Role of variable beta-hairpin loop in determining biological specificities in neurotrophin family

The neurotrophins are members of a family of structurally and functionally related neurotrophic factors that control the development and maintenance of vertebrate neurons. The crystal structure of nerve growth factor (NGF), the prototypic member of this family, contains three pairs of anti-parallel beta-strands connected by beta-hairpin loops, which contain most of the variable residues among the four neurotrophin proteins. Recently, amino acid residues in these variable loop regions have been implicated in the interaction between NGF and its signal-transducing receptor TrkA. In NGF, residues 40-49 (variable region II) span a very flexible and solvent-accessible beta-hairpin loop that is highly variable between different neurotrophins. To investigate the role of this domain in determining biological specificities in the neurotrophin family, we constructed a series of chimeric molecules by exchanging this variable region among three neurotrophins (i.e. NGF, neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)) that bind and activate three different Trk receptors (i.e. TrkA, -C, and -B, respectively). The chimeric molecules were analyzed for their ability to activate different Trk receptor subtypes and to promote the survival of subpopulations of peripheral neurons expressing specific types of Trk mRNAs. Exchange of the 40-49 variable loop region between NGF and NT-3 resulted in molecules capable of activating both TrkA and TrkC receptors and of rescuing neurons containing TrkA and TrkC mRNAs, indicating that this loop plays an important role in determining biological specificities in these two neurotrophins. Furthermore, variable region II from NT-4 conferred the ability to differentiate nnr5 PC12-TrkB cells to a chimeric NT-3 molecule that was originally incapable of eliciting a response in these cells. In contrast, exchanges between NGF and NT-4 did not suffice to generate molecules with broader biological specificities, suggesting that other regions in these molecules are also required. Our results support the evolutionary relationships between the three polypeptides deduced from structural comparisons.