The Putative Chloride Channel hCLCA2 Has a Single C-terminal Transmembrane Segment

Abstract

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.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). 3The 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 ␤4 integrin (19 -21).A common feature of the CLCA family is proteolytic cleavage....

mCLCA4 ER processing and secretion requires luminal sorting motifs

“…As shown in Fig. 1D, prominent nuclear rim staining was observed along with plasma membrane localization, consistent with ER localization of the full-length fragment and similar to findings for hCLCA1 and hCLCA2 (3,7).…”

“…More recent structural analysis indicates a single COOH-terminal transmembrane segment (22) and a conserved NH 2 -terminal hydrolase domain (19). This prediction has been supported by experimental data indicating the secretion of a CLCA fragment in the extracellular space (3,6,14), although differences exist as to whether the NH 2 -terminal fragment (6) or both the NH 2 -and COOH-terminal fragments (3) are secreted extracellularly, with the latter data inconsistent with a single transmembrane domain. For mCLCA3, the two proteins are reported to remain physically associated and secreted in the extracellular space (14), whereas the COOH-terminal fragment of the highly related human homolog hCLCA1 does not appear to be associated with the secreted NH 2 -terminal cleaved peptide and is not found in the extracellular media (6).…”

“…Because previous reports indicated that NH 2 -and COOH-terminal fragments from some CLCAs are secreted (3,6,8), we attempted to immunoprecipitate Myc-tagged mCLCA proteins from media surrounding transfected cells. As shown in Fig.…”

“…Based on results from hCLCA2, Elble et al (3) have suggested a general model of CLCA processing in which the full-length precursor is transported to the cell surface where it undergoes cleavage by an endopeptidase, resulting in release of the NH 2 -terminal fragment, whereas the COOH-terminal fragment with its transmembrane segment remains as an integral membrane protein. Our data are in general consistent with this model, although it appears that both proteins remain associated with the plasma membrane and that the full-length precursor of mCLCA4 does not traffic to the plasma membrane, since the 125-kDa fragment is sensitive to Endo H digestion (Fig.…”

“…Since their initial cloning in bovine epithelium (1) and endothelium (4), numerous mammalian CLCA genes have been described with distinct, tissue-specific expression patterns (2, 13, 18). Originally described as integral membrane proteins that may be chloride channels, CLCAs have recently been shown to undergo extensive processing, including proteolytic cleavage and secretion (3,6,14), suggesting a more complex role than the regulation of chloride permeability and mucous secretion. In the mouse, the highly homologous murine (m) CLCA genes are tightly clustered on chromosome 3 (H3) in a manner that may enable coordinated transcriptional regulation, and the marked alteration in expression of CLCA genes in several disease states has provoked further interest in their function (12,20,23).…”