Due to their membrane location, G protein-coupled receptors (GPCRs) are subject to regulation by soluble and integral membrane proteins as well as membrane components, including lipids and sterols. GPCRs also undergo a variety of post-translational modifications, including palmitoylation. A recent article by Zheng et al. in BMC Cell Biology demonstrates cooperative roles for receptor palmitoylation and cholesterol binding in GPCR dimerization and G protein coupling, underlining the complex regulation of these receptors

Alan D Goddard

Anthony Watts

B Chini

H Zheng

HH Patel

J Oates

JF White

MR Whorton

P Maurice

PJ Harding

SJ Hill

V Cherezov

English

PubMed

Abstract Due to their membrane location, G protein-coupled receptors (GPCRs) are subject to regulation by soluble and integral membrane proteins as well as membrane components, including lipids and sterols. GPCRs also undergo a variety of post-translational modifications, including palmitoylation. A recent article by Zheng et al. in BMC Cell Biology demonstrates cooperative roles for receptor palmitoylation and cholesterol binding in GPCR dimerization and G protein coupling, underlining the complex regulation of these receptors. See research article http://www.biomedcentral.com/1471-2121/13/6</p

Goddard Alan D

Watts Anthony

Directory of Open Access Journals

BMC Biology

Regulation of G protein-coupled receptors by palmitoylation and cholesterol

Crossref

Due to their membrane location, G protein-coupled receptors (GPCRs) are subject to regulation by soluble and integral membrane proteins as well as membrane components, including lipids and sterols. GPCRs also undergo a variety of post-translational modifications, including palmitoylation. A recent article by Zheng et al. in BMC Cell Biology demonstrates cooperative roles for receptor palmitoylation and cholesterol binding in GPCR dimerization and G protein coupling, underlining the complex regulation of these receptors.See research article http://www.biomedcentral.com/1471-2121/13/6

Goddard, Alan D.

