Basolateral targeting and efficient consumption of transforming growth factor-alpha when expressed in Madin-Darby...

To investigate the trafficking of transforming growth factor-alpha precursor (pro-TGF-alpha) in polarized epithelial cells, wild type and membrane-fixed human pro-TGF-alpha were introduced into Madin-Darby canine kidney (MDCK) cells. We show that wild type pro-TGF-alpha was synthesized and processed normally to release mature 5.6-kDa TGF-alpha into the basal medium while membrane-fixed pro-TGF-alpha remained cell-associated. Antibody (mAb-528) receptor blockade experiments demonstrated the efficient consumption of basally released TGF-alpha by basolateral epidermal growth factor receptors, indicating that TGF-alpha can act in an autocrine manner in these polarized epithelial cells. Biochemical analysis showed pro-TGF-alpha was expressed on the basolateral surface as either a 17- or 30-kDa species; the 17-kDa forms of both pro-TGF-alpha constructs had basolateral/apical ratios of > 20:1. By confocal microscopy, membrane-fixed pro-TGF-alpha was immunolocalized to lateral membrane surfaces. In pulse-chase experiments combined with cell surface immunoprecipitation, we demonstrated that newly synthesized wild type and membrane-fixed pro-TGF-alpha are delivered directly to the basolateral surface with 94 and 96% efficiency, respectively. These results also provide direct evidence for sequential cleavage of pro-TGF-alpha at the basolateral membrane surface. Thus, pro-TGF-alpha is sorted intracellularly and vectorially targeted to the basolateral membrane domain in these polarized epithelial cells. The MDCK cell line provides an ideal in vitro model to examine the molecular basis for trafficking of pro-TGF-alpha and other epidermal growth factor-like growth factors in polarized epithelial cells and their potential interactions with basolateral epidermal growth factor receptors.