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Macromolecular complexes of cystic fibrosis transmembrane conductance regulator and its interacting partners

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF). CFTR is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. CFTR chloride channel belongs to the superfamily of the ATP-binding cassette (ABC) transporters, which bind ATP and use the energy to drive the transport of a wide variety of substrates across extra- and intracellular membranes. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, or transporters, in addition to its role as a chloride conductor. The molecular assembly of CFTR with these interacting proteins is of great interest and importance because several human diseases are attributed to altered regulation of CFTR, among which cystic fibrosis is the most serious one. Most interactions primarily occur between the opposing terminal tails (N- or C-) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. These dynamic interactions impact the channel function as well as the localization and processing of CFTR protein within cells. This review focuses on the recent developments in defining the assembly of CFTR-containing complexes in the plasma membrane and its interacting proteins.

Introduction

The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF), and this lethal genetic disease affects 1 in every 2500 Caucasians in the United States. CFTR is an integral membrane glycoprotein composed of 1480 amino acids (Welsh et al., 1995).

CFTR is primarily localized to the lumen-facing, or apical, membranes of epithelial cells in the airway, intestine, reproductive tissues, and exocrine glands (such as sweat glands, exocrine pancreas, and salivary glands). It functions as a cAMP-regulated Cl channel that is responsible for transepithelial salt and water transport (Quinton, 1983, Anderson et al., 1991, Bear et al., 1992). As its name implies, in addition to functioning as a conductor of Cl, CFTR also acts as a conductance regulator, exerting modulatory influences over a plethora of other ion channels, transport proteins, and processes, such as the epithelial Na+ channel (ENaC; Knowles et al., 1983, Boucher et al., 1986, Stutts et al., 1995), the outwardly rectifying chloride channel (Gabriel et al., 1993, Jovov et al., 1995, Schwiebert et al., 1995), apical K+ channels from renal epithelial cells renal outer medullary potassium channel (ROMK) 1 and ROMK2 (McNicholas et al., 1996, McNicholas et al., 1997, Cahill et al., 2000), aquaporin water channels (Schreiber et al., 1999), Cl/HCO3 exchangers (Lee et al., 1999), and ATP release mechanisms (Reisin et al., 1994, Sugita et al., 1998; for review, see Schwiebert et al., 1999).

CFTR is a symmetrical, polytopic protein that belongs to the superfamily of the ATP-binding cassette (ABC) transporters, which bind ATP and use the energy to drive the transport of a wide variety of substrates across extra- and intracellular membranes (Dean et al., 2001). CFTR consists of 2 repeated motifs, each composed of a hydrophobic membrane-spanning domain (MSD) containing 6 helices (transmembranes [TM]) and a cytosolic hydrophilic region for binding with ATP, that is, nucleotide binding domain (NBD; also called nucleotide binding fold [NBF]; Fig. 1; Riordan et al., 1989). These 2 motifs are linked by a cytoplasmic regulatory (R) domain, which contains a number of charged residues and multiple consensus phosphorylation sites (substrates for various protein kinases, such as PKA, PKC, cGMP-dependent protein kinase II [cGKII], etc.). Both the amino (N) and carboxyl (C) terminal tails of this membrane protein are cytoplasmically oriented and mediate the interaction between CFTR and a variety of binding proteins, as will be discussed in Section 3.

Attempts have been made to solve the 3-dimensional structure of CFTR. Recently, the high-resolution crystal structures for mouse NBD1 (mNBD1) have been determined (Lewis et al., 2004). The crystal structures of mNBD1 have features that distinguish them from other ABC proteins in that they have added regulatory segments, a foreshortened subdomain interconnection, as well as an unusual nucleotide conformation (Lewis et al., 2004). Human NBD1 is expected to be very similar in structure to mNBD1, as these molecules share 78% sequence identity. More recently, a low-resolution (∼ 2 nm) structure of recombinant human full-length CFTR has been reported using conventional purification techniques and the formation of 2-dimensional crystalline arrays (Rosenberg et al., 2004). The crystallized CFTR shows an overall architecture for 2 different conformational states and demonstrates a strong structural homology to another eukaryotic ABC transporter (P-glycoprotein). The 2 conformational states of the human CFTR can be observed in the presence of nucleotide, suggesting the open and closed states of the chloride channel (Rosenberg et al., 2004). However, the high-resolution structure of human full-length CFTR by X-ray diffraction is yet to be determined before further insight can be gained into the mechanism of CFTR action.

The biological significance of the CFTR chloride channel is demonstrated by the fact that several human diseases are attributed to altered function of CFTR, among which cystic fibrosis and secretory diarrhea are the 2 major disorders.

