title: Identification of a novel mono-leucine basolateral sorting motif within the cytoplasmic domain of amphiregulin.
abstract: Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand, amphiregulin (AREG), are transmembrane proteins delivered to the BL membrane in polarized epithelial cells. Herein, we show that the cytoplasmic domain of AREG (ACD) contains dominant BL sorting information; replacement of the cytoplasmic domain of apically targeted nerve growth factor receptor with the ACD redirects the chimera to the BL surface. Using sequential truncations and site-directed mutagenesis of the ACD, we identify a novel BL sorting motif consisting of a single leucine C-terminal to an acidic cluster (EEXXXL). In adaptor protein (AP)-1B-deficient cells, newly synthesized AREG is initially delivered to the BL surface as in AP-1B-expressing cells. However, in these AP-1B-deficient cells, recycling of AREG back to the BL surface is compromised, leading to its appearance at the apical surface. These results show that recycling, but not delivery, of AREG to the BL surface is AP-1B dependent.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Gephart
authors.forename: Bhuminder
authors.initials: B
authors.lastname: Singh
authors.forename: James N
authors.initials: JN
authors.lastname: Higginbotham
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Franklin
authors.forename: Alfonso
authors.initials: A
authors.lastname: Gonzalez
authors.forename: Heike
authors.initials: H
authors.lastname: Fölsch
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Coffey
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D066258:Amphiregulin; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism*; D004285:Dogs; D066255:EGF Family of Proteins; D004847:Epithelial Cells / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D018374:LLC-PK1 Cells; D007930:Leucine / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D013552:Swine
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D017475:Receptors, Nerve Growth Factor; D066246:ErbB Receptors; D007930:Leucine