The mode of anchorage to the cell surface determines both the function and the membrane location of Thy-1 glycoprotein.
The surface glycoprotein, Thy-1, when expressed by transfection in NG115/401L neural cells, inhibits their neurite outgrowth over astrocytes. We have investigated the role of the glycosylphosphatidylinositol anchor of Thy-1 in this inhibition. Hybrid molecules, in which the lipid anchor was replaced by polypeptide transmembrane domains, were expressed by transfection. Lines expressing Thy-1 with the transmembrane and full cytoplasmic domains of NCAM-140, or with the transmembrane and truncated cytoplasmic domain of CD8, were not inhibited in their ability to extend neurites over astrocytes. Truncation of the cytoplasmic domain of NCAM-140 to just two amino acids, however, produced a transmembrane form of Thy-1 that, when expressed at high levels, inhibited neurite outgrowth. All forms of Thy-1 were concentrated in clusters that occurred primarily on fine filopodia. In double transfectants expressing normal Thy-1 and Thy-1 with the full NCAM cytoplasmic tail, the clusters of each form were separate, with no instances of the transmembrane form being found within the clusters of lipid-anchored Thy-1. Thy-1 with the two-amino-acid cytoplasmic domain of NCAM also occurred in clusters separate from those occupied by lipid-anchored Thy-1, but substantial 'invasion' of the clusters of normal Thy-1 by this transmembrane construct occurred. We suggest that the ability of this hybrid protein to enter the lipid-anchored clusters enables it to activate the signalling pathways that normal Thy-1 uses. Thus the membrane anchor, in targetting Thy-1 to different microdomains on the cell surface, determines its ability to inhibit neurite outgrowth on astrocytes.
Tiveron
M C
MC
Laboratory of Neurobiology (Norman and Sadie Lee Centre), National Institute for Medical Research, Mill Hill, London, UK.
Nosten-Bertrand
M
M
Jani
H
H
Garnett
D
D
Hirst
E M
EM
Grosveld
F
F
Morris
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D001483:Base Sequence; D002448:Cell Adhesion; D015816:Cell Adhesion Molecules, Neuronal / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D005910:Glioma; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006822:Hybrid Cells; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016501:Neurites / Q000502:physiology*; D009447:Neuroblastoma; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D015183:Restriction Mapping; D018800:Thy-1 Antigens / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection
D015816:Cell Adhesion Molecules, Neuronal; D017261:Glycosylphosphatidylinositols; D009838:Oligodeoxyribonucleotides; D018800:Thy-1 Antigens