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Originally published In Press as doi:10.1074/jbc.M605919200 on July 27, 2006

J. Biol. Chem., Vol. 281, Issue 40, 29448-29454, October 6, 2006
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The Putative Chloride Channel hCLCA2 Has a Single C-terminal Transmembrane Segment*

Randolph C. Elble{ddagger}1, Vijay Walia{ddagger}, Hung-chi Cheng§, Che J. Connon, Lars Mundhenk||, Achim D. Gruber||, and Bendicht U. Pauli**

From the {ddagger}Department of Pharmacology and Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9629, §Department of Biochemistry and Molecular Biology, Medical College, National Cheng Kung University, Tainan 70101, Taiwan, School of Optometry and Vision Sciences, Cardiff University, Cardiff CF23 9BD, United Kingdom, ||Department of Veterinary Pathology, Free University of Berlin, Robert-von-Ostertag-Strasse 15, D-14163 Berlin, Germany, and **Department of Molecular Medicine, Cornell University, Ithaca, New York 14853

Received for publication, June 20, 2006 , and in revised form, July 20, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl– channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680. Earlier investigations of transmembrane geometry suggested five membrane passes. However, analysis by the more recently derived simple modular architecture research tool algorithm predicts that a C-terminal 22-amino-acid hydrophobic segment comprises the only transmembrane pass. To resolve this question, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. Cell surface biotinylation and endoglycosidase H analysis revealed a 128-kDa precursor confined to the endoplasmic reticulum and a maturely glycosylated 141-kDa precursor at the cell surface by 48 h post-transfection. By 72 h, 109-kDa N-terminal and 35-kDa C-terminal cleavage products were detected at the cell surface but not in the endoplasmic reticulum. Surprisingly, however, the 109-kDa product was spontaneously shed into the medium or removed by acid washes, whereas the precursor and 35-kDa product were retained by the membrane. Two other CLCA family members, bCLCA2 and hCLCA1, also demonstrated preferential release of the N-terminal product. Transfer of the hCLCA2 C-terminal hydrophobic segment to a secreted form of green fluorescent protein was sufficient to target that protein to the plasma membrane. Together, these data indicate that hCLCA2 is mostly extracellular with only a single transmembrane segment followed by a short cytoplasmic tail and is itself unlikely to form a channel.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The calcium-activated chloride channel (CLCA)2 family was first identified nearly simultaneously by independent laboratories as either a calcium-activated chloride channel expressed in bovine respiratory epithelium (1) or as a cell adhesion molecule expressed in bovine vascular endothelium (2). Homologs were subsequently identified in other species, with at least six in mouse and four in human (3–12). With the sequencing of numerous genomes, it has become apparent that CLCA proteins are found throughout the chordates and possibly in some invertebrates (13).3

The exact function of these proteins remains unclear. When transfected into 293T cells, CLCA isoforms from mouse, human, bovine, pig, and rat produce a chloride current in response to calcium ionophores or calcium release from the endoplasmic reticulum (ER) (14, 15). Genetic studies link the CLCA family to the secretory disorders cystic fibrosis and asthma (16, 17). On the other hand, disruption of CLCA expression in cancer has been reported for several CLCA genes, especially hCLCA2 and its mouse ortholog mCLCA5 (5, 7, 18). In addition, several CLCA proteins have been found to interact with beta4 integrin (19–21).

A common feature of the CLCA family is proteolytic cleavage. Extensive analysis of cDNA and protein products of the 903-amino-acid bovine archetype Lu-ECAM-1/bCLCA2 revealed two cleavage sites, one following the 21-amino-acid N-terminal signal sequence and the other preceding amino acid 703 (2). The latter converted the 120-kDa precursor glycoprotein into 90-kDa N-terminal and 35-kDa C-terminal products, apparently at the cell surface (2). The functional significance of this cleavage event is unknown, but all CLCA isoforms tested have been processed similarly (3, 4, 7, 9, 10). For example, cleavage of hCLCA2 produces a deglycosylated product of 75 kDa, consistent with cleavage near amino acid position 680 (10).

