The role of the nerve growth factor carboxyl terminus in receptor binding and conformational stability.
The role of the nerve growth factor (NGF) carboxyl terminus in the function of NGF is not well understood. Previous work showed that deletion of residues 112-120 abolished NGF bioactivity. Several mutagenesis studies, however, have localized the binding sites of the two NGF receptors, p75 and TrkA, to other regions of the NGF molecule. To investigate the role of the NGF COOH terminus, we performed a detailed structure-function analysis of this region by deleting stepwise each of the nine COOH-terminal residues as well as constructing six point mutants. We found that point mutations within the 111-115 region, but not deletion of residues 116-120, significantly decreased NGF bioactivity, as determined by TrkA tyrosine phosphorylation and neurite outgrowth from PC12 cells. Mutation of the absolutely conserved Leu112 led to severely disrupted p75 binding on A875 cells but had only a modest effect on TrkA binding to MG87-TrkA fibroblasts. This suggests that the p75 binding surface is more extended than previously believed and includes not only charged residues within loops 1 and 5 but also spatially discontinuous, uncharged residues in a region where the NH2 and COOH termini are in close proximity. Unexpectedly, deletion of COOH-terminal residues beyond Ala116 led to significantly decreased stability. These results demonstrate that residues 111-115, but not residues 116-120, are important for both the structural stability and biological activity of NGF.
Krüttgen
A
A
Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5401, USA.
Heymach
J V
JV
Kahle
P J
PJ
Shooter
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D017384:Sequence Deletion
D009414:Nerve Growth Factors; D017475:Receptors, Nerve Growth Factor