MUC1 Membrane Trafficking Is Modulated by Multiple Interactions

Abstract

MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [35 S]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr 20 and Tyr 60 were both required for efficient endocytosis. Mutation of Tyr 20 significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y 20 HPM is recognized as a YXX motif by the 2 subunit. The tyrosinephosphorylated Y 60 TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr 60 blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1.MUC1 is a mucin-like type 1 transmembrane protein normally expressed on the apical surface of epithelial cells (for review, see Refs. 1 and 2). It is synthesized as a single propeptide and cleaved while in the endoplasmic reticulum to yield the large amino-terminal subunit containing O-glycosylated near-perfect tandem repeats, and the smaller carboxyl-terminal subunit containing the membrane anchor and cytoplasmic tail (3). The resulting subunits remain tightly associated; the heterodimer is SDS-labile but is resistant to boiling, urea, sulfhydryl reduction, peroxide, high salt, or low pH (4). In carcinomas, cells often lose polarity, and MUC1 is found on all surfaces of the plasma membrane. In general, MUC1 expression in tumors from breast, lung, kidney, and thyroid correlates with an aggressive tumor and increased metastasis (5-11). However, a recent immunohistological study of 71 breast carcinomas, coupled with a review of the literature, indicates that it is actually the aberrant localization of intracellular MUC1 or MUC1 in a non-apical pattern that is associated with a worse prognosis for the patient (11).The reason for the intracellular (cytoplasmic) MUC1 staining in breast carcinomas is not clear. Schroeder et al. (12) found a tumor-specific complex between MUC1 and ␤-catenin in the cytoplasm and nucleus in metastatic lesions of breast cancer patients; aberrant cytoplasmic and nuclear levels of activated ␤-catenin in breast tumors also correlates with a poor prognosis for the patient (13). ␤-Catenin binding to the cytop...

Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors

“…Because Pseudomonas aeruginosa requires efficient internalization and activation along a multistep pathway for cell killing, the intracellular routing of the receptor/toxin complex rather than the binding to the target receptor on the cell surface may be rate limiting (28). The underglycosylation in MUC1 alters its internalization and subcellular localization due to truncated O-glycans (13). As toxin requires internalization for its cytotoxic effects, the membrane trafficking rate of the underglycosylated MUC1 in cancer cells would limit the efficacy of the immunotoxin.…”

“…Consequently, this immunogenic peptide sequence is well suited as a target for cancer immunotherapy (9 -11). In addition, aberrant glycosylation of MUC1 also plays an important role in enhancing the internalization of MUC1 into the cytoplasm (12,13), making MUC1 a very attractive therapeutic target for immunotherapeutic reagents, including those that require delivery.…”

Lipid rafts: signaling and sorting platforms of cells and their roles in cancer

“…During this process a follow-up sialylation of glycan chains occurs [8]. In epithelial cells, the endocytosis and vesicular transport of MUC1 follows a dynamin-independent, clathrin-mediated pathway [9] requiring two specific tyrosine motifs in the highly conserved cytosolic MUC1 domain (Tyr20, Tyr60) as binding sites for the adaptor protein complex 2 and Grb2, respectively [10], whereas the re-entry of endocytosed MUC1 into the secretory pathway (recycling) was dependent on S-palmitoylation near the transmembrane domain [11]. In cancer cells, we found that MUC1 is contained in plasma membranous lipid rafts as platforms (B) Lipid rafts (dark blue bars) represent nanoscale assemblies of proteins that organize these proteins into platforms for targeted traffic within cells.…”

MUC1 oncoprotein is targeted to mitochondria by heregulin-induced activation of c-Src and the molecular chaperone HSP90

“…With transformation of normal epithelia to carcinomas, MUC1 is aberrantly overexpressed in the cytosol and over the entire cell membrane (Kufe et al, 1984;Perey et al, 1992). Cell membrane-associated MUC1 is targeted to endosomes by clathrin-mediated endocytosis (Kinlough et al, 2004). In addition, MUC1-C, but not MUC1-N, is targeted to the nucleus (Huang et al, 2003;Li et al, 2003a, b, c;Wen et al, 2003;Baldus et al, 2004;Wei et al, 2005) and mitochondria (Ren et al, 2004).…”