The transmembrane topology of ion channels is difficult to predict due to the inclusion of charged residues in the ion-conductive pore. Cunningham et al. (1) originally proposed four membrane passes for the founding isoform bCLCA1, although only one segment satisfied established criteria (22). However, subsequent biochemical and molecular analyses of hCLCA1 supported this model. Epitope tagging coupled with immunocytochemistry on nonpermeabilized cells suggested that certain segments were internal (9). Moreover, studies of hCLCA2 glycosylation site utilization and protease protection analysis of in vitro translation products were consistent with five transmembrane passes for this distantly related protein (10).

However, a major shortcoming of these models, pointed out by Whittaker and Hynes (23), is the prediction of transmembrane passes within a von Willebrand A protein-protein interaction domain, leading them to propose only one C-terminal transmembrane segment for hCLCA2. In addition, Gibson et al. (24) demonstrate that the N-terminal processing product of the distantly related hCLCA1 can be readily removed from the cell surface, suggesting that it lacks any transmembrane segment. To resolve these issues, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. We report here that the hCLCA2 N-terminal processing product is peripherally associated with the plasma membrane but that the C-terminal product is integral. Although we cannot exclude the possibility that such a protein can itself form a channel, these results militate against that possibility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bioinformatics—Analysis of hCLCA2 and other CLCA proteins was performed using the simple modular architecture research tool (SMART) software package (25) hosted by the European Molecular Biology Laboratory (Heidelberg, Germany). Additional analysis was performed using TopPred (26, 27), TMPred (28), SOSUI (29), DAS (30), and HMMTOP (31, 32), accessed through the Expert Protein Analysis System (ExPASy).

Antibodies—Anti-hCLCA2 antibody H2A, raised against the peptide TVEPETGDPVTLRL, has been described previously (33). Protein A-Sepharose fractions were used for immunoprecipitation, and peptide affinity-purified fractions were used for immunoblotting. Anti-Myc monoclonal antibody 9E10 was produced from the hybridoma (American Type Culture Collection, Manassas, VA). Anti-beta4 integrin H101 was from Santa Cruz Biotechnology and anti-tubulin from Upstate%20Biotechnology">Upstate Biotechnology. Anti-Lu-ECAM-1 6D3 and CU8 have been described previously (2). Secondary antibodies donkey anti-rabbit and goat anti-mouse horseradish peroxidase conjugates were from Invitrogen.

DNAs and Transfection—pcDNA3.1 clones of hCLCA2, hCLCA2-Myc, hCLCA1-Myc, and RcCMV-beta4 have been described previously (9, 10, 34). Plasmids were transfected into 293T cells using Lipofectamine 2000 and OptiMEM (Invitrogen).

Immunoprecipitation—Cells were scraped from 10-cm plates into 2 ml of lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1% bovine serum albumin, 0.5 units/ml aprotinin). After centrifugation, the lysates were precleared with protein A-Sepharose beads for 2 h followed by incubation with the appropriate antibodies overnight. The medium collected from the plates was centrifuged at 40,000 x g for 30 min to remove vesicles and debris. After preclearing, antibodies were added directly to 5 ml of medium. The bound proteins were recovered by incubating for 2 h with protein A beads. The beads were washed once with lysis buffer and twice with 0.1 M Tris, pH 7.5, and then heated in sample buffer at 95 °C for 4 min.

Gel Electrophoresis and Immunoblots—High molecular weight proteins were analyzed on Prosieve gels (Pierce) run in Tris-glycine buffer for exceptional resolution, and low molecular weight proteins were analyzed on 10 or 14% acrylamide. The gels were blotted electrophoretically onto polyvinylidene difluoride membranes (Millipore). Blots were blocked in 5% milk dissolved in phosphate-buffered saline (PBS). Antibody incubation was overnight followed by a 2-h incubation with secondary antibodies and development with West Pico reagent (Pierce). Size markers included prestained ladder 7B (Sigma), unstained ladder, and Magic Mark (Invitrogen). Molecular weights were calculated using Alpha Imager software (Alpha Innotech).

Deglycosylation—Immunoprecipitates collected on protein A beads were denatured and incubated with either PNGase F or endoglycosidase H (Endo H) (Glyko) as directed by the manufacturer.

