MUC1 membrane trafficking is modulated by multiple interactions.
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 YXXphi motif by the mu2 subunit. The tyrosine-phosphorylated 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.
Laboratory of Epithelial Cell Biology, Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Carol L
CL
Kinlough
Paul A
PA
Poland
James B
JB
Bruns
Keri L
KL
Harkleroad
Rebecca P
RP
Hughey
D033962:Adaptor Protein Complex 2 / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism / Q000502:physiology*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002966:Clathrin / Q000737:chemistry; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006031:Glycosylation; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D018909:src Homology Domains
D033962:Adaptor Protein Complex 2; D048868:Adaptor Proteins, Signal Transducing; D002966:Clathrin; D018076:DNA, Complementary; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D018396:Mucin-1; D011993:Recombinant Fusion Proteins; D014443:Tyrosine