(NAS Colloquium) Proteolytic Processing and Physiological Regulation (1999) / Chapter Skim
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Sonic hedgehog protein signals not as a hydrolic enzyme but as an apparent ligand for Patched
Sonic hedgehog protein signals not as a hydrolic enzyme but as an apparent ligand for Patched
Pages 10992-10999
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Shh protein is synthesized in the notochord and can induce differentiation of ventral cell types such as floor plate cells and motor neurons from neural plate explants in vitro (12~; a similar role for Shh in vivo is confirmed by a loss of these cell types in mice lacking Shh gene function (4~. Shh protein thus appears to constitute the inductive patterning signal from the notochord, and in vitro explant experiments have demonstrated a concentration-dependent response, with low concentrations of Shh-N protein inducing motor neuron differentiation and higher concentrations inducing increasing numbers of floor plate cells, ultimately to the exclusion of motor neurons (12, 13~.
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From page 10993... ...
We also used structure-based systematic mutagenesis to produce Shh-N proteins with alterations in evolutionarily conserved surface residues and then compared the signaling activity of these altered proteins in neural plate explants to their capacity for binding to Ptc-expressing cultured cells. We found that Shh-N signaling does not require catalytic activity and instead correlates critically with the ability of Shh-N to bind directly to Ptc.
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(A-C) Chicken intermediate neural plate explants double stained for expression of the motor neuron marker Islet-l~blue)
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, retention of signaling activity by Shh-N hydrolase mutant proteins indicates that catalytic activity is not required for signaling. The reduced potency for EH and EA in signaling may reflect a destabilization of folded protein structure, as might be expected from substitution of Ala for the largely buried side chains of the Glu-177 and His-135 residues.
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, each with alterations in two amino acid residues that comprise distinct subsets of the six residues altered in SC (see Table 1~. All three of these mutant proteins displayed signaling activity in the explant culture assay, but only at significantly reduced levels.
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6C, there is an excellent correlation between Ptc binding and signaling activity in all altered proteins for which these properties can be measured, and these results strongly suggest that Ptc binding may be a critical requirement for signaling. Antibody Recognition and Heparin Binding of Altered Proteins.
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DISCUSSION The Putative Zinc Hydrolase in Shh-N. Alterations in residues that should be critical for the putative zinc hydrolase activity of Shh-N did not disrupt its ability to induce ventral neural cell types or to suppress dorsal markers, suggesting that catalytic activity is not required for Shh signaling in the neural plate.
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From page 10999... ...
At the other extreme, the SC altered protein displays a complete loss of Ptc binding, and this is reflected in a complete loss of neural plate signaling activity. Even more telling, proteins carrying distinct subsets of the residues altered in SC result in intermediate levels of Ptc-binding activity and corresponding intermediate levels of neural plate signaling activity (Fig.
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