Surface Biotinylation—Transfected cells were washed three times with PBS, incubated for 30 min at 4 °C with 0.1 mg/ml sulfonated biotin (LHS-SS-Long Arm (Pierce) or Long-Arm-NHS (Vector Laboratories)), and then quenched with 50 mM ammonium chloride containing 0.1% bovine serum albumin. Cells were scraped, lysed, and analyzed as described above. Blots were probed with avidin-horseradish peroxidase (Invitrogen). To ensure that biotin labeling was specific for the cell surface, the cell lysates were treated with avidinagarose beads (Sigma), and precipitated proteins were analyzed by immunoblot for the presence of tubulin.

Acid Release—Cells were treated as described by Gibson et al. (24). Transfected cells were washed from a 10-cm dish by pipetting with Dulbecco's modified Eagle's medium, collected by centrifugation, and washed twice with Dulbecco's modified Eagle's medium. Cell pellets were resuspended in either PBS, pH 7.5 or 0.9% NaCl equilibrated to pH 2.5 with acetic acid. The cells were rotated end-over-end for 20 min at 4 °C and then collected by brief centrifugation. The supernatants were spun at 40,000 x g for 30 min at 4 °C to remove vesicles and membrane fragments and then subjected to trichloroacetic acid precipitation. Precipitates were resuspended in 0.1 ml of sample buffer plus 10 µl of 1 M Tris, pH 7.5, and heated prior to electrophoresis.

Green Fluorescent Protein (GFP) Chimeras—To direct GFP to the ER, the cleavable 24-amino-acid signal sequence from hCLCA2 (MTQRSIAGPICNLKFVTLLVALSS) was added to the N terminus by PCR. The initial M codon of GFP was converted to N, creating an N-glycosylation site. In addition, a Myc epitope tag was inserted after the signal sequence, although the tag was not utilized in this study. After verifying the sequence of a GEM-T clone (Promega), the insert was transferred to pcDNA3.1Zeo (pSS-GFP). To test the ability of the C-terminal hydrophobic segment of hCLCA2 to direct the protein to the plasma membrane, that sequence (LILKGVLTAMGLIGIICLIIVVTHHTL) was added to the last residue of GFP followed by a termination codon (pSS-GFP-Pho). 293T cells were transfected with these plasmids or a control encoding GFP (Pinco) (35) and then surface-biotinylated 48 h later. Proteins were immunoprecipitated and detected by immunoblotting with anti-GFP antibody (Abcam). The blot was then stripped and treated as described above for detection of surface biotinylation. For confocal microscopy, transfected cells were seeded onto poly-L-lysine-coated coverslips and fixed in 4% paraformaldehyde/PBS. Coverslips were mounted onto slides in ProLong mounting medium (Molecular Probes). GFP fluorescence was detected with an argon laser (Olympus), and central focal planes of the cells were observed and photographed with a 100x objective. To test the hCLCA1 C terminus, its last 28 amino acids were appended to secreted GFP.


Figure 1
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  		FIGURE 1.
A, revised topology of hCLCA2. An adaptation of the SMART model is shown. The vertical bar represents the transmembrane segment. The locations of the H2A and Myc epitopes relative to the cleavage site (lightening bolt) are indicated. ss, signal sequence; VWA, von Willebrand A domain. B, schematic attribution of protein bands observed in this study to precursor and cleavage products, glycosylated (gly+) or deglycosylated (gly–).

 


Figure 2
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  		FIGURE 2.
Characterization of products detected in 293T cells transfected with hCLCA2 bearing a C-terminal Myc tag at amino acid 725. A, left, anti-hCLCA2 H2A detects 141- and 128-kDa precursor bands and 109-kDa processing product. Right, anti-Myc 9E10 detects only the precursor bands but not the 109-kDa product from which the Myc tag has been cleaved. mock, cells transfected with pcDNA3.1 vector. B, an antibody against the larger processing product co-precipitates the smaller product. Thus, cleavage does not itself trigger dissociation and the products remain complexed for a time. 9E10 or H2A antibodies were used to immunoprecipitate (ip) from transfected 293T cells followed by immunoblotting with 9E10. -x, IgG or nonspecific background bands.

