Neurotrophic factors and their receptors structure-function relationships and signalling mechanisms

Background: The development, survival and maintenance of the vertebrate nervous system requires the continuous supply of a set of polypeptides termed neurotrophic factors. Among these, the neurotrophins (NTs) and members of the transforming growth factor-B (TGF-B) superfamily have raised substantial hopes for future clinical applications. Mammalian NTs comprise four members to date, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). NTs interact with two distinct classes of cell-surface receptors: members of the receptor tyrosine kinase family Trk, and p75NTR a smaller receptor that lacks intrinsic catalytic activity. While p75NTR binds all neurotrophins with equal affinity, activation of Trks is specific in that NGF activates TrkA, BDNF and NT-4 interact with TrkB and NT-3 activates TrkC. In addition, NT-3 can signal, albeit to a lesser extent, through TrkA and TrkB. While the function of Trks as signalling receptors is well established, the role of p75NTR is less clear. In contrast to the restricted number of neurotrophins, members of the TGF-B superfamily comprise a heterogenous collection of pleiotropic proteins grouped into several subfamilies. These include the prototypic TGF-Bs, activins and bone morphogenetic proteins (BMP). Most members have been shown to signal through a unique heteromeric complex comprised of two classes of serine threonine kinase receptors termed type I and II. Type II receptors are constitutively active kinases with the ability to bind ligand on their own, while type I receptors require type II receptors in order to interact with ligand efficiently.

Aims: In the first part of the present thesis we focused our attention on the enigmatic p75NTR. We set out to determine residues in neurotrophins responsible for interaction with p75NTR and to define functional roles for p75NTR in vertebrate neurons. In the second part, we studied receptors expressed in different neuronal tissues. We sought to investigate the expression levels of neurotrophin receptors in neuronal tumours and embarked on a project aimed at isolating and characterizing novel receptors for the TGF-B superfamily expressed in the nervous system.

Results: By site-directed mutagenesis we generated recombinant neurotrophin molecules where specific residues were replaced by alanine. Using a range of in vitro assays we compared the activity of mutant molecules with native protein. This allowed us to assess the importance of each individual residue in receptor binding and activation. In the first study we mapped a major functional epitope in BDNF, NT-3 and NT-4 involved in binding to p75NTR, This is located in two spatially close loop regions in one end of the molecules and is formed by only 2-3 positively charged residues. Albeit similar, this epitope is clearly different in its precise conformation between the different neurotrophins. Furthermore it appears to be dispensable for binding and activation of Trks. Interestingly, on cells co-expressing p75NTR and cognate Trks, NT-4, but not BDNF or NT-3 seems to require p75NTR binding in order to activate TrkB efficiently, indicating that p75NTR may modulate neuronal responsiveness to NT-4. In the second study we were able to determine that the major determinant in NT-3 responsible for its interaction with the non-cognate receptors TrkA and TrkB proved to be the same epitope used for p75NTR binding, demonstrating that the interaction between NT-3 and non-cognate Trks is very localized. In the third report we assessed the functional role of p75NTR in NGF responsive vertebrate neurons with the use of a mutant NGF selectively deficient in p75NTR binding.

We conclude that binding to p75NTR modulates TrkA function, by enhancing TrkA mediated neuronal survival in response to NGF when the factor is present in limiting amounts or when cell-surface receptor expression is altered. In the last mutagenesis study we assessed the role of a conserved loop region situated in one end of the NGF-molecule. Positively charged residues were shown to be important for p75NTR binding, in line with the previously shown involvement of basic residues in binding to p75NTR, In the last two reports we focused our attention on receptors for neurotrophic factors. Neurotrophins and their receptors are believed to play a role in childhood neuroblastoma tumours (NB). We analyzed the expression pattern of mRNA coding for TrkC and found that high levels of expression correlated with favourable tumour stage and clinical outcome. This indicates that determination of TrkC mRNA may be of clinical significance in the evaluation of patients with NB. In our final work, we isolated a novel type I receptor (ALK-7) for the TGF-B superfamily expressed in the nervous system. ALK-7 is predominantly expressed in adult neurons of the central nervous system and can interact with type ll receptors for activin and TGF-B in a ligand dependent manner, although we were unable to demonstrate physical interactions between ALK-7 and the ligands. These results suggest that ALK 7 may be the receptor for a novel member of the TGF-B superfamily with neurotrophic activities.