NCAM requires a cytoplasmic domain to function as a neurite outgrowth-promoting neuronal receptor.
The neural cell adhesion molecule (NCAM) promotes axonal growth via a homophilic binding mechanism by acting both as a neuronal receptor and a substratum ligand. We have previously shown that the GPI-linked 120-kDa isoform of NCAM, which lacks a cytoplasmic domain, is effective at promoting neurite outgrowth as a cellular ligand. To test its ability to function as a neuronal receptor, we have transfected PC12 cells with a cDNA encoding human GPI-linked NCAM and tested clones displaying stable cell surface expression of this isoform for their ability to respond to NCAM in a cellular substratum. Although they continued to express endogenous transmembrane rat isoforms of NCAM (140 and 180 kDa), PC12 cells expressing the GPI-linked NCAM lost their ability to extend neurites in response to substratum associated NCAM. However, their outgrowth response to N-cadherin and other activators of axonal growth was undiminished. Removal of GPI-linked NCAM from the surface of these clones using phosphatidylinositol-specific phospholipase C (PIPLC) fully restored their responsiveness to NCAM, indicating that the inhibition was a direct consequence of cell surface expression of this "dominant negative" isoform of NCAM. We have previously shown that expression of transfected 140- and 180-kDa isoforms of human NCAM in PC12 cells does not result in a loss of the neurite outgrowth response to NCAM. However, we show that deletion of the cytoplasmic domain of the 140-kDa isoform has the same effect as expression of GPI-linked NCAM. We conclude that the cytoplasmic domain of NCAM is required for an appropriate neurite outgrowth response.
Saffell
J L
JL
Department of Experimental Pathology, UMDS, Guy's Hospital, London, United Kingdom.
Doherty
P
P
Tiveron
M C
MC
Morris
R J
RJ
Walsh
F S
FS
D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology; D015820:Cadherins / Q000494:pharmacology; D002999:Clone Cells; D018920:Coculture Techniques; D003593:Cytoplasm / Q000502:physiology; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D019006:Neural Cell Adhesion Molecules / Q000096:biosynthesis / Q000502:physiology*; D016501:Neurites / Q000502:physiology* / Q000648:ultrastructure; D016716:PC12 Cells; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D015820:Cadherins; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D019006:Neural Cell Adhesion Molecules; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase