Abstract
BackgroundSonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. However, the influences of the lipid moieties on cellular reception and signal response are not well understood.Methodology/Principal FindingsWe analyzed fully lipidated Shh and mutant forms to eliminate one or both adducts in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We demonstrate that lipid modification is required for cell reception, that either adduct is sufficient to confer cellular association, that the cholesterol adduct anchors ligand to the plasma membrane and that the palmitate adduct augments ligand internalization. We further show that signaling potency correlates directly with cellular concentration of Shh ligand.Conclusions/SignificanceThe findings of this study demonstrate that lipid modification of Shh determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues.
Highlights
The Hedgehog (Hh) family of signaling proteins are secreted from localized sources and elicit concentration-dependent cellular responses to specify tissue pattern during development and homeostasis [1,2]
Conclusions/Significance: The findings of this study demonstrate that lipid modification of Sonic hedgehog (Shh) determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues
To quantify the contribution of each lipid modification to cellular association, NIH3T3 fibroblasts were co-transfected with expression vectors for recombinant Shh and EGFP, and the concentrations of recombinant Shh protein in culture media and cell lysates were measured by Enzyme-linked immunosorbent assay (ELISA) (Figure 1)
Summary
The Hedgehog (Hh) family of signaling proteins are secreted from localized sources and elicit concentration-dependent cellular responses to specify tissue pattern during development and homeostasis [1,2]. A unique biochemical property of the secreted Hh signaling domain is covalent modification by cholesterol and palmitate. The second hydrophobic modification is catalyzed by the acyl-transferase Skinny hedgehog, which results in the amide linkage of palmitate to the amino-terminal Cys residue of the signaling domain [12,13,14]. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. The influences of the lipid moieties on cellular reception and signal response are not well understood
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