Abstract
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.
Highlights
An elongated dimer interface and are held together by three disulfide bridges uniquely arranged in a characteristic cystine knot motif
Eight stepwise deletions covering the region between residues 113–120 of mature nerve growth factor (NGF), as well as five point mutants covering the stretch between residue 111–115 and a double point mutant in which residues Arg118 and Arg119 were exchanged to serines (R118S/R119S)
In this study we have used site-directed mutagenesis to analyze the role of the COOH terminus in NGF bioactivity as well as its binding to the NGF receptors p75 and TrkA
Summary
Cell Lines—COS-7 cells, A875 cells, and PC12 cells were maintained in Dulbecco’s modified Eagle’s medium containing 6% bovine calf serum and 6% horse serum. Stability Assay—Conditioned media from transfected COS-7 cells expressing wt NGF and mutants were incubated at 4 °C versus 37 °C for 3 days and subjected, in triplicate, to a previously described ELISA [23] using the monoclonal MC-1 as capture and a polyclonal anti-NGF antiserum (Sigma) as detection antibody. This ELISA recognizes only native NGF and not reduced or denatured NGF (data not shown). Amino acid residues with relevance to this report were highlighted in color, and the van der Waals’ surface of the polypeptides was calculated
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