CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.
INTRODUCTIONEpithelial cells have distinct apical and basolateral membrane domains with different protein and lipid compositions. The asymmetry is essential for the multiple vectorial functions they perform (Rodriguez-Boulan and Nelson, 1989;Yeaman et al., 1999). The polarized protein distribution results from sorting mechanisms operating in the biosynthetic and recycling pathways that recognize specific sorting signals in plasma membrane proteins. Structural features believed to operate as apical sorting signals include glycosylphophatidylinositol anchors (Lisanti et al., 1989), N-glycans (Scheiffele et al., 1995), O-glycans (Yeaman et al., 1997;Jacob et al., 2000), and protein sequences in the transmembrane (Kundu et al., 1996;Lin et al., 1998), or cytoplasmic domains (Chuang and Sung, 1998;Rodriguez-Boulan and Gonzalez, 1999;Nelson and Yeaman, 2001). On the other hand, basolateral signals are formed by short peptide sequences usually found in the protein domain facing the cytosol (Le Gall et al., 1995;Yeaman et al., 1999;Mostov et al., 2000). They mainly include tyrosine motifs (consensus motif NPXY or YXXF) as well as di-leucine and di-hydrophobic residues (Matter et al., 1992;Aroeti et al., 1993;Hunziker and Fumey, 1994;Simonsen et al., 1997;Bonifacino and Dell'Angelica, 1999;Rodionov et al., 2000).Different types of epithelial cells vary widely in the final distribution of plasma membrane proteins or in the pathways that these proteins follow to the cell surf...