C Terminal End

C TERMINAL END

TLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFP

HRNSSKCKSKPQIAALKEETEEEVQDTRL

(amino acids 1387 thru 1480)

The C-terminus of CFTR has come under intense scrutiny in the last couple of years, largely as a result of the discovery of a PDZ-protein binding motif called TRL. These last three amino acids are located at the far C-terminal end of CFTR, and deletion of this motif has resulted in the mislocalization of CFTR in human airway cells epithelial cells. It is considered likely that these PDZ-containing proteins help couple CFTR to other regulatory proteins in the cell, such as phosphatases, cytoskeletal proteins, or kinases. This domain binds the PDZ-protein NHE-RF, which is a sodium/proton exchanger regulatory factor. CFTR also binds a cytosolic phosphoprotein with as yet unknown binding partners. PDZ domains on accessory proteins such as NHE-RF usually bind to these short linear C-terminal sequences like on membrane associated proteins like CFTR. There has been growing evidence in recent years that these PDZ domain containing proteins interact with ion channels. For example, expression of PDZ domain proteins in cells has been correlated with the clustering of ion channels at cell surfaces. PDZ proteins may help couple CFTR to the cytoskeleton. PDZ gets it's name from the PDZ-containing proteins PSD-95, discs large, and ZO-1. PDZ domain-containing proteins usually have more than one of these domains in their structures and are therefore considered multivalent. Multivalency allows these proteins to provide scaffold-like places for protein-protein interactions, usually seen with signal transduction machinery, or the clustering of protein (ion channels, for example) at the cell membrane. It has been suggested that the PDZ proteins are the long-sought mechanism CFTR uses to regulate other ion channels. PDZ proteins may form scaffolds for CFTR and other proteins to interact with each other.

RECENT RESEARCH:

The acid-terminus of NBD2 could possibly be considered as the beginning of the C-tail.

The C-terminal residue responsible for endosomal targeting is a Tyr(1424)-based internalization signal. It uses an adaptor protein called AP-2 for internalization. In September, 2001 Weixel and Bradbury reported finding an adaptor protein called "m2", which is able to bind CFTR's sequence YDSI in the C-terminal tail. They performed crosslinking experiments using photoreactive peptides containing the YDSI motif and purified adaptor complexes, and found that CFTR peptides crosslinked a 50 kDa subunit of purified AP-2 complexes, the apparent molecular weight of m2. They also found that isolated m2 bound to the sorting motif, YDSI, both in crosslinking experiments and GST-pulldown experiments. They concluded that this confirmed that m2 mediates the interaction between CFTR and AP-2 complexes. They also induced overexpression of a dominant-negative form of m2 in HeLa cells, which resulted in AP-2 complexes that failed to interact with CFTR. In addition, internalization of CFTR in mutant cells was "greatly reduced compared to wild type HeLa cells." The concluded that these results "...indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the m2 subunit of AP-2 and mutations in m2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway." J Biol Chem 2001 Sep 17

Other membrane proteins besides CFTR which bind to PDZ proteins include the beta2-adrenergic receptor and the purinergic P2Y1 receptor.

EBP-50 is an ezrin-binding protein found exclusively at the apical membranes of epithelia cells. It is possible EBP-50 anchors CFTR to the apical membrane cytoskeleton.

In February 2000, Weixel found using cross-linking and in vitro pull-down studies that CFTR binds to endocytic adaptor complex AP-2. Fusion proteins containing the carboxy-terminal end of CFTR (amino acids 1404 thru 1480) were also able to bind it. He speculated that the acid terminus has a tyrosine-based sequence internalization signal that interacts with the endocytic adaptor complex to facilitate entry of CFTR into clathrin-coated vesicles.

Poly-Histidine tags have been engineered into the carboxy terminal of CFTR with no apparent effects on channel activity, however it's ability to retain binding of PDZ-domain-containing proteins was not assessed.

The VSV-G epitope was engineered into the C-terminal end by Costa de Beauregard et al. (Nov,2000). CFTR-VSV-G had activity that was indistinguishable from the wild-type CFTR. Addition of the VSV-G epitope did not impair the localization and function of CFTR.

There are no severe CF-causing mutations in the C-terminal tail. Neither S1455X and Q1476X cause severe CF.

Inhibition of the sodium channel ENaC by CFTR can still occur even without the acid-terminal tail of CFTR.

There is a well-conserved negatively-charged motif within the C-terminal tail (E1469 to E1474).

An example of a PDZ-domain containing protein coupling membrane proteins is the INAD complex of the Drosophila retina. This complex contains at least two ion channels coupled to multiple signaling molecules by a scaffolding protein with 5 PDZ domains Xu et al. J. Cell Biol. 142: 545-555

More Recent Evidence: Pieces to the Puzzle?

In September, 2000 Wang, Yue, Derin and Guggino (of Johns Hopkins University) identified a novel hydrophilic CFTR binding protein, CAP70, and found that it was localized to apical cell membranes. CAP70 has four known PDZ domains, three of which were found to bind to this end of CFTR. They linked two CFTR molecules via cytoplasmic C-terminal binding using either multivalent CAP70 or a bivalent monoclonal antibody which was found to potentiate the channel activity. They concluded that the CFTR channel can be switched to a more active conducting state by way of a modification of intermolecular CFTR-CFTR contact that is enhanced by an accessory protein.

