GPI-anchored proteins are largely localized to detergent-insoluble sphingolipid and cholesterol-rich lipid microdomains that exist as phase-separated so-called “lipid rafts” in the plasma membrane, where downstream signaling components (e.g., Src family kinases, see Fig. 4) are enriched (Simmons and Ikonen, 1997; Brown and London, 1998). Mobilization of RET to lipid rafts leads to more efficient intracellular signaling. Tansey and coworkers showed that only GPI-anchored but not soluble or transmembrane-anchored GFRα1 could recruit RET to lipid rafts (Tansey et al., 2000). However, another report finds that RET is recruited to lipid rafts by cis as well by trans mechanisms (Paratcha et al., 2001). The recruitment in cis is mediated by the GPI-anchored GFRα1 receptor in the cytoplasmic membrane, whereas upon activation in trans, it is mediated by intracellular proteins that normally reside in these rafts. RET can be activated in trans by exogenous GFRα1 when present either as a soluble or immobilized molecule in vitro. After sciatic nerve lesion, there is a marked up-regulation of mRNA expression of both gdnf and gfrα1 in Schwann cells. The increased expression occurs in a gradient with the highest levels of expression found proximal to the site of transection in the distal nerve (Naveilhan et al., 1997; Trupp et al., 1997). These findings suggested that GFRα1 might be involved in nerve regeneration. Supporting evidence for this notion was found in a recent study where cultured neuronal cells, Schwann cells, and injured sciatic nerve were shown to release biologically active soluble GFRα1 to the extracellular space, which indicates a naturally occurring release of this molecule in vivo and a biological relevance of trans activation by GFRα1 (Paratcha et al., 2001). Both soluble and immobilized GFRα1 potentiated neuronal differentiation and survival in response to GDNF independently of GPI-linked GFRα1 (Paratcha et al., 2001). Exogenous GFRα1 in the presence of GDNF, provides positional information and guides the directional growth of chick nodose and sympathetic nerves in culture (Ledda et al., 2002). The ability of soluble GFRα1 to activate the receptor might explain the different tissue distribution of the transcripts in vivo: gfrαs are more widely expressed than ret in the nervous system (Trupp et al., 1996; Yu et al., 1998; Fundin et al., 1999; Baudet et al., 2000; Mikaels et al., 2000), and there is a rapid and dynamic regulation of GFRα1 expression in terminal Schwann cells and targets of innervation that coincide with nerve invasion and formation of specific sets of sensory endings (Fundin et al., 1999). Our results confirm and extend these findings and show that GFRα1 presented as an artificial target in either soluble or bound form markedly affects neurite outgrowth independent of GDNF. Thus, there is biochemical and functional evidence that GFRα1 presented from the target could both in soluble and membrane bound form influence neurite growth. In the present study, we also investigated the GDNF-independent signaling pathways activated by soluble GFRα1 in cultured DRG neurons by the addition of known specific inhibitors to PI-3 kinase (LY294002), MAPK (PD98059), Src family kinases (PP2), and PLCγ (ET-18-OCH3), respectively. We found that presence of ET-18-OCH3 specifically blocked the effect of sGFRα1-induced neurite growth response in a dose-dependent manner. It has been shown in vitro that GFRα1, to some degree, can interact with RET in the absence of GDNF (Eketjäll et al., 1999). It is possible that GFRα1 plays an active role in axonal growth and guidance by binding to Ret, but we cannot exclude an interaction with partners different from Ret. For instance, the nerve growth-regulating transmembrane glycoprotein L1 was shown recently to interact with neuropilin-1 and to be required for sema3A to elicit a response on DRG axons (Castellani et al., 2000). Analogous to our results, the repulsion of sema3A can be converted to attraction by presenting L1 in a soluble form instead of bound. Candidates for GFRα1 cross-talk with other receptors are the integrins that transduces the axonal growth signal promoted by both fibronectin and laminin.