Watts, Anthony

Aston Publications Explorer

CommentaryG protein-coupled receptors (GPCRs) represent the largest family of integral membrane proteins encoded by the human genome and detect a wide variety of ligands, including peptides, hormones, lipids and nucleotides. They transduce extracellular signals across the membrane to activate intracellular signaling cascades via hetero-trimeric G proteins. These consist of a Gα, Gβ and Gγ subunit and the precise combination of these subunits determines the cellular response to receptor activation. Additional non-G protein effectors add layers of com-plexity and diversity to GPCR signaling cascades. Due to their involvement in a wide variety of physiological pro-cesses, GPCRs represent excellent pharmaceutical targets [1] and are the target for approximately 30% of drugs.GPCRs do not exist in isolation in lipid bilayers, but instead interact with components of the bilayer, such as lipids and sterols, as well as with other GPCRs to form dimers and higher order oligomers. It is likely that, at least in some cases, these dimers are of functional signifi-cance and can affect the ligand binding and signaling properties of GPCRs [2]. Much recent effort has been directed to elucidating GPCR crystal structures and a number of the structures obtained seem to indicate the presence of receptor dimers. Intriguingly, the β2-adrenergic receptor (β2AR) crystalized with cholesterol molecules and a post-translationally added palmitate group from each protomer forming most of the dimer interface [3], suggesting a role for lipids and sterols, in addition to protein-protein interactions, in GPCR dimeri zation. However, it was not clear whether this accurately repre-sented the conformation of the dimer within native lipid bilayers. A recent study in BMC Cell Biology by Zheng et al. [4] has revealed a complex interplay between choles-terol, palmitate, receptor dimeri zation and G protein activation. Their study showed that reducing cholesterol levels or preventing palmitoylation of the μ-opioid receptor (OPRM1) reduced receptor dimerization and Gα association. Additionally, preventing palmitoylation reduced the association of OPRM1 with cholesterol, suggesting a functional complex of receptor, palmitate and cholesterol.GPCRs and cholesterolIt has been thought for some time that cholesterol may play an important role or roles in GPCR signaling. Cholesterol is an amphiphilic sterol possessing a small -OH head group and a rigid four-ringed backbone; its size enables it to span approximately half a lipid bilayer, interacting with the hydrophobic tails of phospholipids. Cholesterol can alter the packing of lipids and thus increase membrane order. This has led to the hypothesis that cholesterol is responsible for the formation of lipid rafts and caveolae, regions of the membrane enriched in cholesterol and sphingolipids with higher order than the bulk of the membrane. GPCRs have been found to associate with both regions, and it is believed that this leads to an increased local concentration of receptors and G proteins in the laterally separated regions, increasing the efficiency of signal transduction [5]. In addition to affecting the properties of the membranes in which GPCRs reside, it has been demonstrated that a number of AbstractDue to their membrane location, G protein-coupled receptors (GPCRs) are subject to regulation by soluble and integral membrane proteins as well as membrane components, including lipids and sterols. GPCRs also undergo a variety of post-translational modifications, including palmitoylation. A recent article by Zheng et al. in BMC Cell Biology demonstrates cooperative roles for receptor palmitoylation and cholesterol binding in GPCR dimerization and G protein coupling, underlining the complex regulation of these receptors.Regulation of G protein-coupled receptors by palmitoylation and cholesterolAlan D Goddard and Anthony Watts*See research article  www.biomedcentral.com/content/1471-2121/13/6 CO M M E N TA RY  Open Access*Correspondence: anthony.watts@bioch.ox.ac.uk Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Rd, Oxford, OX1 3QU, UK© 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 http://www.biomedcentral.com/1741-7007/10/27receptors interact directly with cholesterol (Figure  1), which has led to the discovery of consensus cholesterol 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 analogues can greatly increase GPCR stability upon purification and also improve crystal quality. e ability 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 should be noted that the effects of cholesterol on a particular receptor are unpredictable, and it has been demonstrated that certain GPCRs can function in Escherichia coli membranes, which lack cholesterol [6].GPCR palmitoylationGPCRs are post-translationally modified in a number of ways, including glycosylation, phosphorylation and pal-mitoy lation (Figure  1). Palmitoylation is the addition of palmitic acid (a 16C saturated fatty acid) and occurs on one or more cysteines on the intracellular side of GPCRs. e thioester bond that links the palmitate to the cysteine is cleavable and thus the palmitoylation state of a receptor can be used to regulate its activity. It should be noted that, in rare cases, other lipids can be attached to GPCRs and that palmitoylation can occur on residues other than cysteine [7]. Again, the effects of palmitoylation are un-predictable and GPCR-dependent.Palmitoylation influences all aspects of GPCR signaling. e palmitoylation state of certain receptors can preferentially direct signaling through particular G 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, which may select for certain G protein interactions [7]. Palmitoylation can influence the phosphorylation state of the receptor, modulating desensitization and internali-zation, and can also control internalization independent of phosphorylation. It has been suggested that palmitate binding in the endoplasmic reticulum ensures correct processing and trafficking of receptors, and, once at the cell membrane, may target GPCRs to lipid rafts. However, not all palmitoylated receptors associate with rafts and not all raft-associated GPCRs are palmitoylated [7]. In the case of OPRM1, it appears that palmitoylation and cholesterol association (and presumably raft interactions) are intrinsically linked [4].Packed with fatAn intriguing suggestion from the structure of β2AR was the ability of cholesterol and palmitate to act in concert to mediate GPCR dimer formation [3]. It is possible that this effect was largely observed due to crystal packing conditions but such an arrangement could influence GPCR activity in vivo.e recent study by Zheng et al. [4] systematically investigates the effects of cholesterol and palmitoylation on OPRM1 dimerization and G protein coupling using a variety of colocalization techniques. Cholesterol can be depleted by statins and Zheng et al. used