 


Figure 3
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  		FIGURE 3.
Secretion of the N-terminal 109-kDa processing product (but not the C-terminal product) into the medium. A, products immunoprecipitated from lysate (lys.) or medium (med.) from hCLCA2-transfected 293T cells and detected with H2A antibody. B, products immunoprecipitated by H2A from hCLCA2-Myc-transfected cells or medium and detected by anti-Myc monoclonal antibody.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Topology Prediction—The transmembrane topology of hCLCA2 was investigated using the TMHMM2 program of SMART (25). After the N-terminal signal peptide, only a single C-terminal transmembrane pass of 22 amino acids was predicted, spanning residues 906–928 of 943 (Fig. 1A). The predicted protein is almost entirely extracellular, with only a 15-amino-acid tail. Hence, upon cleavage, only the C-terminal processing product would be integral to the membrane (Fig. 1A). Other programs employing strict criteria for transmembrane segments (a transfer energy of 20 kcal/mol (22)), such as HMMTOP and SOSUI, agree with this prediction, differing only in the limits of the segment (data not shown). However, TopPred, TMPred, and DAS employ more relaxed criteria and allow for three, four, or five transmembrane segments, respectively (data not shown).

Processing and Association—An antibody (H2A) was raised against a peptide spanning amino acids 643–656 in the N-terminal processing product (Fig. 1) (33). To characterize the antibody and to confirm the previously reported behavior of hCLCA2, H2A was used to immunoprecipitate hCLCA2 from the lysates of 293T cells transfected with hCLCA2 bearing a Myc tag near the C terminus at amino acid 725 (Fig. 1) (10). Immunoblotting with H2A detected three major species of 141, 128, and 109 kDa, whereas anti-Myc antibody 9E10 detected only the 141- and 128-kDa species, implying that the 109-kDa species must represent the N-terminal processing product (Fig. 2A). To detect the C-terminal processing product, H2A and 9E10 immunoprecipitates were immunoblotted with 9E10 antibody. A 35-kDa band was detected in both immunoprecipitates, indicating that the 35-kDa product remains associated with the 109-kDa product for a time following cleavage (Fig. 2B). H2A did not detect the 35-kDa protein when the blot was reprobed, demonstrating its specificity for the N-terminal product (data not shown). A schematic interpretation of these data is presented in Fig. 1B.

Ectodomain Shedding—To test whether the N-terminal 109-kDa cleavage product really lacked a transmembrane segment, medium from transfected cells was subjected to immunoprecipitation and blotting with H2A. The 109-kDa product, but not the precursors, was detected in the medium, indicating that the product is devoid of transmembrane segments and that cleavage of the precursor removes a C-terminal transmembrane anchor (Fig. 3A). Consistent with this model, the 35-kDa product was not detected in the medium (Fig. 3B).


Figure 4
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  		FIGURE 4.
Resistance to Endo H indicates that the 141-kDa precursor and the 109- and 35-kDa products are expressed on the cell surface, whereas the 128-kDa precursor is confined to the ER. A, left, products immunoprecipitated 48 h post-transfection, before processing occurs. Treatment with PNGase F (F) reduced both precursor bands to 105 kDa, but only the 128-kDa precursor was sensitive to Endo H (H), indicating the immature glycosylation characteristic of the ER. Center panel, by 72 h, most precursor had been cleaved, and a 75-kDa band appeared upon treatment with PNGase F (but not Endo H), indicating cleavage occurs only at the cell surface. Right panel, the 105-kDa product immunoprecipitated from medium shows mature glycosylation. B, the C-terminal 35-kDa product was also fully resistant to Endo H, indicating surface expression. Proteins were immunoprecipitated by H2A antibody from hCLCA2-Myc-transfected cells and treated as described above. Immunoblot probed with anti-Myc monoclonal antibody.

 


Figure 5
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  		FIGURE 5.
Surface biotinylation of transfected cells indicates surface expression of all products except the 128-kDa precursor. Cells were biotinylated, lysed, and products immunoprecipitated with H2A antibody. Nonbiotinylated proteins were run in adjacent lanes as size markers. Products were detected with avidin or by immunoblotting with H2A or anti-Myc 9E10.

