Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched

Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched

^*Department of Molecular Biology and Genetics and ^§Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Abstract

The amino-terminal signaling domain of the Sonic hedgehog secreted protein (Shh-N), which derives from the Shh precursor through an autoprocessing reaction mediated by the carboxyl-terminal domain, executes multiple functions in embryonic tissue patterning, including induction of ventral and suppression of dorsal cell types in the developing neural tube. An apparent catalytic site within Shh-N is suggested by structural homology to a bacterial carboxypeptidase. We demonstrate here that alteration of residues presumed to be critical for a hydrolytic activity does not cause a loss of inductive activity, thus ruling out catalysis by Shh-N as a requirement for signaling. We favor the alternative, that Shh-N functions primarily as a ligand for the putative receptor Patched (Ptc). This possibility is supported by new evidence for direct binding of Shh-N to Ptc and by a strong correlation between the affinity of Ptc-binding and the signaling potency of Shh-N protein variants carrying alterations of conserved residues in a particular region of the protein surface. These results together suggest that direct Shh-N binding to Ptc is a critical event in transduction of the Shh-N signal.

Footnotes

↵ † N.F. and T.M. contributed equally in this work.
↵ ‡ Present address: Ontogeny, Inc., Cambridge, MA 02138.
↵ ¶ Present address: National Institute on Environmental and Health Sciences, Research Triangle Park, NC 27709.
↵ ‖ To whom reprint requests should be addressed at: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 North Wolfe Street PCTB-714, Baltimore, MD 21205. E-mail: pbeachy{at}jhmi.edu.
This paper was presented at the National Academy of Sciences colloquium “Proteolytic Processing and Physiological Regulation,” held February 20–21, 1999, at the Arnold and Mabel Beckman Center in Irvine, CA.
ABBREVIATIONS:

Shh-N,

the amino-terminal signaling domain of the Sonic hedgehog secreted protein;

HNF-3β,

hepatocyte nuclear factor 3β;

Ptc-CTD,

Ptc with a truncation resulting in a 140-residue carboxyl-terminal deletion