Mobility of TrkA is regulated by phosphorylation and interactions with the low-affinity NGF receptor.
The nerve growth factor (NGF) receptor is a complex of two proteins, gp75 and the tyrosine kinase TrkA. Using fluorescence recovery after photobleaching, we have studied the diffusion properties of the TrkA receptor. For PC12 cells that express both gp75 and TrkA, TrkA was relatively immobile with only 28 +/- 1% of receptor molecules free to diffuse with D = (3.64 +/- 0.23) x 10(-9) cm2/s. Addition of NGF decreased the mobile fraction to 21 +/- 1% with D = (4.11 +/- 0.18) x 10(-9) cm2/s. Using the Sf9 baculovirus expression system, we were able to study TrkA in the absence and presence of gp75. On Sf9 cells, TrkA showed a mobile fraction of 46 +/- 2% with D = (2.64 +/- 0.21) x 10(-9) cm2/s in the absence of gp75 and 43 +/- 2% with D = (2.31 +/- 0.25) x 10(-9) cm2/s in its presence. Thus, gp75 did not alter TrkA mobility. Addition of NGF to the medium approximately halved the mobile fraction for TrkA in both the absence and presence of gp75. However, using a kinase-deficient mutant of TrkA, we found that ligand-induced immobilization requires an active kinase in the absence of gp75 but not in its presence. In addition, using point mutations at specific TrkA autophosphorylation sites, we determined that mobility is controlled by multiple phosphorylation sites, but the SHC binding site at Y490 may be particularly important for ligand-induced immobilization of TrkA. Therefore, two mechanisms lead to NGF-induced immobilization of TrkA--the first resulting from autophosphorylation of TrkA and the second occurring through TrkA's association with gp75.
Wolf
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Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA. Wolf@Sci.Wfbr.edu
McKinnon-Thompson
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Daou
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Stephens
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Kaplan
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Ross
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D000818:Animals; D005453:Fluorescence; D006801:Humans; D016716:PC12 Cells; D010766:Phosphorylation; D010777:Photochemistry; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D020800:Receptor, Nerve Growth Factor; D020917:Receptor, trkA / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D018411:Spodoptera
D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D020917:Receptor, trkA