Urfer R, Tsoulfas P, Soppet D, Escandón E, Parada L F, Presta L G
Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080.
EMBO J. 1994 Dec 15;13(24):5896-909. doi: 10.1002/j.1460-2075.1994.tb06935.x.
Survival and maintenance of vertebrate neurons are influenced by neurotrophic factors which mediate their signal by binding to specific cell surface receptors. We determined the binding sites of human neurotrophin-3 (NT-3) to its receptors trkC and gp75 by mutational analysis and compared them to the analogous interactions of nerve growth factor (NGF) with trkA and gp75. The trkC binding site extends around the central beta-strand bundle and in contrast to NGF does not make use of non-conserved loops and the six N-terminal residues. The gp75 epitope is dominated by loop residues and the C-terminus of NT-3. A novel rapid biological screening procedure allowed the identification of NT-3 mutants that are able to signal efficiently through the non-preferred receptors trkA and trkB, which are specific for NGF and BDNF respectively. Mutation of only seven residues in NT-3 resulted in a human neurotrophin variant which bound to all receptors of the trk family with high affinity and efficiently supported the survival of NGF-, BDNF- and NT-3-dependent neurons. Our results suggest that the specificity among neurotrophic factors is not solely encoded in sequence diversity, but rather in the way each neurotrophin interacts with its preferred receptor.
神经营养因子通过与特定细胞表面受体结合来介导信号，从而影响脊椎动物神经元的存活和维持。我们通过突变分析确定了人神经营养因子-3（NT-3）与其受体trkC和gp75的结合位点，并将它们与神经生长因子（NGF）与trkA和gp75的类似相互作用进行了比较。trkC结合位点围绕中央β-链束延伸，与NGF不同的是，它不利用非保守环和N端的六个残基。gp75表位主要由NT-3的环残基和C端组成。一种新型的快速生物学筛选方法能够鉴定出能够通过非首选受体trkA和trkB有效发出信号的NT-3突变体，trkA和trkB分别对NGF和BDNF具有特异性。NT-3中仅七个残基的突变就产生了一种人神经营养因子变体，它与trk家族的所有受体都具有高亲和力，并能有效支持依赖NGF、BDNF和NT-3的神经元的存活。我们的结果表明，神经营养因子之间的特异性并非仅由序列多样性编码，而是由每种神经营养因子与其首选受体相互作用的方式决定。