Cystic fibrosis (CF) is a lethal autosomal recessive human genetic disease and is most common among the Caucasians. CF is caused by the loss or dysfunction of the CFTR Cl channel activity resulting from the mutations (sequence alterations) that decrease either the biosynthesis or the ion channel function of the protein (Cheng et al., 1990, Welsh & Smith, 1993). The absence or dysfunction of CFTR chloride channel leads to aberrant ion and fluid homeostasis at epithelial surfaces in a variety of tissues or organs, including the lung, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive ducts (Zielenski & Tsui, 1995). In the lung, the defect in chloride transport is coupled with hyperabsorption of sodium, as well as the generation of thick and dehydrated mucus and subsequent chronic bacterial infections (such as Pseudomonas aeruginosa). This leads to bronchiectasis and progressive airway destruction. Other symptoms include, but are not limited to, pancreatic insufficiency, meconium ileus, and infertility (Welsh et al., 1995). The pulmonary manifestations of CF are responsible for substantial morbidity, and more than 90% of CF-related mortality. Currently, lung transplantation is the only effective therapy for these patients.

The most common CF mutation is the deletion of 3 nucleotides, resulting in the deletion of a single phenylalanine (F) residue at position 508 (ΔF508) on the protein molecule (Kerem et al., 1989, Davis et al., 1996), and is responsible for ∼ 70% of CF alleles (Tsui, 1995). The ΔF508 CFTR mutant is associated with a severe form of the disease, with more than 90% of CF patients having at least 1 ΔF508 allele. It is estimated that approximately half of the CF patients are homozygous for the mutation ΔF508. This allele encodes an unstable and inefficiently folded CFTR protein, the major consequence being the failure of the mutant protein to be correctly processed and delivered to its proper cellular location in the plasma membrane (Cheng et al., 1990, Thomas et al., 1992, Welsh & Smith, 1993). As a result, the mutant protein is retained in the endoplasmic reticulum (ER) and rapidly targeted for degradation (Cheng et al., 1990, Ward et al., 1995). The investigations into the mechanisms underlying the biosynthesis, trafficking, and degradation of ΔF508 CFTR have already provided a unique opportunity to understand the pathogenesis of this inherited disorder at the molecular and cellular levels (for review, see Kopito, 1999).

Another major disorder involving CFTR is secretory diarrhea, which is caused by excessive activation of this chloride channel in the gut (Field et al., 1972, Gabriel et al., 1994). The importance for the role of CFTR in secretory diarrhea is demonstrated by the fact that bacterial toxins fail to induce secretory diarrhea in CF mice (Gabriel et al., 1994, Cuthbert et al., 1995). Intestinal colonization by pathogenic microorganisms is a major cause for acquired secretory and inflammatory diarrhea (Hyams, 2000, Paton et al., 2000). Several species of bacteria induce secretory and inflammatory diarrhea, including E. coli, Shigella flexneri, Salmonella typhimurium, and Vibrio cholerae. CFTR resides at the apical membranes of secretory epithelial cells lining the lumen of the gut, where it is normally inactive (Frizzell & Halm, 1990). Due to the exposure to enterotoxins, intracellular second messengers (cAMP and/or cGMP) are generated, that lead to overstimulation of the secretory pathway by activating luminal CFTR (for reviews, see Sears & Kaper, 1996, Barrett & Keely, 2000). The activation of the CFTR channel by excessive phosphorylation (due to excessive cAMP and/or cGMP) leads to Cl secretion across the epithelium, which, in turn, increases the electrical and osmotic driving forces for the parallel flows of Na+ and water, respectively. Therefore, the net result is the robust secretion of fluid and electrolytes across the epithelium into the gut lumen, namely, secretory diarrhea and the resultant dehydration, which can be fatal if untreated (Fig. 2; Field, 1971, Clarke et al., 1992). In parallel, electroneutral absorption by Na+/H+ exchanger and electrogenic absorption by ENaC are inhibited (Yun et al., 1997, Kunzelmann & Schreiber, 1999). Cholera toxin and heat-labile E. coli toxin induce intestinal fluid secretion by excessive increase in intracellular cAMP, due to irreversible activation of the adenylate cyclase resulting from the ADP ribosylation of the α-subunit of a stimulatory G protein, Gsα, by the toxins (Kimberg et al., 1971, Kantor, 1975). Moreover, cholera toxin increases intracellular cAMP in both crypts and villus epithelial cells, and thus, both compartments are likely to contribute to the generation of secretory diarrhea (De Jonge, 1975). Other toxins, such as heat-stable E. coli toxin or Y. enterocolitica toxin, enhance intracellular cGMP, leading to the stimulation of cGMP-dependent protein kinase II (cGKII), an apical membrane-targeted kinase that efficiently phosphorylates CFTR, resulting in the activation of Cl secretion in crypts and apical membranes of the intestine (Fig. 2; French et al., 1995, Swenson et al., 1996, Lohmann et al., 1997, Vaandrager et al., 1998, Vaandrager et al., 2000).