the synthetic statin simvastatin to probe the effects of cholesterol on OPRM1. Simvastatin reduced the amount of cholesterol associated with OPRM1 but also decreased receptor dimerization and receptor-G protein interactions. e authors also demonstrate that mutagenesis of the pal-mitoy lated cysteine residue in OPRM1 has no effect on ligand binding but does decrease signaling efficiency, probably by impairing GPCR-G protein association. e same mutant had significantly reduced dimerization, and it was proposed that this was responsible for the reduced G protein coupling. Although it has been suggested that a Figure 1. Schematic of G protein-coupled receptor post-translational modications and interactions. GPCRs have seven transmembrane domains (TMs) with an extracellular amino terminus and intracellular carboxyl terminus and alternating intracellular and extracellular loops. They undergo a variety of post-translational modications and interact with various components of the membrane. (1) The amino terminus and extracellular loops of receptors are often glycosylated, which can be vital for correct cell-surface localization. (2) The carboxy-terminal tail undergoes reversible phosphorylation, which is important in signal desensitization and receptor internalization. (3) Reversible palmitoylation of cysteine residues within intracellular loops and the carboxy-terminal tail results in dierent loop conformations as the palmitate groups penetrate into the lipid bilayer. (4) Cholesterol can interact at specic sites within the TM helices of GPCRs, in this case TM2 and TM4. (5) GPCRs also may also interact with specic lipid components of the membrane. These interactions and modications act in concert to modulate the activity of GPCRs.P P P Extracellular Intracellular (1) (2) (3) (4) (5) Goddard and Watts BMC Biology 2012, 10:27 http://www.biomedcentral.com/1741-7007/10/27Page 2 of 3G protein heterotrimer is too large to signal via a mono-meric GPCR, monomeric receptors have been shown to signal efficiently [8], although this ability may be receptor-specific. Finally, Zheng et al. demonstrate that the palmitate-free mutant associated more weakly with cholesterol [4], suggesting a functional interaction between the palmitate group and cholesterol.It is interesting to note that, in contrast to the β2AR crystal structure [3], the palmitate group on OPRM1 is not located on the carboxy-terminal tail but instead on the intracellular side of transmembrane domain (TM) 3 [4]. In β2AR, the dimer interface involves TM1 and helix 8 (in the carboxy-terminal tail) whereas the OPRM1 dimer interface is predicted to be between TM4 of each protomer, with the palmitate bound to the carboxy-terminal side of TM3. This difference in receptor interface may be driven by the location of palmitoylation and it is possible that regulation of palmitoylation states could dynamically influence the dimerization interfaces of GPCRs. A model of the OPRM1 dimer in which cholesterol and palmitate pack together to facilitate receptor dimerization reveals that cholesterol inter actions contribute approximately 25% of the total interaction energy at the homodimer interface [4]. Dimerization of OPRM1 appears to be regulated by palmitate, which recruits cholesterol to stabilize the dimer. Most GPCR dimers are transient, and anything that affects the weak association between protomers may have dramatic effects on the population of dimers at equilibrium.The complex interplay between receptor, cholesterol and palmitate illustrated by Zheng et al. [4] lends support to the model suggested by the crystal structure of β2AR [3] by demonstrating a role for sterols and lipids in GPCR dimerization in vivo. It remains to be seen if other GPCRs require such modulation to support dimerization. For example, neurotensin receptor 1 can dimerize in artificial lipid membranes even in the absence of palmitoylation [9] and also in low concentrations of detergent (no lipid bilayer) [10]. An interesting discussion remains - what truly constitutes a GPCR dimer? How close do the recep-tors have to be to functionally interact? If a dimer is required solely to provide sufficient surface area for G protein coupling, then the proximity provided by such a lipid bridge seems sufficient, but if allosteric regulation by protomers within the dimer is to occur, more direct protein-protein interactions may be required.These studies remind us that it is vital to consider membrane proteins in the context of their environment. They do not exist in isolation and are heavily influenced by lipids, sterols and other proteins. Cholesterol and palmitoylation can affect all aspects of GPCR activity and their precise roles are likely to be receptor-dependent, although only with more data can we hope to determine any consensus. Membrane environments are challenging to control and probe, but such studies are vital if we are to gain a complete understanding of GPCR function.AcknowledgementsThis work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; grant number BB/G019738/1).Published: 19 March 2012References1. Hill SJ: G-protein-coupled receptors: past, present and future. Br J Pharmacol 2006, 147 Suppl 1:S27-37.2. Maurice P, Kamal M, Jockers R: Asymmetry of GPCR oligomers supports their functional relevance. Trends Pharmacol Sci 2011, 32:514-520.3. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC: High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 2007, 318:1258-1265.4. Zheng H, Pearsall EA, Hurst DP, Zhang Y, Chu J, Zhou Y, Reggio PH, Loh HH, Law P-Y: Palmitoylation and membrane cholesterol stabilize μ-Opioid receptor homodimerization and G protein coupling. BMC Cell Biol 2012, 13:6.5. Patel HH, Murray F, Insel PA: G-protein-coupled receptor-signaling components in membrane raft and caveolae microdomains. Handb Exp Pharmacol 2008:167-184.6. Oates J, Watts A: Uncovering the intimate relationship between lipids, cholesterol and GPCR activation. Curr Opin Struct Biol 2011, 21:802-807.7. Chini B, Parenti M: G-protein-coupled receptors, cholesterol and palmitoylation: facts about fats. J Mol Endocrinol 2009, 42:371-379.8. Whorton MR, Bokoch MP, Rasmussen SG, Huang B, Zare RN, Kobilka B, Sunahara RK: A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc Natl Acad Sci U S A 2007, 104:7682-7687.9. Harding PJ, Attrill H, Boehringer J, Ross S, Wadhams GH, Smith E, Armitage JP, Watts A: Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers. Biophys J 2009, 96:964-973.10. White JF, Grodnitzky J, Louis JM, Trinh LB, Shiloach J, Gutierrez J, Northup JK, Grisshammer R: Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction. Proc Natl Acad Sci U S A 2007, 104:12199-12204.doi:10.1186/1741-7007-10-27Cite this article as: Goddard AD, Watts A: Regulation of G protein-coupled receptors by palmitoylation and cholesterol. BMC Biology 2012, 10:27.Goddard and Watts BMC Biology 2012, 10:27 http://www.biomedcentral.com/1741-7007/10/27Page 3 of 3

