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 s urvival through a receptor complex composed of the c-Ret proto-oncogen e 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 pos sible significance for c-Ret activation is presently unclear. Using ch emical crosslinking we demonstrate here a specific interaction between GDNF and GFR alpha-2 expressed in COS cells, albeit of a lower affini ty 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 st imulation of c-Ret tyrosine phosphorylation. We also describe the isol ation of a novel, more divergent member of the GFR alpha family, GFR a lpha-3, which did not bind GDNF directly, but was able to mediate cros slinking of GDNF to c-Ret when both receptors were coexpressed in COS cells. Thus, all three GFR alpha receptors mediate GDNF binding to c-R et with efficiencies GFR alpha-1>GFR alpha-2>GFR alpha-3. c-Ret showed high levels of constitutive tyrosine autophosphorylation upon overexp ression in COS cells, which was inhibited in a dose-dependent manner b y coexpression with any of the GFR alpha receptors, suggesting that GF R alpha s may also provide a gain control mechanism to increase the si gnal-to-noise ratio of the response to ligand. GFR alpha-2 showed a dy namic pattern of expression in rat brain, distinct from that of GFR al pha-1, characterized by high expression in cortex, basal forebrain, an d specific layers of the olfactory bulb, and low or no expression in s ubstantia nigra, cerebellum, and motor nuclei. GFR alpha-2, but not GF R alpha-3 mRNA expression was highly induced in several nuclei after s timulation with kainic acid. In contrast to GFR alpha-1 and GFR alpha- 2, GFR alpha-3 expression in postnatal and adult brain was highly rest ricted. Developmentally regulated expression of GFR alpha-3 was, howev er, detected in several peripheral organs and ganglia. Together, these results indicate complementary roles for GFR alpha receptors in the r egulation of c-Ret activity and the maintenance of distinct neuronal c ircuits in the central and peripheral nervous systems.