 
Endo H Analysis and Surface Biotinylation—An alternative explanation for the lack of precursor shedding is that the precursors might never reach the cell surface. To determine whether the precursor species were present on the cell surface, endoglycosidases were employed to characterize their glycosylation profiles. Endo H recognizes and removes only immature high-mannose glycosylation typical of the ER, whereas cell surface proteins are fully resistant to this enzyme. PNGase F removes all glycosyl groups. To analyze the precursor species in isolation, hCLCA2 was immunoprecipitated from cells 48 h after transfection, a time by which little processing had occurred. PNGase F reduced both the 141- and 128-kDa species to 105 kDa, confirming that they are alternate glycoforms (Fig. 4A, 1st and 2nd lanes). A shift of this magnitude is consistent with the prediction of 12–14 glycosylation sites in the primary sequence (10). In contrast to these results, only the 128-kDa species was sensitive to Endo H, indicating ER retention, whereas the 141-kDa species was resistant and therefore must be a surface form (Fig. 4A, 3rd lanes).

If processing occurs only at the cell surface, then the 109 kDa N-terminal processing product should be fully resistant to Endo H. To answer this question, the 109-kDa species was immunoprecipitated from cell lysate or medium 72 h post-transfection. Treatment with PNGase F reduced its apparent molecular mass to 75 kDa, consistent with a cleavage near amino acid 680, whereas Endo H had no effect (Fig. 4A, center and right panels), indicating that processing occurs only after the precursor reaches the cell surface.

If the C terminus is responsible for anchoring hCLCA2 to the plasma membrane and cleavage occurs only at the surface, then the C-terminal processing product should also exist only on the cell surface. Indeed, the 35-kDa product was fully resistant to Endo H, whereas PNGase F reduced its apparent molecular mass to 30 kDa (Fig. 4B). These results were confirmed by surface biotinylation. Both the 141- and 109-kDa, but not the 128-kDa, proteins were labeled by a membrane-impermeable biotin compound (Fig. 5A). The 35-kDa product was equally susceptible to surface biotinylation as the higher molecular weight species (Fig. 5B).

Acid Elution—Treating cells with a strong acid or base can distinguish between proteins bearing a true transmembrane segment and those that are only tightly associated by hydrogen bonding to another surface protein (36). This method had been employed to show that the N-terminal processing product of hCLCA1 lacks a transmembrane segment (22). Here, the 109-kDa ectodomain was readily eluted from transfected cells by acid but not PBS (Fig. 6). However, neither the 141-kDa precursor nor the 35-kDa protein was eluted nor was the control, the integral membrane protein beta4 integrin (Fig. 6).

The Hydrophobic Segment Targets GFP to the Plasma Membrane—If the hCLCA2 C-terminal hydrophobic segment really constitutes a transmembrane segment, it should be able to direct a heterologous protein to the plasma membrane. We chose GFP as a normally cytoplasmic protein whose localization could be easily tracked. Two modifications were effected. To target GFP to the secretory compartment, a cleavable signal sequence was appended to the N terminus (Fig. 7, construct B). The putative transmembrane segment from hCLCA2 was then added to the C terminus (Fig. 7, construct C). Human embryonic kidney 293T cells were transfected and surface-biotinylated, and lysates were immunoprecipitated with anti-GFP. A Western blot revealed bands of the expected sizes for GFP and for the secreted, glycosylated GFP constructs (Fig. 7, center left panel). However, detection of the surface-biotinylated protein with avidin produced a band only for construct C, corresponding in mobility to the higher band detected by anti-GFP (Fig. 7, center right panel). Biotin labeling was specific for the cell surface, as evidenced by the lack of labeling of cytoplasmic GFP (Fig. 7), and an additional control protein, tubulin (data not shown). Confocal microscopy of cells transfected with construct C confirmed GFP fluorescence predominantly in the plasma membrane, whereas construct B produced a pattern consistent with targeting to the secretory compartment, and cytoplasmic GFP produced a bright uniform fluorescence (Fig. 7, bottom).


