Our previous studies showed immunological and functional similarities, as well as partial sequence homology, between the enzymatically inactive alternatively spliced variant of human ␤-galactosidase (S-gal) and the 67-kDa elastin/laminin-binding protein (EBP) from sheep. To define the genetic origin of the EBP further, a full-length human S-gal cDNA clone was constructed and subjected to in vitro transcription/translation. The cDNA was also transfected into COS-1 cells and into the EBP-deficient smooth muscle cells (SMC) from sheep ductus arteriosus (DA). In vitro translation yielded an unglycosylated form of the S-gal protein, which immunoreacted with anti-␤-galactosidase antibodies and bound to elastin and laminin affinity columns. S-gal cDNA transfections into COS-1 and DA SMC increased expression of a 67-kDa protein that immunolocalized intracellularly and to the cell surface and, when extracted from the cells, bound to elastin. The S-gal-transfected cells displayed increased adherence to elastin-covered dishes, consistent with the cell surface distribution of the newly produced S-gal-encoded protein. Transfection of DA SMC additionally corrected their impaired elastic fiber assembly. These results conclusively identify the 67-kDa splice variant of ␤-galactosidase as EBP.Virtually all cell types, including tumor cells, interact with the extracellular matrix (ECM) 1 during certain stages of their development. Such contacts may be strictly adhesive or can transduce signals from the ECM to the intracellular machinery (1). These significant cell matrix interactions are mediated through specialized cell surface receptors (2, 3). Interactions between cells and elastin are mediated by a non-integrin cell surface receptor complex consisting of three protein subunits (4 -6). Two of these subunits (61-and 55-kDa subunits) are cell membrane-associated proteins that immobilize the third, a 67-kDa peripheral subunit called the elastin-binding protein (EBP). The EBP binds predominantly to the repeating VGVAPG hydrophobic domains on elastin, but it may also bind to other similar hydrophobic domains on elastin (7), and to the LGTIPG sequence on laminin (5, 8). Moreover, the EBP also interacts with moieties containing ␤-galactosugars through a separate "lectin-like" binding domain. However, binding of ␤-galactosugar-bearing moieties to the lectin domain of the EBP causes such conformational changes in the 67-kDa protein that it loses its affinity for elastin and separates from the other subunits of the elastin receptor. Thus, the EBP can be shed from the cell surface by interactions with galactosugars (galactose, lactose) or with N-acetylgalactosamine-containing glycosaminoglycans (chondroitin sulfate, dermatan sulfate), which bind to its lectin site (9-11). The EBP appears to be directly involved in the generation of intracellular signal transmission after contact with its matrix ligands (6). Binding of elastin-derived peptides to the EBP, when present on the cell surface, resulted in a rapid and transient increase in free ...