EPAC1 leads to Rap1 activation, a Ras‐related small GTPase, which in turn could elicit cytoskeleton changes through RhoA, Rac1, and Ca²⁺ signaling, the latter being associated with INF2 activity modulation and cytoskeleton remodeling capability. (B) Since INF2 is a negative regulator of CFTR and its depletion increases core‐glycosylated CFTR levels, INF2 could influence CFTR's trafficking similarly to how it influences other integral membrane proteins. Also, its known role in CaAR could potentially be linked to how it promotes cytoskeleton remodeling through Ca²⁺ signaling.

INF2, in turn, is a peculiar formin containing typical FH2 and FH1 domains, an N‐terminus DID and a C‐terminal DAD that presents a greater homology to the WH2 actin‐binding domain than other formins [102, 103]. Interestingly, INF2 can sever F‐actin and this activity has been associated with its capacity for side binding actin polymers through INF2 oligomerization with F‐actin; the WH2/DAD domain then wedges between actin monomers, weakening monomer binding and disrupting the actin polymer [104]. Given INF2's negative regulation of CFTR, it is possible that EPAC1 activation inhibits its activity in the cortical region, where it could potentially disassemble the F‐actin structures. One process associated with INF2 activity is the calcium‐mediated actin reset (CaAR), in which, upon an intracellular Ca²⁺ spike, an actin ridge is formed led by INF2‐dependent actin polymerization around the nucleus and throughout the ER, with a consequent decrease in cortical actin [105]. Yet, whether and how EPAC1 activation specifically leads to INF2 regulation is unknown, and there is a major knowledge gap in how these two processes might be related. Interestingly, INF2 KD increases core‐glycosylated levels of F508del‐CFTR, also promoting an increase in the complex glycosylated form after CFTR rescuing in CFBE cells [100]. This suggests that INF2 might act early in CFTR biogenesis, perhaps targeting core‐glycosylated, ER‐localized CFTR (Fig. 4B). Indeed, INF2 has two main isoforms, one of which is ER‐localized due to the presence of a C‐terminal CAAX domain that becomes prenylated [106] and could potentially interfere with CFTR trafficking. Also, INF2 variants with altered DID domains have been linked to abnormal trafficking of membrane proteins, suggesting that it might regulate vesicle transport processes that influence CFTR ER‐exit and trafficking through the secretory pathway [107]. Thus, a hypothesis in which INF2 influences CFTR steady‐state levels at the ER is plausible, but further studies dissecting this mechanism are yet necessary to understand the interplay between these two proteins.

Regulation of CFTR membrane stability by phosphorylation

Phosphorylation of CFTR is a major regulator of its channel function but also of its PM stability (Fig. 5).

Lemur tyrosine kinase 2

Lemur tyrosine kinase 2 (LMTK2) has been reported to phosphorylate CFTR at Ser737 [108]. This protein is, despite its name, a serine/threonine kinase with an N‐terminal transmembrane domain, a cytosolic catalytic domain and a long C‐terminal cytosolic tail that participates in TGF‐β signal transduction [109]. It was shown to colocalize and co‐immunoprecipitate with CFTR at the apical side of polarized primary cultures of human bronchial epithelial (HBE) cells. Depletion of LMTK2, or inhibition of its kinase activity, increased both PM CFTR levels and CFTR‐dependent currents, and decreased the channel's endocytosis rate, showing that LMTK2 works as a negative regulator of CFTR PM stability. Later, it was shown that TGF‐β1 increases apical levels of LMTK2 in polarized CFBE41o‐ cells and in primary HBE cells without increasing mRNA levels [110]. TGF‐β1 specifically increases Rab11‐mediated transport of LMTK2 to the PM, thereby increasing CFTR phosphorylation and LMTK2‐mediated surface level decrease of the channel. Interestingly, LMTK2 was described as a major TGF‐β1 pathway activator by inhibiting TβR‐II dephosphorylation by protein phosphatase 1 (PP1). LMTK2 phosphorylates the catalytic subunit PP1c and inhibits it, which allows TGF‐β1 signal transduction through SMAD3 phosphorylation and nuclear translocation [111]; TGF‐β signaling has been shown to have a critical role in lung fibrosis in chronic obstructive pulmonary disease, also aggravating CF [112, 113]. Additionally, TGF‐β1 has been shown to decrease CFTR mRNA and protein levels, impairing functional rescue of F508del‐CFTR [114]. Thus, LMTK2 inhibition, which plays a role both directly in CFTR PM levels and in TGF‐β1 signaling, is a potential therapy target to increase CFTR PM levels of rescued disease‐causing CFTR variants, such as F508del‐CFTR.

