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Regulation of G Protein-Coupled Receptors by Palmitoylation and Cholesterol Alan D Goddard and Anthony Watts*

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Regulation of G Protein-Coupled Receptors by Palmitoylation and Cholesterol Alan D Goddard and Anthony Watts* Goddard and Watts BMC Biology 2012, 10:27 http://www.biomedcentral.com/1741-7007/10/27 COMMENTARY Open Access Regulation of G protein-coupled receptors by palmitoylation and cholesterol Alan D Goddard and Anthony Watts* See research article www.biomedcentral.com/content/1471-2121/13/6 directed to elucidating GPCR crystal structures and a Abstract number of the structures obtained seem to indicate the Due to their membrane location, G protein-coupled presence of receptor dimers. Intriguingly, the β2-adrenergic receptors (GPCRs) are subject to regulation by soluble receptor (β2AR) crystalized with cholesterol molecules and integral membrane proteins as well as membrane and a post-translationally added palmitate group from components, including lipids and sterols. GPCRs also each protomer forming most of the dimer interface [3], undergo a variety of post-translational modifications, suggesting a role for lipids and sterols, in addition to including palmitoylation. A recent article by Zheng et protein-protein interactions, in GPCR dimeri zation. al. in BMC Cell Biology demonstrates cooperative roles However, it was not clear whether this accurately repre- for receptor palmitoylation and cholesterol binding in sented the conformation of the dimer within native lipid GPCR dimerization and G protein coupling, underlining bilayers. A recent study in BMC Cell Biology by Zheng et the complex regulation of these receptors. al. [4] has revealed a complex interplay between choles- terol, palmitate, receptor dimeri zation and G protein activation. Their study showed that reducing cholesterol Commentary levels or preventing palmitoylation of the μ-opioid G protein-coupled receptors (GPCRs) represent the receptor (OPRM1) reduced receptor dimerization and largest family of integral membrane proteins encoded by Gα association. Additionally, preventing palmitoylation the human genome and detect a wide variety of ligands, reduced the association of OPRM1 with cholesterol, including peptides, hormones, lipids and nucleotides. suggesting a functional complex of receptor, palmitate They transduce extracellular signals across the membrane and cholesterol. to activate intracellular signaling cascades via hetero- trimeric G proteins. These consist of a Gα, Gβ and Gγ GPCRs and cholesterol subunit and the precise combination of these subunits It has been thought for some time that cholesterol may determines the cellular response to receptor activation. play an important role or roles in GPCR signaling. Additional non-G protein effectors add layers of com- Cholesterol is an amphiphilic sterol possessing a small plexity and diversity to GPCR signaling cascades. Due to -OH head group and a rigid four-ringed backbone; its their involvement in a wide variety of physiological pro- size enables it to span approximately half a lipid bilayer, cesses, GPCRs represent excellent pharmaceutical targets interacting with the hydrophobic tails of phospholipids. [1] and are the target for approximately 30% of drugs. Cholesterol can alter the packing of lipids and thus GPCRs do not exist in isolation in lipid bilayers, but increase membrane order. This has led to the hypothesis instead interact with components of the bilayer, such as that cholesterol is responsible for the formation of lipid lipids and sterols, as well as with other GPCRs to form rafts and caveolae, regions of the membrane enriched in dimers and higher order oligomers. It is likely that, at cholesterol and sphingolipids with higher order than the least in some cases, these dimers are of functional signifi- bulk of the membrane. GPCRs have been found to cance and can affect the ligand binding and signaling associate with both regions, and it is believed that this properties of GPCRs [2]. Much recent effort has been leads to an increased local concentration of receptors and G proteins in the laterally separated regions, increasing *Correspondence: [email protected] the efficiency of signal transduction [5]. In addition to Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, affecting the properties of the membranes in which South Parks Rd, Oxford, OX1 3QU, UK GPCRs reside, it has been demonstrated that a number of © 2012 Goddard and Watts; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Goddard and Watts BMC Biology 2012, 10:27 Page 2 of 3 http://www.biomedcentral.com/1741-7007/10/27 receptors interact directly with cholesterol (Figure 1), which has led to the discovery of consensus cholesterol (1) binding sites in almost