BASOLATERAL TARGETING AND EFFICIENT CONSUMPTION OF TRANSFORMING GROWTH FACTOR-ALPHA WHEN EXPRESSED IN MADIN-DARBY CANINE KIDNEY-CELLS

To investigate the trafficking of transforming growth factor-alpha pre cursor (pro-TGF-alpha) in polarized epithelial cells, wild type and me mbrane-fixed human pro-TGF-alpha were introduced into Madin-Darby cani ne kidney (MDCK) cells. We show that wild type pro-TGF-alpha was synth esized and processed normally to release mature 5.6-KDa TGF-alpha into the basal medium while membrane-fixed pro-TGF-alpha remained cell-ass ociated. Antibody (mAb-528) receptor blockade experiments demonstrated the efficient consumption of basally released TGF-alpha by basolatera l epidermal growth factor receptors, indicating that TGF-alpha can act in an autocrine manner in these polarized epithelial cells. Biochemic al analysis showed pro-TGF-alpha was expressed on the basolateral surf ace as either a 17- or 30-kDa species; the 17-kDa forms of both pro-TG F-alpha constructs had basolateral/apical ratios of >20:1. By confocal microscopy, membrane-fixed pro-TGF-alpha was immunolocalized to later al membrane surfaces. In pulse-chase experiments combined with cell su rface immunoprecipitation, we demonstrated that newly synthesized wild type and membrane-fixed pro-TGF-alpha are delivered directly to the b asolateral surface with 94 and 96% efficiency, respectively. These res ults also provide direct evidence for sequential cleavage of pro-TGF-a lpha at the basolateral membrane surface. Thus, pro-TGF-alpha is sorte d intracellularly and vectorially targeted to the basolateral membrane domain in these polarized epithelial cells. The MDCK cell line provid es an ideal in vitro model to examine the molecular basis for traffick ing of pro-TGF-alpha and other epidermal growth factor-like growth fac tors in polarized epithelial cells and their potential interactions wi th basolateral epidermal growth factor receptors.