THE NEUROTROPHIN RECEPTOR, GP75, FORMS A COMPLEX WITH THE RECEPTOR TYROSINE KINASE TRKA

The high-affinity NGF receptor is thought to be a complex of two recep tors, gp75 and the tyrosine kinase TrkA, but direct biochemical eviden ce for such an association has been lacking. In this report, we demons trate the existence of such a gp75-TrkA complex by a copatching techni que. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribut ion of TrkA is probed with an anti-TrkA antibody and fluorescent secon dary antibody. We utilize a baculovirus-insect cell expression system which allows high level expression of wild-type and mutated NGF recept ors. TrkA and gp75 copatch in both the absence and presence of NGF. Th is association is specific, since gp75 does not copatch with other tyr osine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA ar e required for copatching, we used a series of TrkA-Tor chimeric recep tors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains shows partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copa tching. A point mutation which inactivates the TrkA kinase has no effe ct on copatching, indicating that this enzymatic activity is not requi red for association with gp75. Hence, although interactions between th e gp75 and TrkA extracellular domains are sufficient for complex forma tion, interactions involving other receptor domains also play a role.