Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium

Abstract

<p id="d10049164e331">The Hedgehog pathway is critical in development and disease, but how cells respond to the secreted Hedgehog signal remains mysterious. A key step involves the regulation of the seven-transmembrane oncoprotein Smoothened by the 12-pass transporter-like Hedgehog receptor Patched1. We investigate the model that Patched1 is an ion-driven transporter of an endogenous lipidic Smoothened ligand. Whereas Patched–Smoothened regulation has traditionally been studied through indirect, downstream pathway readouts, we developed rapid, direct functional assays to dissect this step in simplified cell-based and in vitro systems. Cholesterol, a major membrane lipid, constitutively activates purified Smoothened by engaging its membrane-spanning region. Patched1 activity depends on extracellular Na ⁺, suggesting that transmembrane Na ⁺ gradients, universal among metazoans, might power Patched1 transporter-like activity in Smoothened regulation. </p><p class="first" id="d10049164e340">Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1–Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1–Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na ⁺ to regulate Smoothened in our assays, raising the possibility that a Na ⁺ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na ⁺ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation. </p>