It is still not known for certainty which PDZ domain protein(s) interact with CFTR. In September, 2000 Sun, et.al stated based on confocal immunofluorescence microscopy studies that "we [were able to] show that CFTR associates with Na(+)/H(+) exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/NHE regulatory factor (NHERF)-related PDZ domain protein." The authors concluded their findings with the following: "We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl(-) currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actin-binding protein, the formation of a CFTR.E3KARP.ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells." J Biol Chem 2000 Sep 22;275(38):29539-46

"Weixel and Bradbury used in vivo cross-linking and in vitro pull-down assays to show that full-length CFTR binds to the endocytic adaptor complex AP2. Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ability of AP2 to bind the C terminus of CFTR. However, mutation to a phenylalanine residue, which is normally found in dogfish CFTR at this position, did not perturb AP2 binding. Taken together, these data suggest that the C terminus of CFTR contains a tyrosine-based internalization signal that interacts with the endocytic adaptor complex AP2 to facilitate efficient entry of CFTR into clathrin-coated vesicles". J Biol Chem 2000 275: 3655-3660

"Wang (2000) identified a hydrophilic CFTR-binding protein, CAP70, which is concentrated on the apical surfaces. CAP70 had previously been identified by Kocher (1998) as PDZK1. The protein contains 4 PDZ domains, 3 of which are capable of binding to the CFTR C terminus. Linking at least 2 CFTR molecules via cytoplasmic C-terminal binding by either multivalent CAP70 or a bivalent monoclonal antibody potentiates the CFTR chloride channel activity. Thus, the CFTR channel can be switched to a more active conducting state via a modification of intermolecular CFTR-CFTR contact that is enhanced by an accessory protein." Cell 103: 169-179, 2000

Dimerization of CFTR via CAP70 does not change the conductance state of single CFTR molecules, however it does increase the open probability. It is therefore theorized that CFTR may form transitional dimers of higher Cl- channel activity. This could provide a new mechanism for regulation of ABC proteins like CFTR.

PDZ-interacting domains usually consist of between 3 and 5 amino acids at the C-terminal end of proteins. In CFTR it consists of Q-N-T-R-L. When the leucine (L) is replaced by an alanine, there was no longer localization of CFTR to the cell surface. It also interrupted association of CFTR with EBP-50. Changing threonine (T) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chloride secretion. By contrast, individual point substitution of any of the other amino acids in the PDZ domain had no effect on measured parameters. (Moyer 2000) concluded that mutations that delete the C terminus of CFTR may cause cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells."

Moyer, et al found that TRL deletion mutants results in the mislocalization of CFTR in airway and kidney epithelial cells. It was speculated that one reason for PDZ-domain mediated interactions could be to keep CFTR in the apical rather than basolateral cell membranes of polarized epithelial cells. J. Clin. Invest. 104: 1353-1361, 1999

Mutagenesis experiments indicate an endosomal targeting of CFTR mediated via a Tyr(1424)-based internalization signal. The mutant form was less likely to undergo endocytosis, as well as interact with the adapter protein AP-2. Hu, Howard, and Lukacs, (of the Hospital for Sick Children: Toronto, Canada) used site-directed mutagenesis (a chimera with the Il-2 receptor alpha chain and the C-terminal tail of CFTR, as well as full-length CFTR) in an attempt to reveal additional endosomal targeting signals. They claim to have found "..multiple internalization motifs at the C-terminus, consisting of a phenylalanine-based motif (Phe(1413)) and a bipartite endocytic signal, comprising a tyrosine (Tyr(1424)) and a di-Leu-based (Leu(1430)-Leu) motif." They found that replacement of any one of the three internalization motifs with an alanine "..prevented the endocytosis of the chimaera, mutagenesis of Phe(1413)-Leu impaired the biosynthetic processing of CFTR, indicating that Phe(1413) is indispensable for the native structure of CFTR. In contrast, replacement of Leu(1430)-Leu- and Tyr(1424)-based signals with alanine increased the cell-surface density of both the chimaeras and CFTR in an additive manner. These results suggest that the internalization of CFTR is regulated by multiple endocytic sorting signals." : Biochem J 2001 Mar 15;354(Pt 3):561-572

In February 2001, Milewski et al. write that "..the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains." These researchers coexpressed the C terminus alone along with full-length CFTR and found CFTR redistributed from apical to basolateral membranes, providing evidence that both proteins interacted with the same target at the apical membrane. It was found that two additional previously unknown C-terminal regions, from amino acid residues 1370-1394 and 1404-1425 of CFTR, are also required for localizing to the apical plasma membrane. They concluded that "based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif." Milewski M, Mickle J, Forrest J, Stafford D, Moyer B, Cheng J, Guggino W, Stanton B, Cutting G: J Cell Sci 2001 Feb;114(Pt 4):719-26

In June, Karthikeyan et al (Harvard Medical School) reported on a study concerning the mechanism of interaction between teh PDZ1 domain and CFTR. In their own words: "The PDZ1 domain of the Na(+)/H(+) exchanger regulatory factor (NHERF) binds with nanomolar affinity to the carboxyl-terminal sequence QDTRL of the cystic fibrosis transmembrane conductance regulator (CFTR) and plays a central role in the cellular localization and physiological regulation of this chloride channel. The crystal structure of human NHERF PDZ1 bound to the carboxyl-terminal peptide QDTRL has been determined at 1.7-A resolution. The structure reveals the specificity and affinity determinants of the PDZ1-CFTR interaction and provides insights into carboxyl-terminal leucine recognition by class I PDZ domains. The peptide ligand inserts into the PDZ1 binding pocket forming an additional antiparallel beta-strand to the PDZ1 beta-sheet, and an extensive network of hydrogen bonds and hydrophobic interactions stabilize the complex. Remarkably, the guanido group of arginine at position -1 of the CFTR peptide forms two salt bridges and two hydrogen bonds with PDZ1 residues Glu(43) and Asn(22), respectively, providing the structural basis for the contribution of the penultimate amino acid of the peptide ligand to the affinity of the interaction." J Biol Chem 2001 Jun 8;276(23):19683-6

Some references used for this page....