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Glial cell line-derived neurotrophic factor (GDNF) has been shown to signal through a multicomponent receptor complex consisting of the Ret receptor tyrosine kinase and a member of the GFRalpha family of glycosylphosphatidylinositol-anchored receptors. In the current model of GDNF signaling, Ret delivers the intracellular signal but cannot bind ligand on its own, while GFRalphas bind ligand but are thought not to signal in the absence of Ret. We have compared signaling pathways activated by GDNF in two neuronal cell lines expressing different complements of GDNF receptors. In a motorneuron-derived cell line expressing Ret and GFRalphas, GDNF stimulated sustained activation of the Ras/ERK and phosphatidylinositol 3-kinase/Akt pathways, cAMP response element-binding protein phosphorylation, and increased c-fos expression. Unexpectedly, GDNF also promoted biochemical and biological responses in a line of conditionally immortalized neuronal precursors that express high levels of GFRalphas but not Ret. GDNF treatment did not activate the Ras/ERK pathway in these cells, but stimulated a GFRalpha1-associated Src-like kinase activity in detergent-insoluble membrane compartments, rapid phosphorylation of cAMP response element-binding protein, up-regulation of c-fos mRNA, and cell survival. Together, these results offer new insights into the dynamics of GDNF signaling in neuronal cells, and indicate the existence of novel signaling mechanisms directly or indirectly mediated by GFRalpha receptors acting in a cell-autonomous manner independently of Ret.

Nerve growth factor (NGF) supports the survival and differentiation of distinct populations of peripheral and central neurons. NGF binds to two classes of cell-surface receptors, the protein tyrosine kinase TrkA and the smaller p75 receptor lacking intrinsic catalytic activity. It has been suggested that both receptors are required for NGF high affinity binding, although TrkA appears to be sufficient for transducing most of the biological effects of NGF. Some evidence suggests that p75 could play a modulatory role on TrkA activation by an as yet unknown mechanism. In this study, we have investigated functional roles of p75 using a purified triple mutant NGF (triNGF) deficient in p75 binding but retaining significant TrkA binding and activation. The mutant was found to be as potent as wild type NGF at promoting survival of serum-deprived TrkA-expressing fibroblasts. On developing chick sensory neurons, survival responses to mutant and native NGF were indistinguishable when assayed at nanomolar concentrations. However, triNGF was 3- to 4-fold less potent than wild type NGF at lower concentrations (i.e. 10(-11) M). Interestingly, in PC12 cells coexpressing TrkA and p75, no high affinity binding sites for triNGF could be detected. The reduced responsiveness to triNGF in sensory neurons was increasingly evident at later developmental stages; late embryonic neurons did not respond at all to concentrations of triNGF that were saturating at earlier developmental stages. Likewise, although no difference could be seen between wild type and mutant NGF on the survival responses of embryonic rat superior cervical ganglion sympathetic neurons, the mutant was much less potent than native NGF on postnatal sympathetic neurons. In sensory neurons, the decrease in responsiveness to triNGF correlated with a developmental reduction in the expression of both p75 and TrkA. Thus, NGF binding to p75 enhances responsiveness to ligand, particularly when this is present at limiting concentrations. During development, p75 modulates responsiveness to NGF so that binding to p75 becomes increasingly important in neurons undergoing a down-regulation of NGF receptors. These results support a ligand-dependent modulatory role for p75 in NGF-mediated neuron survival consistent with p75 functioning as a TrkA regulator and/or signaling receptor.

Receptor serine-threonine kinases (RSTK) mediate inhibitory as well as stimulatory signals for growth and differentiation by binding to members of the transforming growth factor-beta (TGF-beta) superfamily. Over 12 different RSTKs have been isolated so far, displaying wide expression in peripheral tissues and in the nervous system. Here we report the isolation and characterization of a novel type I RSTK termed activin receptor-like kinase-7 (ALK-7) that, unlike other members of this receptor family, is predominantly expressed in the adult central nervous system. The ALK-7 gene encodes a 55-kDa cell-surface protein that exhibits up to 78% amino acid sequence identity in the kinase domain to previously isolated type I receptors for TGF-beta and activin. In the extracellular domain, however, ALK-7 is more divergent, displaying comparable similarities with all members of the ALK subfamily. RNase protection and in situ hybridization studies demonstrated a highly specific mRNA distribution restricted to neurons in several regions of the adult rat central nervous system, including cerebellum, hippocampus, and nuclei of the brainstem. Receptor reconstitution and cross-linking experiments indicated that ALK-7 can form complexes with type II RSTKs for TGF-beta and activin in a ligand-dependent manner, although direct binding of ALK-7 to ligand in these complexes could not be demonstrated. The specific expression pattern of ALK-7, restricted to the postnatal central nervous system, indicates that this receptor may play an important role in the maturation and maintenance of several neuronal subpopulations.

