Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling

Abstract

Smoothened (SMO), a class-Frizzled G protein-coupled receptor (class-F GPCR), transduces the Hedgehog signal across cell membrane. Sterols can bind to its extracellular cysteine rich domain (CRD) and to several sites in the 7 transmembrane helices (7-TMs) of SMO. However, the mechanism how sterols regulate SMO via multiple sites is unknown. Here we determined structures of SMO–G i complexes bound to the synthetic SMO agonist (SAG) and to 24( S ),25-epoxycholesterol (24( S ),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMO D384R and SMO G111C/I496C , showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.

Multiple Targets for Oxysterols in Their Regulation of the Immune System

“…A year later, Qi et al [135] solved more structures of WT and SMO-Gi complexes with several oxysterols and an agonist, SAG, bound (PDB:6XBM). In this way they discovered more, interconnected binding sites, which seem to allow for transfer through the receptor, from the deep 7TM pocket outwards to the CRD.…”

Insights into the Role of Membrane Lipids in the Structure, Function and Regulation of Integral Membrane Proteins

“…Cholesterol Activity regulation [8,9] Activity and changes in conformation [10], Modulation of substrate binding affinity [11] Channel inhibition [12] Activation [13], Signalling [14,15], Stability [16,17], Allosteric regulation [18], Lower affinity ligand binding [17], Oligomerisation [19][20][21][22][23] PA Activity [24] Stabilisation [25] PE Dimer formation and function [26], Dimer formation [27], conformer stability [28] Conformer stabilisation and channel desensitisation [29] Agonist and antagonist binding affinities [30], Increase G protein coupling [31,32] Protein assembly [33] PC Dimerisation [34], β-arrestin interaction and function [35], Increase G protein coupling [31] PG Oligomerisation and function [36] Conformer stabilisation and channel desensitisation [37] Increase G protein coupling [30,38], Active conformer stability [39], β-arrestin interaction and function [35], Decrease G protein coupling [31] PS Dimer formation [27], Stabilisation [25], Dimerisation [34], Decrease G protein coupling [31] PI Dimer formation and function…”

“…Cholesterol directly affects the ligand-binding ability of several GPCRs, including the subtype 2 galanin receptor and the serotonin 1A receptor [95], and there is evidence that cholesterol also plays a role in GPCR signalling, for example, increasing basal activity of the cannabinoid 2 receptor [14]. In the recent study of the class F GPCR, Smoothened, cholesterol is revealed to traffic through a channel in the receptor and play a fundamental role in receptor activation [13].…”

A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

“…In the pathway "on" state, Hh ligands bind to and inactivate the 12-transmembrane sterol transporter PATCHED1 (PTCH1), which releases SMO from PTCH1-mediated inhibition 4 . This process allows SMO to access its endogenous sterol ligands and undergo an activating conformational change [11][12][13][14] . Once activated, SMO blocks PKA-C-mediated phosphorylation of GLI [15][16][17][18] , a key step that likely occurs within the tiny cell-surface compartment formed by the primary cilium [19][20][21] .…”