TrkA glycosylation regulates receptor localization and activity.
The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, we show here that both conserved and variable N-glycosylation sites are used during maturation of TrkA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrkA. Unglycosylated TrkA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-gamma. Second, glycosylation is required to localize TrkA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, we show that unglycosylated active Trk receptors are trapped intracellularly. Furthermore, the unglycosylated active TrkA receptors are unable to activate kinases in the Ras-MAP kinase pathway, MEK and Erk. Consistent with these biochemical observations, unglycosylated TrkA core protein does not promote neuronal differentiation in Trk PC12 cells even at high levels of constitutive catalytic activity.
Watson
F L
FL
Harvard Medical School and Department of Neurology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115.
Porcionatto
M A
MA
Bhattacharyya
A
A
Stiles
C D
CD
Segal
R A
RA
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009474:Neurons / Q000737:chemistry* / Q000201:enzymology*; D016716:PC12 Cells; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D011506:Proteins; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D020703:ras GTPase-Activating Proteins; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma