Cloning and Characterization of Lung-Endothelial Cell Adhesion Molecule-1 Suggest It Is an Endothelial Chloride Channel

Abstract

Lung-endothelial cell adhesion molecule-1 (Lu-ECAM-1) is an endothelial cell surface molecule that mediates adhesion of metastatic melanoma cells to lung endothelium. Here we analyze the organization of the Lu-ECAM-1 protein complex, report the sequence of Lu-ECAM-1 cDNAs, and reveal a novel function of the protein. Lu-ECAM-1 immunopurified from bovine aortic endothelial cells (BAEC) consists of tightly associated glycoproteins of 90, 38, and 32 kDa, with minor components of 130 and 120 kDa. We present evidence that all of these protein species are encoded by a single open reading frame whose initial translation product is proteolytically processed to yield the other products. Correct processing in vitro was demonstrated by transfection of the longest cDNA into human embryonic kidney 293 cells; immunoblot analysis showed that the ϳ120-kDa precursor gave rise to 90-and 38-kDa products. RNA blots of BAEC mRNA detected messages in agreement with the sizes of the cDNA clones in addition to several of high molecular weight. DNA blot analysis showed that Lu-ECAM-1 is conserved throughout its length in all mammals tested, usually as a single or low copy gene. In the bovine, Lu-ECAM-1 protein is 88% identical to a calcium-dependent chloride channel described recently in tracheal epithelium, Ca-CC. Probes for Lu-ECAM-1 mRNA and protein confirmed the presence of a homolog in this tissue. We show that messages for both proteins are present in lung while only Ca-CC is present in trachea and only Lu-ECAM-1 is present in BAEC. These results suggest that endothelial cells express a chloride channel that is related to, but distinct from, that expressed in tracheal epithelium. They further suggest that an adhesion molecule can also be a chloride channel.The preference of metastasizing tumor cells for certain organs may be explained if such cells fortuitously recognize and adhere to organ-specific, endothelial cell-surface molecules. In studying this hypothesis, much emphasis has been placed on the role of members of the classic families of cell-cell adhesion molecules including selectins, the immunoglobulin superfamily, and integrins (1-3). The contribution of these adhesion molecules to organ preference of metastasis was suggested by a number of reports describing the presence of such molecules on endothelia of various tissues and vessel calibers and denoting corresponding ligands on malignant cells of tumors of various tissue origins (1-7). In our laboratory, a different approach was chosen to testing the contribution of specific endothelial cell adhesion molecules to organ preference of metastasis. It relied on an endothelial cell culture system that could be modulated by growing unspecific, large vessel-derived endothelial cells (e.g. bovine aortic endothelial cells (BAEC) 1 ) on organ-specific matrix to express phenotypic traits of the microvasculature of that organ (8, 9). Tumor cells with distinct metastatic dissemination patterns were then evaluated for adhesion to these endothelial cells, observing that tumor cells only...

Re-expression of Detachment-inducible Chloride Channel mCLCA5 Suppresses Growth of Metastatic Breast Cancer Cells

“…1B). In addition to the higher primary sequence identity, mCLCA5 shares two other distinct properties of hCLCA2: the spacing of the symmetrical multicysteine motif, CX 9 CX 4 CX 4 -CX 9 C, as opposed to CX 12 CX 4 CX 4 CX 12 C in the rest of the family; and a frameshift mutation that results in an ϳ40-amino-acid carboxyl-terminal extension (20,24). In addition, the conservation of most of the putative N-linked glycosylation and protein kinase C phosphorylation sites suggests structural and functional conservation.…”

“…The family is deeply divergent, some members sharing as little as 36% identity, yet all share certain signatory features. The prototypical CLCA family member is a type I transmembrane protein about 900 amino acids in length with a proteolytic cleavage near amino acid 680 that results in products of 90 kDa and 30 -40 kDa that are found in close association on the exterior surface of the plasma membrane (19,24,26,28). Another common feature is a symmetrical multiple-cysteine motif, CX 12 CX 4 CX 4 CX 12 C, in the amino-terminal tail.…”

“…hCLCA2 belongs to a recently discovered family of proteins, currently comprising about a dozen members, that are widely expressed in mammals but apparently not beyond (19 -28). Various family members are found in epithelia, endothelia, and smooth muscle of multiple organs (21,24,25,27,28). CLCAs do not resemble any other known Cl Ϫ channel (i.e.…”

Tumor Suppression by a Proapoptotic Calcium-activated Chloride Channel in Mammary Epithelium

“…The CLCA 1 family of calcium-activated chloride channels comprises about a dozen members thus far expressed in a variety of organ and tissue types in mammals (1)(2)(3)(4)(5)(6)(7)(8). Although the family is deeply divergent, some members sharing as little as 36% identity, all share several features.…”

Association of the hCLCA1 gene with childhood and adult asthma

“…The hCLCA1 enhances mucus secretion by mediating the active transport of chloride ions. 10 Currently, the family includes two bovine homologues (bCLCA1 and bCLCA2), [17][18][19] four murine homologues (mCLCA1, mCLCA2, mCLCA3 (gob-5), mCLCA4), [20][21][22][23][24] and four human homologues (hCLCA1, hCLCA2, hCLCA3, hCLCA4 (hCaCC2)). 15,16,25,26 All human CLCA genes identified are clustered on the short arm of chromosome 1 (1p22-31).…”