Spleen tyrosine kinase

Spleen tyrosine kinase (SYK) has also been implicated in modulating CFTR levels at the PM. It co‐immunoprecipitates with CFTR and phosphorylates it at Y512 within a SYK recognition motif in NBD1 (consensus Y(D/E)(D/E)X). Phosphorylation at this residue leads to both decreased maturation and steady‐state surface levels of the channel [115, 116]. Interestingly, Y512E‐CFTR, a phosphomimetic mutation, is enough to reduce the channel's surface steady‐state levels indicating that this phosphorylation works as a signal cue that decreases CFTR PM residency [115]. This effect of SYK was confirmed in CFBE41o‐ cells, showing it influences CFTR PM levels in a physiologically relevant model. Later, the phosphotyrosine‐based (pY) motif phosphorylated by SYK was indicated to be recognized by SHC1, a SH2 and PTB domain‐containing protein that binds to pY cues in several membrane proteins [117]. A CFTR‐context pY512 motif pull‐down assay identified several putative interactors of CFTR that could regulate the SYK‐phosphorylated channel, among which ACTB and SHC1 were the most relevant interactors identified [118]. SHC1 KD, then, counteracted SYK phosphorylation by reverting the effect of SYK and sustaining PM CFTR levels [118]. Also, depletion of both SHC1 and SYK doubled CFTR‐dependent iodide influx rate, and SCH 1 depletion increased PM CFTR levels by a similar extent, strongly suggesting it participates in the SYK pathway.

SYK‐dependent phosphorylation of CFTR was inhibited in vitro by the presence of WNK4 (with‐no‐lysine 4), a serine/threonine kinase that has kinase‐independent roles in regulating several ion channel levels. Furthermore, WNK4 overexpression increases CFTR PM levels and Cl⁻ current in BHK cells, suggesting it inhibits SYK to increase surface levels of CFTR [115, 119]. Its effects on the channel were largely kinase‐independent, since a kinase‐dead mutant also promoted increased CFTR steady‐state surface levels. Co‐immunoprecipitation experiments showed that WNK4 interacts with SYK, suggesting that the former could sequestrate SYK and block CFTR phosphorylation by it. Interestingly, WNK proteins WNK1 and WNK4 were shown to inhibit CFTR Cl⁻ currents in Xenopus leavis oocytes; however, through different mechanisms: WNK1 did not affect CFTR PM levels and its inhibition was kinase‐dependent, suggesting a direct role in phosphorylating either CFTR or one of its upstream regulators; conversely, WNK4's inhibition was largely kinase‐independent and reduced CFTR PM levels [120]. These results directly contrast the reversal of SYK modulation of CFTR caused by WNK4 and could be a consequence of the cellular model used. Beyond that, it has been reported that individuals bearing WNK4 disease‐causing variants exhibit changes in nasal potential differences and sweat conductivity similar to the ones observed in individuals with CF, suggesting an inhibitory role of WNK4 in CFTR activity [121]. Thus, even though current data on WNK4 implications in CFTR activity are unclear, it definitely participates in the regulation of surface levels of CFTR which highlights it as a potential target for increased CFTR levels of disease‐causing variants.