Figure 6
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  		FIGURE 6.
Acid treatment releases the 109-kDa product (but not the precursor or the 35-kDa product) from the cell surface. Cells transfected with hCLCA2-Myc were surface-biotinylated and then washed with acid to release peripherally associated proteins from the plasma membrane or with PBS. Eluted material was concentrated by trichloroacetic acid precipitation before electrophoresis. The transfected beta4 integrin served as an integral membrane protein control. Lysates were immunoprecipitated with H2A, 9E10, or anti-beta4 H101.

 
Ectodomain Shedding by Other CLCAs—To address the generality of ectodomain shedding among CLCA family members and whether it occurs in cells that naturally express CLCA proteins, we examined media from bovine aortic endothelial cells for the presence of bCLCA2 (Lu-ECAM-1). Medium immunoprecipitated with monoclonal antibody 6D3 and immunoblotted with CU8, antibodies specific for bCLCA2, revealed the presence of the 90-kDa N-terminal processing product but not the 120–130-kDa precursors (Fig. 8A) (2). Another distant relative of hCLCA2, hCLCA1, has also been reported to shed its ectodomain into cell medium (22). In agreement with those results, we detected the ~90-kDa N-terminal product; however, in contrast, we did not detect the 120-kDa precursor in the medium (Fig. 8B). Nevertheless, when we appended the C-terminal 28 amino acids of hCLCA1 to construct B above, it failed to direct the chimera to the plasma membrane, supporting the contention that hCLCA1 lacks a transmembrane segment (data not shown).


Figure 7
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  		FIGURE 7.
Targeting of GFP to the plasma membrane by the hCLCA2 C-terminal hydrophobic segment. Top, GFP variants that were transfected into 293T cells. SS, signal sequence; Pho, hydrophobic segment of hCLCA2. Left center, detection of GFP variants by immunoprecipitation and immunoblotting with anti-GFP. Right center, detection of surface-biotinylated products by immunoprecipitation with anti-GFP and blotting with streptavidin-horseradish peroxidase. Bottom, the central focal plane of each transfected cell population was photographed with a 100x objective. Arrowheads, positions of the surface-biotinylated chimera.

 


Figure 8
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  		FIGURE 8.
Shedding of the larger processing product by other CLCA family members. A, bovine CLCA2 (Lu-ECAM-1) immunoprecipitated from bovine aortic endothelial cell medium with monoclonal antibody 6D3 and detected by polyclonal antibody CU8. PI, preimmune serum. Positions of size markers are indicated. B, Myc-tagged hCLCA1 immunoprecipitated from transfected 293T cells and detected by 9E10. Deduced sizes are indicated.

 


Figure 9
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  		FIGURE 9.
Model of hCLCA2 maturation. A pool of 128-kDa precursor accumulates in the ER and then transits through the Golgi to the plasma membrane, increasing to 141 kDa as it matures. The protein is then cleaved, but some uncleaved precursor is detectable on the plasma membrane. The cleavage products remain associated until an unidentified event triggers the release of the ectodomain.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results support the following model for hCLCA2 synthesis and processing, summarized in Fig. 9. The protein is secreted into the ER and glycosylated to form a 128-kDa precursor containing a single C-terminal transmembrane anchor. The precursor is transported to the Golgi, where glycosylation is modified and then appears on the cell surface as a 141-kDa species. This protein is then cleaved by an unknown endopeptidase activity. The processing event likely occurs at the cell surface, because the mature precursor appears on the surface, and processing products are resistant to Endo H, reflecting mature glycosylation. A less likely alternative is that the surface precursor is endocytosed, cleaved, and returned to the plasma membrane. Cleavage in itself does not result in immediate dissociation, as the 109- and 35-kDa products co-immunoprecipitate, and strong acid is required to remove the ectodomain from the cell surface. What triggers the spontaneous release of the ectodomain into the medium is unknown. In contrast, neither the 35-kDa product nor the 141-kDa precursor appears in the medium nor is acid-releasable from the plasma membrane, indicating the presence of a bona fide transmembrane segment; thus, hCLCA2 is an integral membrane protein with a single transmembrane segment as predicted by SMART analysis.

