Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.
Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.
Ibáñez
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Department of Medical Chemistry II, Karolinska Institute, Stockholm, Sweden.
Hallböök
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Ebendal
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Persson
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D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D008969:Molecular Sequence Data; D009154:Mutation; D009414:Nerve Growth Factors / Q000502:physiology*; D011972:Receptor, Insulin; D011994:Recombinant Proteins; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship
D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D011972:Receptor, Insulin