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. 2009 Jul 17;284(29):19169-72.
doi: 10.1074/jbc.C109.011957. Epub 2009 May 28.

A sonic hedgehog missense mutation associated with holoprosencephaly causes defective binding to GAS1

David C Martinelli  1 , Chen-Ming Fan
Affiliations

A sonic hedgehog missense mutation associated with holoprosencephaly causes defective binding to GAS1

David C Martinelli et al. J Biol Chem. .

Abstract

Holoprosencephaly (HPE) is a common birth defect predominantly affecting the forebrain and face and has been linked to mutations in the sonic hedgehog (SHH) gene. HPE is genetically heterogeneous, and clinical presentation represents a spectrum of phenotypes. We have previously shown that Gas1 encodes a cell-autonomous Hedgehog signaling enhancer. Combining cell surface binding, in vitro activity, and explant culture assays, we provide evidence that SHH contains a previously unknown unique binding surface for its interaction with GAS1 and that this surface is also important for maximal signaling activity. Within this surface, the Asn-115 residue of human SHH has been documented to associate with HPE when mutated to lysine (N115K). We provide evidence that HPE associated with this mutation can be mechanistically explained by a severely reduced binding of SHH to GAS1, and we predict a similar result if a mutation were to occur at Tyr-80. Our data should encourage future searches for mutations in GAS1 as possible modifiers contributing to the wide spectrum of HPE.

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Figures

FIGURE 1.
FIGURE 1.
SHH-N-AP cell surface binding to COS7 cells expressing each binding partner. A, AP activity was measured in relative luminescence units (RLU) for WT SHH-N-AP binding. Values shown are after subtraction of binding to mock-transfected cells. Less binding to GAS1 is likely due to its higher Kd. Total surface protein levels were not controlled for as they were not relevant to this experiment. B, the binding of each SHH mutant is displayed as a fraction of WT SHH binding for each respective protein. All assays were performed in triplicate; error bars = 1 standard deviation.
FIGURE 2.
FIGURE 2.
The signaling activity defect of the Y81A and N116K SHH mutants is due to a lack of GAS1 activity. A, titration of WT SHH-N-AP in NIH3T3-GLI-Luc cells; the -fold induction of luciferase activity is shown on the Y axis. 20 nm was determined to be the appropriate concentration for subsequent analysis. B, various SHH-N-AP mutants tested in NIH3T3-GLI-Luc cells, which are shown on the X axis. C, diagram depicting how E10.5 forelimb buds were dissected. D, qRT-PCR for Gli1 in WT and Gas1 mutant limb explants. The Y axis shows the -fold induction of Gli1 transcript level over control explants. Samples were normalized by qRT-PCR for Gapdh. All assays were performed at least in triplicate; error bars = 1 standard deviation.
FIGURE 3.
FIGURE 3.
SHH-N structure (from Ref. 17) and specified residues colored using the Cn3D program from NCBI. Residues colored red are important for the SHH-GAS1 interaction. Residues colored yellow are likely not part of the GAS1-SHH interface. Two views are shown.
See this image and copyright information in PMC

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