Because CFTR plays the central role in certain forms of secretory diarrhea as described above, it has been argued that patients heterozygous for the CF defect have a genetic advantage because of their limited secretory responses to infections by some bacteria or viruses (Baxter et al., 1988, Taylor et al., 1988, Cuthbert et al., 1994, Cuthbert et al., 1995, Gabriel et al., 1994, Quinton, 1994, Grubb & Boucher, 1999). On the other hand, it seems reasonable to propose that blocking luminal CFTR Cl channels would be the appropriate treatment for these forms of secretory diarrhea. Several studies reported new approaches to identify promising specific blockers of CFTR by high throughput screening (Schultz et al., 1999, Galietta et al., 2001). A chromanole compound 293 B was reported to block basolateral cAMP-dependent KvLQT1 K+ channels, which play essential roles in maintaining the electrical driving force for luminal Cl secretion (Lohrmann et al., 1995). These blockers could also be useful for the treatment of secretory diarrhea, because they inhibited the equivalent short-circuit current induced by prostaglandin E2, vasoactive intestinal polypeptide, adenosine, cholera toxin, and cAMP in the distal rabbit colon from both the mucosal and the serosal sides of the epithelium and show fairly low IC50 values (Lohrmann et al., 1995). Gabriel et al. (1999) identified a novel inhibitor of cAMP-mediated fluid and chloride secretion, SP-303, which is derived from the latex of the plant Croton lechleri. This naturally occurring latex has been used by the indigenous people of South America to treat various kinds of watery diarrheas, including diarrhea caused by cholera. Gabriel et al. (1999) demonstrated that SP-303 is effective against in vivo cholera toxin-induced fluid secretion and in vitro cAMP-mediated Cl secretion. A potent and selective small-molecule CFTR inhibitor (CFTRinh-172) was identified recently by Verkman's group (Ma et al., 2002, Thiagarajah et al., 2004) by high-throughput screening. CFTRinh-172 inhibited CFTR-mediated chloride transport, as well as intestinal fluid secretion induced by cholera toxin and STa E. coli toxin in animal models. Most recently, we found that lysophosphatidic acid, a naturally occurring phospholipid in blood and food, efficiently inhibited cholera toxin-induced CFTR-dependent secretory diarrhea in mice (Li et al., submitted for publication).

The goal of this article is to review new findings and developments in our understanding of the cellular and molecular aspects of protein–protein interactions involving CFTR chloride channel. It is beyond the scope of this review to discuss all the interactions and their regulation involving CFTR. Therefore, our focus is on the recent progress made in the area of CFTR-containing complexes in the plasma membrane and a few of its interacting proteins.

Section snippets

Cystic fibrosis transmembrane conductance regulator quaternary structure

Controversy remains regarding the functional form of CFTR in the plasma membrane. Investigations applying biochemical techniques (co-immunoprecipitation of different CFTR mutants or CFTR with different tags), electrophysiological techniques (patch-clamp studies), as well as electrophoretic studies (using nondenaturing gels), argued that the functional form of CFTR is a monomer. Marshall et al. (1994) concluded that CFTR existed primarily as a monomer in the membrane because coexpressed

Interactions between cystic fibrosis transmembrane conductance regulator and its binding partners

Accumulating evidence has been documented to demonstrate the existence of direct or indirect interactions between CFTR and a wide variety of proteins, including transporters, ion channels, kinases, phosphatases, and cytoskeletal elements. Among these reported interactions, many are mediated through a physical interaction between these binding proteins with the opposing tails of the CFTR chloride channel, as will be discussed briefly in 3.1 Cystic fibrosis transmembrane conductance regulator

Macromolecular complex assembly of cystic fibrosis transmembrane conductance regulator and its interacting partners

It is now well documented and accepted that the formation of multiprotein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. One interesting feature of epithelial cells is that signals originating at either the apical or basolateral cell surfaces do not always lead to detectable changes in the concentration of specific second messengers (cAMP, cGMP, or Ca2+, etc.), although the cellular response is significantly

Currents limitations and future directions

In summary, CFTR interacts with a wide variety of proteins, physically and functionally. The unique properties of CFTR proteins, being a chloride channel, a regulator of other channels, and important in the pathogenesis of many serious human diseases, make it an important target for structural and functional studies. It is suggested that protein–protein interactions that influence the expression or functional activity of the CFTR channel at plasma membrane would provide additional layers of

Acknowledgments

We thank all the members of our laboratory who have contributed to these projects, and our collaborators and faculty colleagues for helpful discussions. We thank Dr. Donald Thomason (UTHSC), Dr. Kevin Kirk (UAB), and Dr. David Armbruster (UTHSC) for critically reading the manuscript. Chunying Li is a recipient of the Dorothy K. and Daniel L. Gerwin graduate scholarship and the Leonard Share Young Investigator Award from The University of Tennessee Health Science Center. Anjaparavanda Naren is a

References (132)

  • R.A. Frizzell et al.

