Basolateral sorting of transforming growth factor-α precursor in polarized epithelial cells: characterization of cytoplasmic domain determinants

In polarized Madin–Darby canine kidney (MDCK) cells, newly synthesized transforming growth factor-α precursor (proTGFα) is directly sorted to the basolateral cell surface where it is sequentially cleaved and released into the basolateral conditioned medium (Dempsey, P.J., Coffey, R.J., J. Biol. Chem. 269 (1994) 16878–16889). In the present study, the role of the proTGFα cytoplasmic domain in basolateral sorting has been investigated using deletional and site-directed mutagenesis, as well as chimeric analyses of different TGFα constructs stably expressed in MDCK cells. The loss of polarized secretion of a proTGFα secretory mutant (TGFsec88) indicated that the proTGFα transmembrane and/or cytoplasmic domains contain essential basolateral sorting information. Using reporter chimeras with two apically sorted membrane proteins, p75 neurotrophin growth factor receptor and placental alkaline phosphatase, we show that the proTGFα cytoplasmic domain contains dominant basolateral sorting information. Analysis of proTGFα cytoplasmic domain truncation and internal deletion mutants, together with site-directed mutagenesis studies within the full-length proTGFα cytoplasmic domain, revealed redundant basolateral sorting motifs. Importantly, the C-terminal type I PDZ-binding motif was not required for basolateral sorting as determined by the integrity of basolateral sorting in deletion mutants lacking this motif. ProTGFα basolateral sorting may have important consequences for ligand presentation and spatial compartmentalization of epidermal growth factor receptor signaling networks in polarized epithelial cells. [Copyright &y& Elsevier]