In the neurotrophin family, variable regions contain solvent-accessible residues important for receptor binding specificity, whereas many of the conserved residues are buried in hydrophobic cores or in the dimer interface. A stretch of six amino acids (from Asp-72 to Asn-77) in nerve growth factor (NGF) represents an exception to this general rule. These residues are highly conserved and yet form an exposed hydrophilic loop region away from other known determinants of receptor binding. We have investigated the functional importance of this region in NGF using alanine-scanning mutagenesis. Individual mutation of Asp-72, Lys-74, or His-75 to alanine (mutants D72A, K74A, and H75A, respectively) reduced the binding affinity for the p75 neurotrophin receptor by 4-10-fold. Only the D72A mutant showed an additional impairment in binding to the TrkA receptor, which was accompanied by reduced biological activity in PC12 cells, indicating a structural and/or conformational effect of this mutation. Replacement of Ser-73 or Asn-77 with alanine (mutants S73A and N77A, respectively) had no measurable effects on receptor binding. The triple mutant K74A/H75A/N77A exhibited properties that were consistent with the combined effects of the individual mutations, namely impaired binding to p75 without deficits in its interaction with TrkA. In contrast, in the triple mutant D72A/S73A/K74A, the simultaneous replacement of Asp-72 and Lys-74 with alanine had a compensatory effect such that binding to both p75 and TrkA was comparable to that of wild-type NGF, despite the deficits seen in the individual replacements. This molecule, however, was produced at low levels, and its biological activity in sympathetic ganglion explants was reduced, which, together with results from TrkA phosphorylation assays, indicated a reduced stability during prolonged culture conditions. Taken together, these data reveal a second region of interaction with the p75 receptor in NGF with the positively charged residues Lys-74 and His-75 as candidate points of contact. In addition, Asp-72 appears to be a structurally important side chain for stabilizing the conformation of the loop through interactions with neighboring residues.

This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer.

Neurotrophins regulate differentiation and survival of vertebrate neurons through binding to members of the Trk family of receptor tyrosine kinases and to a common low affinity receptor, p75LNGFR. The specificity of neurotrophin action is determined by their selective interaction with the different members of the Trk family; TrkA, TrkB, and TrkC serve as cognate receptors for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3), respectively. Unlike nerve growth factor and brain-derived neurotrophic factor, NT-3 can to some extent also bind and activate non-cognate TrkA and B receptors, although the physiological relevance of these interactions is unclear. Previous studies established that neurotrophins use an extended surface for binding to cognate Trk receptors, while binding to p75LNGFR is mediated by a localized cluster of positively charged residues. Here we show that the binding site of NT-3 to its non-preferred receptors TrkA and TrkB is dominated by two positively charged residues, Arg-31 and His-33, previously shown to constitute a main determinant of binding to p75LNGFR. Simultaneous mutation of these two residues into Ala completely abolished NT-3 binding and signaling through TrkA and greatly diminished binding and activation of TrkB. However, NT-3 binding and signaling through its cognate receptor TrkC was unaffected by the mutation. These results show that binding of NT-3 to p75LNGFR, TrkA, and TrkB is mediated by a common determinant, which is distinct from that recognized by TrkC and also different and more localized than the one recognized by TrkA and TrkB in their cognate ligands. Thus, although homologous regions in all neurotrophins are used for binding to Trk receptors, a given Trk may actually contact different residues in different neurotrophins. The mutant NT-3 described here may be of greater advantage than native NT-3 when a trophic activity needs to be specifically targeted to TrkC-expressing neurons and provides a monospecific neurotrophin for future therapeutic development.