Casein kinase II

Casein kinase II (CK2), a ubiquitous and constitutively active Ser/Thr kinase, was shown to modulate not only CFTR activity but its biogenesis and trafficking. It is a heterotetrametric kinase with two catalytic (α) and two regulatory (β) subunits and it has been shown to colocalize with WT‐CFTR in the apical membranes of epithelial cells, as well as to be co‐immunoprecipitated with the channel in airway epithelial cells [122]. CK2 inhibition with tetrabromobenzotriazole (TBB) led to decreased CFTR‐dependent Cl⁻ currents and prolonged closed reduced open pore probability in Xenopus oocytes and in colonic and airway epithelium; consistently, TBB treatment inhibits cAMP‐induced CFTR‐dependent $HC O_{3}^{-}$ transport in guinea pig pancreatic ducts, indicating that this kinase is necessary for proper CFTR function and positively regulates CFTR [116, 122]. CK2 binds specifically to the WT‐CFTR peptide encompassing the F508 residue, and this peptide inhibits CFTR‐dependent Cl⁻ currents by two‐fold in the presence of CK2 in Xenopus oocytes [122]. Also, CK2 inhibition both increased the turnover of immature WT‐CFTR and decreased its maturation [116]. Previous data suggested that S511 in CFTR, which resides in a CK2 phosphorylation motif (consensus S/TXXE/D), was phosphorylated by this kinase; surprisingly, S511A and S511D did not elicit any change in CFTR function or maturation, demonstrating that CK2 does not act through this residue. Nonetheless, S511D‐CFTR was uncoupled from TBB inhibition, suggesting that this residue might participate in the association of CK2 with CFTR [116, 122]. On another study, mass spectrometry analysis of NBD1 digested peptides after CK2 phosphorylation in vitro revealed that S511 is not a CK2 target, while residues S422, S432, and S627 were identified as being phosphorylated [122, 123]. Interestingly, even though mutations of the S422 residue to either A or D did not elicit any change in CFTR maturation, S422D‐CFTR particularly increased forskolin/IBMX‐activated whole cell currents in CFTR‐expressing Xenopus oocytes and increased TBB sensitivity of CFTR conductance. On the other hand, S422A reversed these effects, suggesting that S422 phosphorylation by CK2 is relevant for CFTR function regulation [116]. Yet another phosphorylation site in CFTR by CK2, T1471, was identified [116]: the T1471A mutation decreased steady‐state levels of band C and decreased CFTR maturation, while T1471D completely abolished band C appearance. Also, T1471A decreased CFTR‐dependent Cl⁻ currents, which agrees with the positive regulation of the channel by CK2. Nonetheless, T1471D did not bypass TBB inhibition of CFTR currents, suggesting additional regulatory layers still play a role in CK2 regulation of CFTR function [116]. In another study, a C‐terminal CFTR peptide containing the T1471 residue was shown to be readily phosphorylated by CK2 holoenzyme, and its phosphorylation needed the regulatory β subunit [124]. Overall, it is not clear how CK2 mechanistically regulates CFTR; however, it directly interferes with several processing steps of the channel, highlighting the potential of this kinase to modulate CFTR function. Noteworthy, the TBB inhibitory effect on CK2 was lost in excised patches experiments, supposedly because the protein–protein interaction architecture around CFTR, which might be necessary for proper CK2 targeting of CFTR, is compromised. [122]. In general, the specific mechanism by which CK2 regulates CFTR has not been established, and whether and how it might modulate intracellular and cell surface CFTR levels and activity might reveal another target for increasing disease‐causing CFTR variant levels at the PM.

F508del‐CFTR: at the basis of CF

Although the processes described above were largely characterized using WT‐CFTR, they collectively define the regulatory framework that governs the post‐Golgi fate of the protein at the plasma membrane. Endocytic turnover, recycling efficiency, ubiquitin‐dependent sorting, cytoskeletal tethering, and phosphorylation‐dependent gating together determine the surface stability and functional output of CFTR. The relevance of this regulatory network becomes particularly evident in the context of disease‐causing variants. Among them, F508del, the most prevalent mutation associated with CF, not only disrupts CFTR early biosynthetic folding but also profoundly alters the post‐Golgi behavior of the channel. Increasing evidence indicates that even when rescued to the cell surface, F508del‐CFTR displays reduced plasma membrane stability, accelerated endocytosis, impaired recycling, and enhanced targeting to ubiquitin‐dependent degradation pathways [125]. In this context, the post‐Golgi regulatory mechanisms discussed in the previous sections become critical determinants of mutant channel fate and represent important targets for therapeutic intervention.