half of the family A GPCRs [6]. e importance of cholesterol for GPCR function is supported by that fact that addition of cholesterol or its Extracellular analogues can greatly increase GPCR stability upon purification and also improve crystal quality. e ability (5) of cholesterol to exert both direct and indirect effects on GPCR activity often complicates interpretation of results, but in a number of cases cholesterol clearly plays an important role in receptor function [6]. However, it (3) should be noted that the effects of cholesterol on a particular receptor are unpredictable, and it has been (4) demonstrated that certain GPCRs can function in Intracellular Escherichia coli membranes, which lack cholesterol [6]. (2) GPCR palmitoylation P P P GPCRs are post-translationally modified in a number of Figure 1. Schematic of G protein-coupled receptor post- ways, including glycosylation, phosphorylation and pal- translational modications and interactions. GPCRs have mitoy lation (Figure 1). Palmitoylation is the addition of seven transmembrane domains (TMs) with an extracellular amino palmitic acid (a 16C saturated fatty acid) and occurs on terminus and intracellular carboxyl terminus and alternating intracellular and extracellular loops. They undergo a variety of post- one or more cysteines on the intracellular side of GPCRs. translational modications and interact with various components e thioester bond that links the palmitate to the cysteine of the membrane. (1) The amino terminus and extracellular loops of is cleavable and thus the palmitoylation state of a receptor receptors are often glycosylated, which can be vital for correct cell- can be used to regulate its activity. It should be noted surface localization. (2) The carboxy-terminal tail undergoes reversible that, in rare cases, other lipids can be attached to GPCRs phosphorylation, which is important in signal desensitization and receptor internalization. (3) Reversible palmitoylation of cysteine and that palmitoylation can occur on residues other than residues within intracellular loops and the carboxy-terminal tail cysteine [7]. Again, the effects of palmitoylation are un- results in dierent loop conformations as the palmitate groups predictable and GPCR-dependent. penetrate into the lipid bilayer. (4) Cholesterol can interact at specic Palmitoylation influences all aspects of GPCR sites within the TM helices of GPCRs, in this case TM2 and TM4. signaling. e palmitoylation state of certain receptors (5) GPCRs also may also interact with specic lipid components of the membrane. These interactions and modications act in concert to can preferentially direct signaling through particular G modulate the activity of GPCRs. proteins and hence give different responses to the same ligand. Up to three palmitate groups can be found on GPCRs and different palmitoylation profiles can result in various conformations of the carboxy-terminal tail, this effect was largely observed due to crystal packing which may select for certain G protein interactions [7]. conditions but such an arrangement could influence Palmitoylation can influence the phosphorylation state of GPCR activity in vivo. the receptor, modulating desensitization and internali- e recent study by Zheng et al. [4] systematically zation, and can also control internalization independent investigates the effects of cholesterol and palmitoylation of phosphorylation. It has been suggested that palmitate on OPRM1 dimerization and G protein coupling using a binding in the endoplasmic reticulum ensures correct variety of colocalization techniques. Cholesterol can be processing and trafficking of receptors, and, once at the depleted by statins and Zheng et al. used the synthetic cell membrane, may target GPCRs to lipid rafts. However, statin simvastatin to probe the effects of cholesterol on not all palmitoylated receptors associate with rafts and OPRM1. Simvastatin reduced the amount of cholesterol not all raft-associated GPCRs are palmitoylated [7]. In associated with OPRM1 but also decreased receptor the case of OPRM1, it appears that palmitoylation and dimerization and receptor-G protein interactions. e cholesterol association (and presumably raft interactions) authors also demonstrate that mutagenesis of the pal- are intrinsically linked [4]. mitoy lated cysteine residue in OPRM1 has no effect on ligand binding but does decrease signaling efficiency, Packed with fat probably by impairing GPCR-G protein association. e An intriguing suggestion from the structure of β2AR was same mutant had significantly reduced dimerization, and the ability of cholesterol and palmitate to act in concert it was proposed that this was responsible for the reduced to mediate GPCR dimer formation [3]. It is possible that G protein coupling.
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