Multiple GPI-anchored receptors control GDNF-dependent and independent activation of the c-Ret receptor tyrosine kinase

Glial cell line-derived neurotrophic factor (GDNF) mediates neuronal survival through a receptor complex composed of the c-Ret proto-oncogene and GFR alpha-1, a member of a family of GPI-anchored receptors. The extent of cross-talk between GDNF and GFR alpha receptors and its possible significance for c-Ret activation is presently unclear. Using chemical crosslinking we demonstrate here a specific interaction between GDNF and GFR alpha-2 expressed in COS cells, albeit of a lower affinity than the one between GDNF and GFR alpha-1. In addition, GFR alpha-2 mediated crosslinking of GDNF to c-Ret as well as ligand-dependent stimulation of c-Ret tyrosine phosphorylation. We also describe the isolation of a novel, more divergent member of the GFR alpha family, GFR alpha-3, which did not bind GDNF directly, but was able to mediate crosslinking of GDNF to c-Ret when both receptors were coexpressed in COS cells. Thus, all three GFR alpha receptors mediate GDNF binding to c-Ret with efficiencies GFR alpha-1>GFR alpha-2>GFR alpha-3. c-Ret showed high levels of constitutive tyrosine autophosphorylation upon overexpression in COS cells, which was inhibited in a dose-dependent manner by coexpression with any of the GFR alpha receptors, suggesting that GFR alpha s may also provide a gain control mechanism to increase the signal-to-noise ratio of the response to ligand. GFR alpha-2 showed a dynamic pattern of expression in rat brain, distinct from that of GFR alpha-1, characterized by high expression in cortex, basal forebrain, and specific layers of the olfactory bulb, and low or no expression in substantia nigra, cerebellum, and motor nuclei. GFR alpha-2, but not GFR alpha-3 mRNA expression was highly induced in several nuclei after stimulation with kainic acid. In contrast to GFR alpha-1 and GFR alpha-2, GFR alpha-3 expression in postnatal and adult brain was highly restricted. Developmentally regulated expression of GFR alpha-3 was, however, detected in several peripheral organs and ganglia. Together, these results indicate complementary roles for GFR alpha receptors in the regulation of c-Ret activity and the maintenance of distinct neuronal circuits in the central and peripheral nervous systems.