F508del‐CFTR causes CF due to the intrinsic structural defect elicited by the deletion of the F508 residue. This residue is important for the TMD2‐NBD1 interface, lying at a socket present in NBD1 that works as a base for TMD2 contact [126]. The end result of F508 deletion is, thus, a CFTR variant that never achieves its native folded state, residing in an intermediate, misfolded one. Studies revealed that F508del‐CFTR NBD1 folding is severely impaired when compared to that of the WT counterpart, and the full‐length protein is unable to achieve a mature conformation that is necessary for progression down the trafficking route. While a great portion of WT‐CFTR is degraded in an intermediate folding at the ER, F508del‐CFTR is unable to leave such conformational state, leading to its increased target for proteasomal degradation and impaired trafficking [5, 127]. The ER quality control (ERQC) is decisive in the fate of the mutated protein: the Hsp70 chaperone family and the co‐chaperone Hsp40 protein family are the main responsible for the early targeting of F508del‐CFTR to proteasomal degradation at the ER [128, 129]. Because of this strict folding itinerary, F508del‐CFTR is precluded from achieving the PM where it could regulate epithelial ion flux. Even though this CFTR variant withholds a folding defect, it still displays a residual channel function that could promote Cl⁻ flux should the folding checkpoints be overcome [130].

Currently, there are ways to tackle the intrinsic folding defect in class II variants of CFTR, specially F508del‐CFTR. These therapies consist of small molecules, termed correctors, that can bind CFTR and correct the folding defect, allowing the protein to bypass ERQC and achieve the PM for proper function [131]. Lumacaftor (VX‐809) was the first corrector approved as a therapy, in combination with potentiator ivacaftor (VX‐770), for people with CF (pwCF) carrying at least one F508del‐CFTR allele [132]. Later, tezacaftor (VX‐661), a similar compound, showed a more favorable safety profile and is used in the current triple therapy involving the correctors elexacaftor (VX‐445) and tezacaftor (double combination, ET) and the potentiator ivacaftor (triple combination, ETI) [133]. Both VX‐809 and VX‐661 bind to TMD1 and are considered type I correctors, a category that refers to pharmacological chaperones that stabilize interdomain interactions (TMDs‐NBDs) [131]. VX‐445, in turn, is considered a type III corrector because of its capacity to stabilize NBD1 folding. This comes from its binding to TM2, TM10, and the lasso motif, which appears to stabilize the interaction between TMD2 and NBD1, directly influencing NBD1's folding [134]. More recently, a novel triple combination including tezacaftor, vanzacaftor and deutivacaftor was approved both in Europe and in the US [135].

Even though these therapies successfully rescue F508del‐CFTR from the ERQC and allow its translocation to the PM, they still fail to restore mutant CFTR levels to those of WT‐CFTR [6]. At the PM, rescued F508del‐CFTR shows a reduced stability and has a half‐life of only 1 h, opposed to the approximately 3‐h half‐life of its WT counterpart [6]. These differences are accounted for by the different regulatory cues that target these two variants. Rescued F508del‐CFTR still presents a defective folding, and this is recognized by the peripheral protein quality control system. The general folding machinery is a major player in this system, including the Hsp70 and Hsp90 chaperone family proteins, and co‐chaperones such as BAG1, AHA1, Hsp40, and HOP that assist in CFTR recognition [38]. These proteins deliver CFTR to the ubiquitination machinery at the cell surface, which is mainly carried out by the E3 ligases CHIP (through Hsp70 proteins), c‐Cbl, and RFFL (which directly recognizes conformationally defective CFTR proteins), which typically ubiquitinate post‐endocytic F508del‐CFTR [37, 41]. This variant is extensively ubiquitinated in the cell periphery when compared to WT‐CFTR, and this leads to a route shift from the recycling pathway to the lysosomal degradation one [36, 37]. As mentioned above, a study proposed that ubiquitination does not influence the CFTR internalization rate [36]. They showed that, like in the WT‐CFTR scenario, AP2 and DAB2 are necessary for its endocytosis in airway epithelial cells, indicating that the endocytic machinery responsible for both channel's internalization is the same; also, CHIP depletion did not change rescued F508del‐CFTR endocytosis, but increased surface stability, suggesting that the variant's endocytosis is largely independent from the ubiquitin machinery. Furthermore, endocytosis inhibition through Dyngo‐4a decreases rescued F508del‐CFTR ubiquitination over time to the same extent as CHIP depletion, strongly suggesting that ubiquitin modifications take place at the endosomal compartment [41]. A later study, however, showed that a CFTR‐Ub construct possesses an increased internalization rate in CFBE cells, which was comparable to that of rescued F508del‐CFTR; also, F508del‐CFTR displayed decreased recycling back to the PM in these cells, suggesting that both processes are relevant to explain the decreased PM half‐life of rescued F508del‐CFTR [37]. Thus, even though some conflicting results might be related to the specific cellular model used, it is not yet a consensus whether both endocytosis and recycling are decisive in modulating the PM half‐life of CFTR. Interestingly, the authors found that different ubiquitin modification patterns are present in rescued F508del‐CFTR at the PM: both K48‐ and K63‐linked polyubiquitin chains were found at F508del‐CFTR molecules, and each of them is associated with distinct cellular processes [37].

