We have studied ligand-independent signaling by the nerve growth factor receptor TrkA in PC12 clones, under conditions of receptor overexpression. Our results indicate that TrkA-overexpressing PC12 clones display constitutive receptor activation, involving both the mature, 140-kDa form and the immature, intracellular 110-kDa form of the receptor. Phosphorylation of Tyr 674/675, located in the activation loop domain and reflecting TrkA kinase activity, appears particularly prominent in the immature form of the receptor. Constitutive receptor activation is able to chronically stimulate the PI-3 kinase/Akt as well as the mitogen-activated protein kinase pathways, leading to ligand-independent neurite extension. Under conditions of overexpression, a significant fraction of the receptor is retained intracellularly by thiol-mediated mechanisms. Exposure of the cells to reducing agents promotes translocation of the intracellular pool of the receptor to the plasma membrane and suppresses ligand-independent neurite outgrowth. Our results suggest that the levels of expression of TrkA, both intracellularly and at the cell surface, may act to modulate its kinase activity and generate ligand-independent downstream signaling. (C) 2002 Elsevier Science (USA).