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or only mildly affect Ptc binding, and these proteins retain normal or nearly normal signaling activity. 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. 6C). Multiple individual residues within the SC surface region that contribute independently to Ptc binding thus also similarly contribute to signaling potency. Although our results cannot exclude a role for interactions with other proteins in reception of the Shh signal, they do strongly suggest that direct binding to Ptc is a critical step, and this information will serve as the basis for further elucidation of downstream events.

We thank Drs. M.Scott and T.Jessell for various cDNAs and antibodies. We also thank J.Wrable for help with circular dichroism analysis, Dr. J.Taipale for suggestions on the crosslinking experiment, Dr. M.Cooper and K.Young for preparation of some recombinant proteins, Dr. O.Sundin for help with chicken embryo experiments, Dr. R.Mann for comments on the manuscript, and members of the Beachy laboratory for discussions and suggestions. N.F. was a postdoctoral fellow of the Human Frontier Science Program. D.J.L. and P.A.B. are investigators of Howard Hughes Medical Institute. This work was supported in part by grants from the American Paralysis Association and the Ara Parseghian Medical Research Foundation.

Under a licensing agreement between Ontogeny, Inc. and the Johns Hopkins University, Dr. Beachy and the University hold equity in Ontogeny and are entitled to a share of royalties from sales of products related to the research described in this article. Dr. Beachy serves on Ontogeny’s Scientific Advisory Board and as a consultant to the company. All financial aspects of these arrangements are managed by the University in accordance with its policies.

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