    Chloride channels in epithelial cells

  • L.V. Galietta et al.

    Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds

    J Biol Chem

    (2001)
  • K.R. Hallows et al.

    Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells

    J Biol Chem

    (2003)
  • T. Hegedus et al.

    C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins

    Biochem Biophys Res Commun

    (2003)
  • A.Y. Hung et al.

    PDZ domains: structural modules for protein complex assembly

    J Biol Chem

    (2002)
  • B. Jovov et al.

    Cystic fibrosis transmembrane conductance regulator is required for protein kinase A activation of an outwardly rectified anion channel purified from bovine tracheal epithelia

    J Biol Chem

    (1995)
  • B.E. Kemp et al.

    Dealing with energy demand: the AMP-activated protein kinase

    Trends Biochem Sci

    (1999)
  • T. Kirchhausen et al.

    Linking cargo to vesicle formation: receptor tail interactions with coat proteins

    Current Opin Cell Biol

    (1997)
  • M.G. Lee et al.

    Regulation of Cl:HCO3 exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells

    J Biol Chem

    (1999)
  • C. Li et al.

    Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane

    J Biol Chem

    (2004)
  • S.M. Lohmann et al.

    Distinct and specific functions of cGMP-dependent protein kinases

    Trends Biochem Sci

    (1997)
  • J. Ma et al.

    Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein

    J Biol Chem

    (1996)
  • J. Marshall et al.

    Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro

    J Biol Chem

    (1994)
  • M.I. Milewski et al.

    PDZ-binding motifs are unable to ensure correct polarized protein distribution in the absence of additional localization signals

    FEBS Lett

    (2005)
  • P.M. Quinton

    Human genetics. What is good about cystic fibrosis?

    Curr Biol

    (1994)
  • D. Reczek et al.

    The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule

    J Biol Chem

    (1998)
  • M.F. Rosenberg et al.

    Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR)

    J Biol Chem

    (2004)
  • A. Ruknudin et al.

    Novel subunit composition of a renal epithelial KATP channel

    J Biol Chem

    (1998)
  • R. Schreiber et al.

    The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells

    J Biol Chem

    (1999)
  • E.M. Schwiebert et al.

    CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP

    Cell

    (1995)
  • L. Aguilar-Bryan et al.

    Toward understanding the assembly and structure of KATP channels

    Physiol Rev

    (1998)
  • M.P. Anderson et al.

    Demonstration that CFTR is a chloride channel by alteration of its anion selectivity

    Science

    (1991)
  • K.E. Barrett et al.

    Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects

    Annu Rev Physiol

    (2000)
  • P.S. Baxter et al.

    Accounting for cystic fibrosis

    Nature

    (1988)
  • I. Bezprozvanny et al.

    PDZ domains: more than just a glue

    Proc Natl Acad Sci U S A

    (2001)
  • R.C. Boucher et al.

    Sodium transport in cystic fibrosis epithelia. Abnormal basal rate and response to adenylate cyclase activation

    J Clin Invest

    (1986)
  • N.A. Bradbury et al.

    Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator

    Am J Physiol

    (1999)
  • S.Y. Chang et al.

    Mechanism of CFTR regulation by syntaxin 1A and PKA

    J Cell Sci

    (2002)
  • J.H. Chen et al.

    CFTR is a monomer: biochemical and functional evidence

    J Membr Biol

    (2002)
  • L.L. Clarke et al.

    Defective epithelial chloride transport in a gene targeted mouse model of cystic fibrosis

    Science

    (1992)
  • E. Cormet-Boyaka et al.

    CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

    Proc Natl Acad Sci U S A

    (2002)
  • E. Cormet-Boyaka et al.

    CFTR interacting proteins

  • E. Cormet-Boyaka et al.

    Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation

    Proc Natl Acad Sci U S A

    (2004)
  • A.W. Cuthbert et al.

    Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice

    Br J Pharmacol

    (1994)
  • A.W. Cuthbert et al.

    The genetic advantage hypothesis in cystic fibrosis heterozygotes: a murine study

    J Physiol (Lond)

    (1995)
  • P.B. Davis et al.

    Cystic fibrosis

    Am J Respir Crit Care Med

    (1996)
  • M. Dean et al.

    The human ATP-binding cassette (ABC) transporter superfamily

    Genome Res

    (2001)
  • D.T. Dransfield et al.

    Ezrin is a cyclic AMP-dependent protein kinase anchoring protein

    EMBO J

    (1997)
  • M.E. Egan et al.

    Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells

    Nat Med

    (2002)
  • S. Eskandari et al.

    Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy

    Proc Natl Acad Sci U S A

    (1998)
  • Cited by (0)

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