A molecular sensor for cholesterol in the human serotonin _1A receptor

Abstract

The function of several G protein–coupled receptors (GPCRs) exhibits cholesterol sensitivity. Cholesterol sensitivity of GPCRs could be attributed to specific sequence and structural features, such as the cholesterol recognition/interaction amino acid consensus (CRAC) motif, that facilitate their cholesterol-receptor interaction. In this work, we explored the molecular basis of cholesterol sensitivity exhibited by the serotonin1A receptor, the most studied GPCR in the context of cholesterol sensitivity, by generating mutants of key residues in CRAC motifs in transmembrane helix 2 (TM2) and TM5 of the receptor. Our results show that a lysine residue (K101) in one of the CRAC motifs is crucial for sensing altered membrane cholesterol levels. Insights from all-atom molecular dynamics simulations showed that cholesterol-sensitive functional states of the serotonin1A receptor are associated with reduced conformational dynamics of extracellular loops of the receptor. These results constitute one of the first reports on the molecular mechanism underlying cholesterol sensitivity of GPCRs.

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

“…Membrane cholesterol has emerged as a key modulator of the function of membrane proteins, such as GPCRs ( 95 ). Modulation of plasma membrane cholesterol offers a useful tool to address cholesterol-dependent organization and function of membrane proteins.…”

Lysine 101 in the CRAC Motif in Transmembrane Helix 2 Confers Cholesterol-Induced Thermal Stability to the Serotonin1A Receptor

“…Cells expressing the K101A mutant serotonin 1A receptor showed similar (~51%) reduction in membrane cholesterol content upon treatment with 10 mM MβCD. Importantly, we previously showed that membrane cholesterol depletion using 10 mM MβCD did not result in a notable effect on the plasma membrane localization of the wild-type and K101A mutant serotonin 1A receptors in HEK-5-HT 1A R cells (Kumar et al 2021).…”

“…Notably, we recently attributed cholesterol-dependence of serotonin 1A receptor signaling to a CRAC motif present in the transmembrane helix 2 of the receptor (CRAC motif I, see Fig. 1) that facilitates a preferential association with membrane cholesterol (Kumar et al 2021). We showed that a key lysine residue (K101) in this CRAC motif of the serotonin 1A receptor establishes polar interaction with the hydroxyl headgroup of cholesterol and thereby acts as a molecular sensor of membrane cholesterol (Kumar et al 2021).…”

“…1) that facilitates a preferential association with membrane cholesterol (Kumar et al 2021). We showed that a key lysine residue (K101) in this CRAC motif of the serotonin 1A receptor establishes polar interaction with the hydroxyl headgroup of cholesterol and thereby acts as a molecular sensor of membrane cholesterol (Kumar et al 2021). In addition to modulating organization, dynamics and function of the serotonin 1A receptor, we previously showed that membrane cholesterol stabilizes the receptor against thermal deactivation (Saxena et al 2012).…”

Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes

“…With this aim we took a closer look at two types of cholesterol binding motifs, the so-called cholesterol recognition/interaction amino acid consensus (CRAC) motif with the L/V -X(1–5)- Y/F -X(1–5)- R/K pattern, and the reverse CARC motif with the R/K- X(1–5)- Y/F -X(1–5)- L/V pattern [ 36 , 37 , 38 ]. CRAC and CARC sites have been identified in transmembrane segments of several membrane proteins, such as G-protein coupled receptors, but also in membrane-interacting segments of several bacterial toxins and viral proteins that interact with cholesterol [ 39 , 40 , 41 , 42 ]. Moreover, in the aforementioned three RTX toxins (LtxA, HlyA and RtxA), CRAC sites have been identified in their pore-forming domains and for two of them, LtxA and RtxA, the interaction with cholesterol has been experimentally demonstrated [ 33 , 34 , 35 ].…”