Figure 1. RET protein.

Schematic representation of the two RET isoforms, RET9 and RET51, with the signal peptide (SP), cadherin-like (CAD), cysteine-rich (CYS), transmembrane (TM), and tyrosine kinase (TK) domains.

comprises four tandemly repeated cadherin-like domains and binds specifically to Ca2+ ions (Anders, J. et al., 2001), supporting the hypothesis of RET as a distant member of the cadherin superfamily. RET is the only member of this superfamily containing an intrinsic tyrosine kinase domain, suggesting that RET may have arisen by the recombination of an ancestral cadherin with a protein-tyrosine kinase (Anders, J. et al.,

Twenty seven of 28 cysteine (Cys) residues in the cysteine-rich domain are conserved among species suggesting a critical role for these residues in formation of intramolecular disulfide bonds and thus in determining the tertiary structure of RET proteins (Takahashi, M. et al., 1988; Iwamoto, T. et al., 1993). A single transmembrane domain of RET is followed by an evolutionary conserved tyrosine kinase (TK) domain, which is interrupted by a 27 amino acids kinase insert (Takahashi, M. et al., 1987).

The RET gene is alternatively spliced to yield two main protein isoforms of 1072 (RET9 or short isoform) or 1114 (RET51 or long isoform) amino acids (Tahira, T. et al., 1990) differing at the C-terminus region, by displaying 9 or 51 unrelated aminoacids (Figure 1). The RET9 and RET51 isoforms are evolutionary highly conserved over a broad range of species, suggesting that distinct isoforms can exert different roles in physiological functions of RET (Carter, M. T. et al., 2001).

RET is the signaling component of a multiprotein receptor complex involving members of two distinct groups of proteins: a soluble ligand belonging to the glial cell line-derived neurotrophic factor (GDNF) family and a glycosyl-phosphatidylinositol (GPI)-membrane anchored co-receptor belonging to GDNF family receptor RET remained an orphan receptor until 1996 when GDNF was identified as the ligand of RET (Durbec, P. et al., 1996; Trupp, M. et al., 1996; Vega, Q. C. et al., 1996; Treanor, J. J. et al., 1996). Four members in the GDNF family ligands have now been characterized: Glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN) (Figure 2). They represent a new subclass of the transforming growth superfamily. They are secreted as disulfide-linked dimers, function as homodimers and are all neuronal survival factors (reviewed in Baloh, R. H. et al., 2000; Saarma, M., 2000). GFRa molecules do not display intracellular domains but are anchored to the cell membrane via a glycosyl-phosphatidylinositol (GPI) linkage. The ligands GDNF, neurturin, artemin and persephin use and as the preferred receptors, respectively (reviewed in Airaksinen, M. S. et al., 2002).



Figure 2. GDNF family ligands with their receptors.

Homodimeric GDNF family ligands activate the transmembrane RET tyrosine kinase by binding with high affinity to different GPI-linked GFRa receptors. Binding of ligand-GFRa complex to RET triggers its homodimerization, phosphorylation and intracellular signaling. The complete lines represent preferred functional binding; dotted lines may not be significant in vivo.

Figure 2. GDNF family ligands with their receptors.

Homodimeric GDNF family ligands activate the transmembrane RET tyrosine kinase by binding with high affinity to different GPI-linked GFRa receptors. Binding of ligand-GFRa complex to RET triggers its homodimerization, phosphorylation and intracellular signaling. The complete lines represent preferred functional binding; dotted lines may not be significant in vivo.

The GDNF and GFRa family members show both distinct and overlapping expression patterns (Baloh, R. H. et al., 2000), suggesting that activation of RET by formation of ligand-receptor complexes is a tightly regulated process. GDNF can also signal via GFRal in a RET-independent manner (Poteryaev, D. et al., 1999; Trupp, M. et al., 1999). It was recently demonstrated that in hippocampal and cortical neurons GDNF/GFRctl make a complex with neural cell adhesion molecule (NCAM) and induce activation of Fyn and FAK promoting migration and axonal growth independently of RET (Paratcha, G. et al., 2003).

Role of RET signaling during development

The RET signaling has a critical role in the development of the enteric nervous system (ENS) and kidney, as attested by the similar and peculiar phenotype of mice with null mutations in RET, GDNF and GFRa. genes. They all show severe defects in enteric innervation and renal differentiation (Schuchardt, A. et al., 1994; Sanchez, M. P. et al., 1996; Cacalano, G. et al., 1998). During vertebrate embryogenesis RET is expressed in the developing excretory system, in all lineages of the peripheral nervous system (PNS) and in motor and catecholaminergic neurons of the central system (CNS), including ventral midbrain dopaminergic neurons (Avantaggiato, V. et al., 1994; Durbec, P. et al., 1996; Marcos, C. et al., 1996; Pachnis, al., 1993; Trupp, M. et al., 1997; Tsuzuki, T.

et al., 1995; Young, H. M. et al., 1998). Despite the widespread expression of RET in the nervous system of vertebrates, mutations of this locus affect, albeit drastically, only a subset of PNS ganglia. Thus, loss of function mutations of RET in humans lead to Hirschsprung's disease, a condition characterized by the absence of enteric ganglia from the terminal colon (Edery, P. et al., 1994; Romeo, G. et al., 1994).

Organ culture experiments and transgenic approaches have shown that RET signaling triggered by GDNF is essential for the initial ureteric budding and subsequent branching of kidney during mammalian embryogenesis (reviewed in Sariola, H. et al., 1999). In the mammalian kidney, the ureteric bud (UB), which expresses RET, induces epithelial differentiation of the nephrogenic mesenchyme, which expresses GDNF and, in turn, promotes branching of the bud. The co-receptor is expressed by both the nephrogenic mesenchyme and the UB. Thus, GDNF/RET signaling regulates the reciprocal inductive interactions between the UB and the nephrogenic mesenchyme. Recent data indicate that only RET9 seems to be critically important for kidney morphogenesis and enteric nervous system development, whereas RET51 appears dispensable (de Graaff, E. et al., 2001). However, RET51 has been suggested to be related to differentiation events in later kidney organogenesis (Lee, D. C. et al., 2002). Besides neuronal tissues and kidney, GDNF was recently implicated in sperm differentiation. GDNF is expressed by Sertoli cells, and RET and are displayed by a subset of spermatogonia including the stem cells for spermatogenesis (Meng, X. et al., 2000). Gene-targeted mice with one GDNF-null allele show depletion of spermatogenic stem cells, whereas mice overexpressing GDNF accumulate undifferentiated spermatogonia. Thus, GDNF contributes to the paracrine regulation of spermatogonial self-renewal and differentiation. The regulatory functions of GDNF/RET signaling in kidney morphogenesis and spermatogenesis indicate that the dosage of GDNF has both quantitative (e.g. number of branches from the UB) and qualitative (e.g. cell lineage determination of spermatogonia) dose-dependent effects in the target tissue. Thus, the expression of RET and GFRa on a cell defines the target cell type for GDNF, while the quality and nature of the response are regulated by the dosage of the ligand (reviewed in Sariola, H., 2001).

RET signaling

Ligand stimulated wild-type RET, as well as constitutive active oncogenic RET mutants, are phosphorylated at specific cytoplasmic tyrosine residues (Liu, X. et al., 1996; Coulpier, M. et al., 2002). Tyrosine autophosphorylation is required for downstream RET signaling. Recent studies have shown differences between the two isoforms, RET9 and RET51, in the intracellular signaling (Lorenzo, M. J. et al., 1997; Borrello, M. G. et al., 2002; Tsui-Pierchala, B. A. et al., 2002) as well as in the ligand-induced activation of RET inside or outside the lipid rafts (Paratcha, G. et al., 2001), which are detergent-insoluble sphingolipid and cholesterol-rich lipid microdomains that exist as phase-separated "rafts" in the plasma membrane (reviewed in Simons, K. et al., 1997; Brown, D. A. et al., 1998). Lipid rafts may be considered highly specialized signaling organelles, which assist to compartmentalize different sets of signaling molecules at both sides of the plasma membrane allowing them to interact a.

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