In patients with medullary thyroid carcinoma (MTC) and type 2A multiple endocrine neoplasia (MEN2A), mutations of cysteine residues in the extracellular juxtamembrane region of the RET receptor tyrosine kinase cause the formation of covalent receptor dimers linked by intermolecular disulfide bonds between unpaired cysteines, followed by oncogenic activation of the RET kinase. The close proximity to the plasma membrane of the affected cysteine residues prompted us to investigate the possible role of the transmembrane (TM) domain of RET (RET-TM) in receptor-receptor interactions underlying dimer formation. Strong self-association of the RET-TM was observed in a biological membrane. Mutagenesis studies indicated the involvement of the evolutionary conserved residues Ser-649 and Ser-653 in RET-TM oligomerization. Unexpectedly, RET-TM interactions were also abrogated in the A639G/A641R double mutant, first identified in a sporadic case of MTC. In agreement with this, no transforming activity could be detected in full-length RET carrying the A639G and A641R mutations, which remained fully responsive to glial cell-line-derived neurotrophic factor (GDNF) stimulation. When introduced in the context of C634R - a cysteine replacement that is prevalent in MEN2A cases - the A639G/A641R mutations significantly reduced dimer formation and transforming activity in this otherwise highly oncogenic RET variant. These data suggest that a strong propensity to self-association in the RET-TM underlies - and may be required for - dimer formation and oncogenic activation of juxtamembrane cysteine mutants of RET, and explains the close proximity to the plasma membrane of cysteine residues implicated in MEN2A and MTC syndromes.

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr 687 in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr 687 and association with components of the Tyr 1062 signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser 696 , a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr 687 as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.Neurotrophic factors regulate neuronal survival, morphology, and function and are essential for the development and maintenance of the nervous system. Together with the neurotrophins, glial cell line-derived neurotrophic factor (GDNF) 4 and related family members are among the most important neurotrophic factors in mammals. Because of the potent prosurvival and neuroprotective effects of GDNF on midbrain dopaminergic neurons, much of the initial interest in GDNF was focused on its possible therapeutic effects in Parkinson disease (1, 2). It has more recently been appreciated that GDNF and related factors affect a host of important processes in mature neurons and their precursors and also outside the nervous system. GDNF signals by binding to a receptor complex formed by the ligand-binding subunit GFR␣1 and the signaling subunit RET, a member of the receptor tyrosine kinase (RTK) family (3). GFR␣1 also associates with the neural cell adhesion molecule, NCAM, to mediate the effects of GDNF independently of RET (4 -6). Some of the activities of GDNF can also be mediated by GFR␣1 in the absence of either RET or NCAM, suggesting alternative mechanisms of transmembrane signaling (7,8). A thorough understanding of the molecular mechanisms by which GDNF functions in different contexts will be essential to elucidating its contribution to nervous system development and exploiting its therapeutic poten...

GDNF (glial cell line-derived neurotrophic factor) promotes the differentiation and migration of GABAergic neuronal precursors of the medial ganglionic eminence (MGE). These functions are dependent on the GPI-anchored receptor GFRα1, but independent of its two known transmembrane receptor partners RET and NCAM. Here we show that soluble GFRα1 is also able to promote differentiation and migration of GABAergic MGE neurons. These activities require endogenous production of GDNF. Although GDNF responsiveness is abolished in Gfra1−/− neurons, it can be restored upon addition of soluble GFRα1, a result that is only compatible with the existence of a previously unknown transmembrane signaling partner for the GDNF-GFRα1 complex in GABAergic neurons. The roles of two candidate transmembrane receptors previously implicated in GABAergic interneuron development - MET, a receptor for hepatocyte growth factor (HGF), and ErbB4, the neuregulin receptor – were examined. GDNF did not induce the activation of either receptor, nor did inhibition of MET or ErbB4 impair GDNF activity in GABAergic MGE neurons. Unexpectedly, however, inhibition of MET or HGF per se promoted neuronal differentiation and migration and enhanced the activity of GDNF on MGE neurons. These effects were dependent on endogenous GDNF and GFRα1, suggesting that MET signaling negatively regulates GDNF activity in the MGE. In agreement with this, Met mutant MGE neurons showed enhanced responses to GDNF and inhibition of MET or HGF increased Gfra1 mRNA expression in MGE cells. In vivo, expression of MET and GFRα1 overlapped in the MGE, and a loss-of-function mutation in Met increased Gfra1 expression in this region. Together, these observations demonstrate the existence of a novel transmembrane receptor partner for the GDNF–GFRα1 complex and uncover an unexpected interplay between GDNF–GFRα1 and HGF–MET signaling in the early diversification of cortical GABAergic interneuron subtypes.