title: Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells).
abstract: Our previous studies have shown that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. GM1 is also known to be a major constituent of caveola or glycosphingolipid-enriched microdomain (GEM) of the plasma membrane. In order to study the effect of the glycosylation of Trk on the formation of GM1-Trk complex and subcellular distribution of this protein, we generated PC12 cells stably overexpressing Trk (PCtrk). Pretreatment of this stable clones with tunicamycin, a potent inhibitor of N-glycosylation, caused the appearance of unglycosylated Trk core protein. These unglycosylated Trk can hardly respond to its ligand, NGF. Sucrose density gradient analysis revealed that unglycosylated Trk core protein was recovered in high density fractions, whereas most of GM1 is present in low density fractions corresponding to caveola or GEM fractions. Moreover, these unglycosylated Trk proteins lose their ability to form a complex with GM1, although GM1 is present in the same high density fractions. These data strongly suggest that spatial segregation of GM1 from the Trk protein by the inhibition of the glycosylation of Trk might be an important molecular mechanism for the unresponsiveness to NGF. Moreover, the binding site of GM1 in the Trk protein might act as an important determinant for the normal trafficking of the Trk protein within the cells.
authors.affiliation: The Second Department of Internal Medicine, Faculty of Medicine, Fukui Medical University, Japan. mutoh@fmsrsa.fukui-med.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Mutoh
authors.forename: T
authors.initials: T
authors.lastname: Hamano
authors.forename: A
authors.initials: A
authors.lastname: Tokuda
authors.forename: M
authors.initials: M
authors.lastname: Kuriyama
mesh_terms: D000818:Animals; D002999:Clone Cells; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006031:Glycosylation; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects; D010766:Phosphorylation; D051381:Rats; D020917:Receptor, trkA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D014415:Tunicamycin; D005677:G(M1) Ganglioside; D020932:Nerve Growth Factor; D020917:Receptor, trkA