Mutagenesis identifies amino-terminal residues of nerve growth factor necessary for Trk receptor binding and biological activity.
Limited proteolysis and site-directed mutagenesis of human nerve growth factor (hNGF) was utilized to determine the role of the NH2 terminus in p140TrkA (TrkA) receptor function. Purified (6-118)hNGF, representing deletion of the first 5 NH2-terminal residues, is 9-fold less potent than (1-118)hNGF in displacing 125I-hNGF from TrkA. The deletion of a further 4 residues to form (10-118)hNGF is nearly 300-fold less potent. (6-118)hNGF is only 2-4-fold less potent than (1-118)hNGF in eliciting TrkA autophosphorylation and PC12 cell neurite outgrowth, suggesting that the first 5 residues of the NH2 terminus are necessary for full TrkA binding activity but may be less critical for potent receptor activation and intracellular signaling. In contrast, purified mutant H4D results in a nearly 1000-fold loss in TrkA binding at 4 degrees C relative to (1-118)hNGF, at least 10-fold less potency of TrkA autophosphorylation, and 30-fold loss of potency in PC12 cell differentiation. NH2-terminal hBDNF/hNGF and hNT3/hNGF chimeric mutants further affected these activities suggesting that the NH2-terminal sequence specificity of hNGF contributes to structural interactions necessary for TrkA receptor binding and ligand-induced signaling. The potency of binding of all hNGF variants to the low affinity NGF receptor p75 was largely unaffected indicating distinct structural contributions of the NH2-terminal region of hNGF to the binding to TrkA versus p75.
Shih
A
A
Department of Neuroscience, Genetech, Inc., South San Francisco, California 94080.
Laramee
G R
GR
Schmelzer
C H
CH
Burton
L E
LE
Winslow
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D001681:Biological Assay; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D016501:Neurites / Q000648:ultrastructure; D016716:PC12 Cells; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA