Two distinct sites in sonic hedgehog combine for heparan sulfate interactions and cell signaling functions

Shu Chun Chang, Barbara Mulloy, Anthony I. Magee, John R. Couchman

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparinbinding domain for signaling was assessed. It was determined that key residues in human (h) Shh involved in heparin and HSPG syndecan-4 binding and biological activity included the well known cationic Cardin-Weintraub motif (lysines 32-38) but also a previously unidentified major role for lysine 178. The activity of Shh mutated in these residues was tested by quantitation of alkaline phosphatase activity in C3H10T1/2 cells differentiating into osteoblasts and hShh-inducible gene expression in PANC1 human pancreatic ductal adenocarcinoma cells. Mutated hShhs such as K37S/K38S, K178S, and particularly K37S/K38S/K178S that could not interact with heparin efficiently had reduced signaling activity compared with wild type hShh or a control mutation (K74S). In addition, the mutant hShh proteins supported reduced proliferation and invasion of PANC1 cells compared with control hShh proteins, following endogenous hShh depletion by RNAi knockdown. The data correlated with reduced Shh multimerization where the Lys-37/38 and/or Lys-178 mutations were examined. These studies provide a new insight into the functional roles of hShh interactions with HSPGs, which may allow targeting this aspect of hShh biology in, for example, pancreatic ductal adenocarcinoma.

Original languageEnglish
Pages (from-to)44391-44402
Number of pages12
JournalJournal of Biological Chemistry
Volume286
Issue number52
DOIs
Publication statusPublished - Dec 30 2011
Externally publishedYes

Fingerprint

Cell signaling
Heparitin Sulfate
Cell Communication
Heparan Sulfate Proteoglycans
Hedgehog Proteins
Lysine
Heparin
Adenocarcinoma
Syndecan-4
Mutation
Molecular modeling
Normal Distribution
Osteoblasts
Normal distribution
Mutant Proteins
RNA Interference
Bioactivity
Gene expression
Biological Assay
Embryonic Development

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Two distinct sites in sonic hedgehog combine for heparan sulfate interactions and cell signaling functions. / Chang, Shu Chun; Mulloy, Barbara; Magee, Anthony I.; Couchman, John R.

In: Journal of Biological Chemistry, Vol. 286, No. 52, 30.12.2011, p. 44391-44402.

Research output: Contribution to journalArticle

Chang, Shu Chun ; Mulloy, Barbara ; Magee, Anthony I. ; Couchman, John R. / Two distinct sites in sonic hedgehog combine for heparan sulfate interactions and cell signaling functions. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 52. pp. 44391-44402.
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