GPI-anchored proteins and detergent-resistant membrane domains.
Glycosylphosphatidylinositol (GPI)-anchored proteins are expressed on the apical surface of polarized epithelial cells. The anchor may act as an apical sorting signal by associating with clusters or rafts of apically directed glycosphingolipids (GSL). We have previously shown that endogenous GPI-anchored proteins and stably transfected placental alkaline phosphatase (PLAP) can be isolated from detergent lysates of cultured epithelial cells in association with a detergent-insoluble membrane that is rich in GSL. Here, we investigate the behavior of a hybrid GPI-anchored protein, GThy, that contains the ectodomain of the vesicular stomatitis virus glycoprotein (VSV-G) and a GPI-anchor from the Thy1 protein. We have previously shown that GThy is efficiently (85-90%) targeted to the apical surface of MDCK cells. Here we show that the protein also becomes insoluble in Triton X-100 as it moves through the secretory pathway of these cells. However, the degree of Triton X-100 insolubility is never as great as that seen for PLAP. This may result from the fact that it is an engineered protein, as the same behavior has been reported for another hybrid GPI-anchored protein. In addition, GThy is rapidly lost from MDCK cells by release into the media, with a t1/2 of about 50 min. This turnover appears to be mediated by a cell-surface protease that may recognize viral glycoproteins.
Brown
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Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215.
D000469:Alkaline Phosphatase; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D010450:Endopeptidases / Q000276:immunology; D058851:GPI-Linked Proteins; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism*
C028391:G protein, vesicular stomatitis virus; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental; D010450:Endopeptidases