In contrast to these results, a prior study of hCLCA2 transmembrane topology using both glycosylation site mutagenesis and protease sensitivity concluded that the protein contained five transmembrane segments (10). In the first approach, glycosylation sites were mutated, and the effects were analyzed by in vitro translation into microsomes and PAGE. By this method, if the mutation causes a change in mobility relative to wild type, then the site must be extracellular. The lack of change is interpreted as evidence of a cytoplasmic localization. A weakness of this method is that potential glycosylation sites may not be used, because they are masked by protein folding, leading to a mistaken conclusion. The second approach relies on accurate interpretation of protease digestion products after in vitro translation into microsomes and digestion with a protease. However, in the absence of a panel of appropriate antibodies to aid in interpretation, results may again be misleading.

All members of the CLCA protein family terminate in a hydrophobic segment, although none is as long as that of hCLCA2 and its mouse ortholog mCLCA5 (1–10, 12, 15). It is yet unclear whether any of the shorter segments are capable of membrane anchoring. Gibson et al. (24) have found that hCLCA1 precursor is spontaneously shed into the medium, although much less efficiently than the N-terminal product. We also readily detected the N-terminal processing products of hCLCA1 and Lu-ECAM-1/bCLCA2 in culture media, but we were unable to detect the respective precursors. However, consistent with the conclusion of Gibson et al. (24), when we appended the C-terminal 28 amino acids of hCLCA1 to signal sequence-GFP, it failed to direct the chimera to the plasma membrane.4 The preferential release of the N-terminal product over the precursor in the apparent absence of a C-terminal transmembrane segment suggests that the C terminus of some CLCA family members must have another means of associating with the membrane, perhaps by binding tightly to widely expressed integral membrane proteins or glycosylphosphatidylinositol anchoring. However, at least one CLCA protein, mCLCA3, does not appear to associate with the membrane at all but rather is shed directly into the cellular environment.5 This lack of conservation of C-terminal association with the membrane reflects the deep phylogenetic divergence within the CLCA family and independent diversification within species.

The functional significance of cleavage and ectodomain shedding for CLCA family members remains unknown. Ectodomain shedding has been described for a variety of cell surface proteins, including cell adhesion molecules, ectoenzymes, and growth factor receptors (37). Its consequences are also varied. Cleavage may be a means of down-regulating the protein (as demonstrated for L-selectin) or activating it (as shown for TNF{alpha}) (38). In many cases, the responsible agents have been identified; for example, epidermal growth factor receptor ligands are released by ADAM17 metalloprotease, and this step is essential for activation of cell migration (39). Similarly, the hCLCA2 ectodomain may act as a diffusible signaling molecule. This possibility is especially interesting given that hCLCA2 and its mouse ortholog mCLCA5 have demonstrated tumor-suppressive activities and both are expressed in mammary epithelium (7, 18). Cleavage of hCLCA2 has been observed in the mammary epithelial cell line MCF10A.6

On the other hand, several lines of evidence are consistent with a role in modulating chloride current across the plasma membrane. First, transfection of any CLCA cDNA into 293T or other heterologous cell types results in a calcium-stimulated chloride current across the plasma membrane (14, 15). Second, genetic modifiers of the severity of cystic fibrosis and asthma, both secretory diseases in which chloride flux plays a prominent role, map to the CLCA gene cluster (16, 17). The identification of interacting proteins should allow a better definition of the functions of this protein family and permit an accurate renaming.


   FOOTNOTES
 
* This work was supported by Philip Morris USA Inc., Philip Morris International, and by United States Army Breast Cancer Research Fund Grant DAMD17-00-1-0219 (to R. C. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 217-545-7381; E-mail: relble2{at}siumed.edu.

2 The abbreviations used are: CLCA, calcium-activated chloride channel; ER, endoplasmic reticulum; SMART, simple modular architecture research tool; GFP, green fluorescent protein; PBS, phosphate-buffered saline; TMHMM, transmembrane-helix Hidden Markov Model. Back

3 A dendrogram of CLCA proteins is available in the Phylogenetically Inferred Groups data base, cluster 211900 (phigs.jgi-psf.org). Back

4 V. Walia and R. C. Elble, unpublished data. Back

5 L. Mundhenk and A. D. Gruber, unpublished observations. Back

6 R. C. Elble, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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