Mutational studies of conserved residues in the dimer interface of nerve growth factor.
An understanding of the structure-function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly full active with 61, 120, 91, and 73% of wild-type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.
Guo
M
M
Department of Biological Chemistry, Finch UHS/Chicago Medical School, Illinois 60064, USA.
Meyer
S L
SL
Kaur
H
H
Gao
J J
JJ
Neet
K E
KE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003196:Computer Graphics; D017124:Conserved Sequence; D017931:DNA Primers / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D017931:DNA Primers; D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA