The epidermal growth factor receptor juxtamembrane domain has multiple basolateral plasma membrane localization determinants, including a dominant signal with a polyproline core

The epidermal growth factor (EGF) receptor is located predominantly in the basolateral membrane of polarized epithelia, where it plays a pivotal role during organogenesis and tissue homeostasis. We have shown previously that a 22-amino acid sequence in the EGF receptor juxtamembrane domain contains autonomous sorting information necessary for basolateral localization using the Madin-Darby canine kidney epithelial cell model. The goal of this study was to determine the molecular basis of EGF receptor basolateral membrane expression using site-directed mutagenesis to modify specific residues in this region. We now show that this sequence has two different, functionally redundant basolateral sorting signals with distinct amino acid requirements: one dependent on residues (LL659)-L-658 conforming to well-characterized leucine-based sorting signals, and a second containing a polyproline core comprising residues Pro(667) and Pro(670) ((PXXP670)-P-667). Our data also suggest that Arg(662) contributes to the function of the proline-based signal. (PXXP670)-P-667 was the dominant signal when both motifs were present and was more effective than (LL659)-L-658 at overriding strong apical sorting signals located in the same molecule. Site-directed mutations at Arg(662), Pro(667), and Pro(670) were also associated with increased apical expression of full-length EGF receptors, demonstrating for the first time that the juxtamembrane region is necessary for accurate polarized expression of the native molecule.