Lipid modification of secreted signaling proteins

Abstract

Proteins of the Hedgehog, Wnt and Epidermal Growth Factor Receptor (EGFR) ligand families are secreted signals that induce concentration-dependent responses in surrounding cells. Although these proteins must diffuse through the aqueous extracellular environment, recent work has shown that hydrophobic lipid modifications are essential for their functions. All three classes of ligands are palmitoylated in the secretory pathway by related enzymes, and Hedgehog also carries a C-terminal cholesterol modification as a result of its autocatalytic cleavage. Palmitoylation is required for Wingless secretion and contributes to the signaling activity of Hedgehog and Wnt3a, but is not required for secretion or receptor activation by the EGFR ligand Spitz. While lipid modifications enhance the long-range activity of Sonic hedgehog, they restrict the range and increase the local concentration of Spitz. We discuss the diverse functions and the possible extent of palmitoylation of secreted ligands.

Figure 1. Different effects of lipid modifications on Spi, Hh and Wg

Ligand-producing cells are shown in the upper row and ligand-receiving cells in the lower row. Porc in the ER palmitoylates Wg on an internal cysteine (A), while Rasp in the Golgi palmitoylates Spi and Hh on their N-terminal residues (B, C). Palmitoylation of Wg enhances its glycosylation, lipid raft targeting and secretion (A). Palmitoylation of Spi increases its association with the plasma membrane of producing cells and raises its local concentration to the threshold necessary to activate the EGFR (B). Palmitate and cholesterol modifications on Hh promote its incorporation into multimers that may contain Apolipophorin (ApoL) and enhance its long-range activity. Palmitoylation is also required for Hh to signal productively when bound to Ptc (C).

Figure 2. Rasp is present in the Golgi in Drosophila S2R+ cells

(A–C) show a cell transfected with actin-GAL4, UAS-rasp-HA and the Golgi marker UAS-dGRASP65-GFP (kindly provided by Henry Chang). Anti-HA staining (A, magenta in C) colocalizes with GFP (B, green in C).

Figure 3. A Hh-Ptc fusion protein shows reduced activity in rasp mutant wing discs

(A) shows a schematic of the wing pouch. The solid line indicates the anterior-posterior (AP) compartment boundary where endogenous dpp is expressed, and the dotted line indicates vestigial (vg)-GAL4 expression at the dorsal-ventral (DV) boundary. (B–D) show third instar wing discs carrying vg-GAL4 and dpp-lacZ and stained with X-Gal. (B–C) rasp^T392/rasp^T802; (D) wildtype. (B) expresses UAS-hh and (C–D) express UAS-hh-ptc. All discs were stained in parallel for the same length of time. The Hh-Ptc fusion activates dpp expression to a slightly greater extent than Hh in the absence of rasp, but its activity is lower than in wildtype discs. Note that endogenous dpp expression at the AP boundary is lost in rasp mutant discs due to the lack of palmitoylation of endogenous Hh–. The expression pattern of vg-GAL4 is not altered in rasp mutant wing discs ( and see Fig. 4).

Figure 4. Dpp is active in the absence of rasp

All panels show third instar wing discs. (A–C) wildtype; (D–F) rasp^T392/rasp^T802; (G–I) rasp^T392/rasp^T802; vg-GAL4/UAS-dpp. The discs were stained to show expression of the Dpp target genes optomotor-blind (Omb; magenta in A, C, D, F, G, I) and spalt (Sal; green in B, C, E, F, H, I). dpp is not expressed in rasp mutant discs due to defective Hh signaling ( and see Fig. 3); however, when misexpressed at the dorsal-ventral boundary of the wing disc it is able to activate sal and omb expression. Transfections and X-gal and antibody stainings were carried out as described.