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, g p75 and the tyrosine kinase TrkA. Using fluorescence recovery after ph otobleaching, we have studied the diffusion properties of the TrkA rec eptor. For PC12 cells that express both gp75 and TrkA, TrkA was relati vely immobile with only 28 +/- 1% of receptor molecules free to diffus e with D = (3.64 +/- 0.23) x 10(-9) cm(2)/s. Addition of NGF decreased the mobile fraction to 21 +/- 1% with D = (4.11 +/- 0.18) x 10(-9) cm (2)/s. Using the Sf9 baculovirus expression system, we were able to st udy TrkA in the absence and presence of gp75. On Sf9 cells, TrkA showe d a mobile fraction of 46 +/- 2% with D = (2.64 +/- 0.21) x 10(-9) cm( 2)/s in the absence of gp75 and 43 +/- 2% with D = (2.31 +/- 0.25) x 1 0(-9) cm(2)/s in its presence. Thus, gp75 did not alter TrkA mobility. Addition of NGF to the medium approximately halved the mobile fractio n for TrkA in both the absence and presence of gp75. However, using a kinase-deficient mutant of TrkA, we found that ligand-induced immobili zation requires an active kinase in the absence of gp75 but not in its presence. In addition, using point mutations at specific TrkA autopho sphorylation sites, we determined that mobility is controlled by multi ple phosphorylation sites, but the SHC binding site at Y490 may be par ticularly important for ligand-induced immobilization of TrkA. Therefo re, two mechanisms lead to NGF-induced immobilization of TrkA-the firs t resulting from autophosphorylation of TrkA and the second occurring through TrkA's association with gp75.