Hedgehog Pathway Modulation by Multiple Lipid Binding Sites on the Smoothened Effector of Signal Response

Abstract

Summary Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo), by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

A Functional and Putative Physiological Role of Calcitriol in Patched1/Smoothened Interaction

“…Nevertheless, a study by Myers et al suggested that neither the CRD nor the 7TM are the major sites of regulation by Ptch (39). Additionally the authors discussed that oxysterols like 20(S)-OHC or 7-ketocholesterol derivatives are unlikely to subserve the regulatory functions of Ptch (39). In accordance with this hypothesis our novel data provide evidence that neither the CRD nor the 7TM are required for calcitriol binding or calcitriol-mediated Smo inhibition.…”

“…This fascinating fact raises the question if this binding pocket might also be a target site for Smo-inhibiting molecules, like calcitriol (17,26). Nevertheless, a study by Myers et al suggested that neither the CRD nor the 7TM are the major sites of regulation by Ptch (39). Additionally the authors discussed that oxysterols like 20(S)-OHC or 7-ketocholesterol derivatives are unlikely to subserve the regulatory functions of Ptch (39).…”

“…Indeed, vitamin D 3 or its derivatives may potentially fulfill the requirements for such a molecule (16, 17, 48, 49). However, VOLUME 290 • NUMBER 32 • AUGUST 7, 2015 the direct proof and evidence for those assumptions are still missing (18,19,39).…”

“…In (18,19,39). This fascinating fact raises the question if this binding pocket might also be a target site for Smo-inhibiting molecules, like calcitriol (17,26).…”

Lipoproteins carry endocannabinoids that inhibit the Hedgehog pathway

“…Hydroxysterols have been shown to stimulate Smo activity [9,10,11] by binding to its Nterminal domain [11,12,13]. Although MS/MS analysis detected hydroxysterols in fractions 33-peer-reviewed) is the author/funder.…”