As mentioned, cytoskeleton anchoring through the CFTR‐NHERF1‐ezrin axis is decisive for CFTR surface levels, and F508del‐CFTR‐expressing airway epithelial cells present defects at this regulatory mechanism. Indeed, F508del‐CFTR was shown to interact with NHERF1 less efficiently than its WT counterpart, and VX‐809 rescuing increased such interaction [136]. Also, CFBE41o‐ cells expressing F508del‐CFTR displayed lower NHERF1 expression levels when compared to 16HBE41o‐ cells expressing WT‐CFTR, and NHERF1 overexpression, as mentioned, improves F508del‐CFTR surface expression and restores CFTR‐dependent Cl⁻ currents [57]. This effect is abolished when the NHERF1‐ezrin interaction is disrupted after NHERF1 overexpression, suggesting that F508del‐CFTR is somehow unable to integrate this regulatory axis which stabilizes its levels at the PM. Even though Rac1 and RhoA were discovered to participate in the NHERF1‐ezrin axis downstream regulation of the cytoskeleton, only Rac1 activity elicited increased F508del‐CFTR cell surface levels [12]. Also, active‐dominant Rac1 increased fully glycosylated CFTR band C levels while no change was observed in levels of the immature CFTR band B, indicating that only post‐Golgi CFTR is being affected by NHERF1 overexpression and consistent with a decreased turnover of fully glycosylated F508del‐CFTR. It could be the case that while Rac1 promotes PM retention, which decreases F508del‐CFTR internalization and subsequent ubiquitination, RhoA, by promoting recycling, is not able to modulate cell surface levels of this variant since F508del‐CFTR is recognized within endosomes and quickly targeted for lysosomal degradation; thus, inhibiting the channel's internalization opens a new therapeutic window to promote increased PM levels of this variant in pwCF under treatment with modulators.

In this sense, many molecular players were identified to have an effect in the PM levels of F508del‐CFTR. CAL depletion, for instance, increases PM F508del‐CFTR, and synthetical peptides that disrupt the F508del‐CFTR‐CAL interaction promote increased Cl⁻ currents and the efficiency of rescuing by pharmacological chaperones [137, 138]. LMTK2 depletion facilitated VX‐809 rescuing of the mutant channel, increasing its processing efficiency and Cl⁻ currents [108]. Another binding partner of F508del‐CFTR that modulates its activity is YAP1: it binds the channel through NHERF1 and recruits the YES kinase which, in turn, phosphorylates SHC1 [139]. Interestingly, interference with one of these components decreases both SHC1 phosphorylation and delays F508del‐CFTR internalization, linking YES1‐mediated phosphorylation of SHC1 to rescued F508del‐CFTR PM stability. Consistently, a dominant‐active Ras protein bypasses the p‐SHC1 promotion of Ras activation and leads to MAPK kinase signaling, which culminates in increased CFTR internalization [139]. The precise mechanism of MAPK‐induced CFTR internalization is unknown, but could be a potential target for increased CFTR PM retention. In F508del‐CFTR, the non‐phosphorylatable variant F508del‐CFTR Y512A displayed increased Cl⁻ currents conductance and was rendered sensitive to CK2 activity, and CK2 inhibition by TBB decreased this variant's conductance in Xenopus oocytes [116]. This contrasts with the known inability of SYK to phosphorylate F508del‐CFTR due to the misfolding caused by F508 deletion, and to the fact that CK2 did not show any regulatory role in F508del‐CFTR‐derived currents, as opposed to the WT counterpart [122]. Interestingly, increased Cl⁻ currents were also observed in CFTR bearing only the Y512A mutation, which suggests that the absence of modulation of this site's phosphorylation state might influence the conductance of both variants [116]. Calpain‐1 (CAPN1), a cysteine protease that belongs to a group of calcium‐sensitive cysteine proteases that are ubiquitously expressed in human cells and associated with subcellular organelles such as the cytoskeletal actin filaments, vesicles, and the PM [140], has been previously shown to modulate rescued F508del‐CFTR steady‐state PM levels by degrading apical ezrin, a known target of this enzyme [141]. CAPN1 depletion increased F508del‐CFTR PM levels and derived Cl⁻ currents and restored the interaction between F508del‐CFTR and ezrin through NHERF1. Modulating the activity of CAPN1 could potentially lead to a therapeutical complementation to the current modulator therapy for improving CFTR function in CF patients. EPAC1 activation was also observed to increase the levels of VX‐809‐rescued fully glycosylated F508del‐CFTR, which was coupled to higher Cl⁻ conductance [94]. As mentioned, the apparent downstream EPAC1 effector INF2 also seems to modulate F508del‐CFTR levels, since its depletion causes a 7‐fold increase in fully glycosylated CFTR, but also in core‐glycosylated CFTR band B [100].

