Abstract
Sigma 1 receptor (S1R) is a 223-amino-acid-long transmembrane endoplasmic reticulum (ER) protein. S1R modulates activity of multiple effector proteins and is a well-established drug target. However, signaling functions of S1R in cells are poorly understood. Here, we test the hypothesis that biological activity of S1R in cells can be explained by its ability to interact with cholesterol and to form cholesterol-enriched microdomains in the ER membrane. By performing experiments in reduced reconstitution systems, we demonstrate direct effects of cholesterol on S1R clustering. We identify a novel cholesterol-binding motif in the transmembrane region of human S1R. Mutations of this motif impair association of recombinant S1R with cholesterol beads, affect S1R clustering in vitro and disrupt S1R subcellular localization. We demonstrate that S1R-induced membrane microdomains have increased local membrane thickness and that increased local cholesterol concentration and/or membrane thickness in these microdomains can modulate signaling of inositol-requiring enzyme 1α in the ER. Further, S1R agonists cause disruption of S1R clusters, suggesting that biological activity of S1R agonists is linked to remodeling of ER membrane microdomains. Our results provide novel insights into S1R-mediated signaling mechanisms in cells.
Highlights
Cholesterol is an essential component of cellular membranes, and levels of cholesterol in cells are tightly controlled (Brown and Goldstein, 1999; Goldstein and Brown, 2015; Radhakrishnan et al, 2008)
In agreement with previous reports (Hayashi and Su, 2003a; Hayashi and Su, 2003b), Sigma 1 receptor (S1R)-GFP formed puncta in the endoplasmic reticulum (ER) (Figure 1A). To determine if these puncta corresponded to mitochondria-associated membranes (MAMs), we co-stained cells with a mitochondrial marker TOM20 and established that S1R gene fused with GFP (S1R-GFP) puncta were frequently found in close opposition to mitochondria (Figure 1A)
Our results suggest that wild-type S1R localizes to and is enriched in ER contact sites such as MAMs and ER-plasma membrane (PM) junctions and that CARC motif is important for S1R targeting to these sites
Summary
Cholesterol is an essential component of cellular membranes, and levels of cholesterol in cells are tightly controlled (Brown and Goldstein, 1999; Goldstein and Brown, 2015; Radhakrishnan et al, 2008). The plasma membrane (PM) is highly enriched in cholesterol and other sterols, containing 30–40% cholesterol and 10–30% sphingolipids based on molar amounts (van Meer et al, 2008). There is limited information about spatial distribution of cholesterol in the ER membrane, but recent studies suggested existence of cholesterol-enriched microdomains in the ER membrane (Area-Gomez et al, 2012; Hayashi and Fujimoto, 2010; King et al, 2020; Montesinos et al, 2020).
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