Molecular mechanisms of Sonic hedgehog release and delivery

Abstract

The Hedgehog signaling pathway numbers among the five highly conserved cell–cell signaling pathways that operate early in metazoan development. The pathway’s activating ligand, the Sonic hedgehog (SHH) morphogen, is posttranslationally modified by palmitate and cholesterol, two lipids that tether SHH strongly to the membrane of producing cells. To effect many of its patterning roles, however, SHH must signal to faraway target cells, raising the question of how lipidated SHH is mobilized to act at a distance. In this dissertation, I investigate the biochemical basis for SHH movement between producing and responding cells by addressing two related questions: (1) how is lipidated SHH released, in a soluble form, from the membrane of producing cells and (2) how is SHH delivered to its receptor, Patched1 (PTCH1), on target cells?

In Chapter 2, we address the first question by employing a quantitative cell-based assay to measure SHH release kinetics as a function of two known SHH release factors: the transporter-like membrane protein Dispatched1 (DISP1) and a secreted chaperone SCUBE2, which binds the lipidated termini of SHH. We determine that transfer of SHH from the producing cell membrane to exogenous SCUBE2 is rate-limited by DISP1 transporter activity, which we find is powered by the plasma membrane Na+ gradient.

In Chapter 3, we address the second question by following the fate of the soluble SCUBE2–SHH complex. Using cell-based experiments and biochemical reconstitution, we discover that SHH transfer from SCUBE2 to PTCH1 is accomplished by the sequential activities of the SHH coreceptors CDON/BOC and GAS1, factors necessary for Hedgehog signaling in vivo but with yet-unclear molecular functions. We find that, first, CDON/BOC bind both SCUBE2 and SHH, to recruit SCUBE2–SHH to the cell surface. Next, GAS1, which we find binds SHH lipid moieties, accepts SHH from SCUBE2. Finally, GAS1 coordinates transfer of SHH lipids to PTCH1, activating the pathway.

Collectively, these studies establish a biochemical framework for the movement of lipidated SHH from the producing cell membrane to the PTCH1 receptor, in which successive handoffs of the SHH lipid appendages along an extracellular chaperone cascade ultimately dictate its tissue distribution and cell-autonomous activity.