Therapeutic opportunities

The large set of information on the intricate processes that regulate CFTR stability (and thus abundance) at the PM—and in many cases how they are impaired in the presence of disease‐causing variants such as F508del—triggers the possibility of identifying additional strategies that can be used in combination with currently available modulators to boost their effect. Below we highlight some of the aspects that, from our perspective, are more realistically targetable and how/if they are being pursued:

Modulation of PKA anchoring. As mentioned above, the use of a PI3Kγ mimetic peptide that blocks the PKA‐PI3Kγ interaction leads to an increase in cAMP levels and can increase CFTR levels and function at the PM through a mechanism that is additive to the modulators [93]. Considering the previous testing of this peptide in animal models, this is a plausible approach to improve CFTR PM levels and function [93].
Activation of EPAC1. Considering that activation of EPAC1 increases CFTR levels and function at the PM and is additive to modulators [94], it is likely that an EPAC1 activator could be beneficial. Although there are currently no activators approved for clinical use, EPAC1 is considered a promising therapeutic target for treating vascular inflammation, endothelial dysfunction, and fibrosis. Several compounds are currently in the preclinical or research phase [142], although caution needs to be taken considering the role of EPAC1 in the heart.
Another possibility is to target kinases that regulate CFTR PM levels. Regarding SYK, fostamatinib is the first‐in‐class FDA‐approved oral SYK inhibitor, primarily used for immune thrombocytopenia [143]. LMTK2 is also a promising target in several conditions including prostate cancer, neurodegenerative diseases (like Alzheimer's), and as a tool for male contraception, and its inhibition could be additive to CFTR modulators (and even regulate inflammatory signaling in CF), but there are currently no inhibitors in clinical use.
Activation of small GTPases of the Rac1 family by hepatocyte growth factor (HGF) has also been proposed to synergize with CFTR modulators [12] but this must be taken with caution considering it can also act as an oncogenic driver.

In the context of exploring CFTR stability to improve current therapies and define novel approaches, it is important to mention that the pathological environment of the CF lung can impair CF regulation at the plasma membrane. Among these, infection with Pseudomonas aeruginosa, the most common CF‐associated pathogen, may have a detrimental effect on CFTR restoration by available therapies. In fact, infection with this pathogen inhibits CFTR recycling, limiting the apical membrane expression of both WT‐ or rescued F508del‐CFTR [144], an effect that may be due to the impact of Cif (CFTR inhibitory factor), a virulence factor secreted by P. aeruginosa, that induces CFTR ubiquitination [145, 146].

Concluding remarks

The regulation of CFTR steady‐state levels at the plasma membrane is a complex process that involves multiple protein partners and connects CFTR to major signaling pathways through the actin cytoskeleton (summarized in Table 1). Although the molecular details of many aspects are not fully understood yet, data from multiple studies allow the “drawing” of a comprehensive picture that can be modulated to regulate PM retention of the protein and thus has the potential to lead to additional therapeutic opportunities by targeting this still unexplored element of CFTR trafficking in epithelial cells.

Author contributions

CMF and JPO conceived the publication and discussed the content. JO wrote the first draft and prepared figures. CMF prepared the final version.

Acknowledgments

Work in C.M.F.'s laboratory is supported by grants from Fondazione Fibrosi Cistica Ricerca (FFC#2/2023) and the Cystic Fibrosis Foundation (FARINH24G0). Additional funding was provided to BioISI by Fundação para a Ciência e a Tecnologia (FCT), Portugal, under center grant UID/04046/2025.

Edited by Felix Wieland

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