Goddard, AD

Watts, A

Oxford University Research Archive (ORA)

Springer - Publisher Connector

Oxford University Research Archive

Goddard, Alan

University of Lincoln Institutional Repository

Commentary
G protein-coupled receptors (GPCRs) represent the 
largest family of integral membrane proteins encoded by 
the human genome and detect a wide variety of ligands, 
including peptides, hormones, lipids and nucleotides. 
They transduce extracellular signals across the membrane 
to activate intracellular signaling cascades via hetero-
trimeric G proteins. These consist of a Gα, Gβ and Gγ 
subunit and the precise combination of these subunits 
determines the cellular response to receptor activation. 
Additional non-G protein effectors add layers of com-
plexity and diversity to GPCR signaling cascades. Due to 
their involvement in a wide variety of physiological pro-
cesses, GPCRs represent excellent pharmaceutical targets 
[1] and are the target for approximately 30% of drugs.
GPCRs do not exist in isolation in lipid bilayers, but 
instead interact with components of the bilayer, such as 
lipids and sterols, as well as with other GPCRs to form 
dimers and higher order oligomers. It is likely that, at 
least in some cases, these dimers are of functional signifi-
cance and can affect the ligand binding and signaling 
properties of GPCRs [2]. Much recent effort has been 
directed to elucidating GPCR crystal structures and a 
number of the structures obtained seem to indicate the 
presence of receptor dimers. Intriguingly, the β2-adrenergic 
receptor (β2AR) crystalized with cholesterol molecules 
and a post-translationally added palmitate group from 
each protomer forming most of the dimer interface [3], 
suggesting a role for lipids and sterols, in addition to 
protein-protein interactions, in GPCR dimeri zation. 
However, it was not clear whether this accurately repre-
sented the conformation of the dimer within native lipid 
bilayers. A recent study in BMC Cell Biology by Zheng et 
al. [4] has revealed a complex interplay between choles-
terol, palmitate, receptor dimeri zation and G protein 
activation. Their study showed that reducing cholesterol 
levels or preventing palmitoylation of the μ-opioid 
receptor (OPRM1) reduced receptor dimerization and 
Gα association. Additionally, preventing palmitoylation 
reduced the association of OPRM1 with cholesterol, 
suggesting a functional complex of receptor, palmitate 
and cholesterol.
GPCRs and cholesterol
It has been thought for some time that cholesterol may 
play an important role or roles in GPCR signaling. 
Cholesterol is an amphiphilic sterol possessing a small 
-OH head group and a rigid four-ringed backbone; its 
size enables it to span approximately half a lipid bilayer, 
interacting with the hydrophobic tails of phospholipids. 
Cholesterol can alter the packing of lipids and thus 
increase membrane order. This has led to the hypothesis 
that cholesterol is responsible for the formation of lipid 
rafts and caveolae, regions of the membrane enriched in 
cholesterol and sphingolipids with higher order than the 
bulk of the membrane. GPCRs have been found to 
associate with both regions, and it is believed that this 
leads to an increased local concentration of receptors and 
G proteins in the laterally separated regions, increasing 
the efficiency of signal transduction [5]. In addition to 
affecting the properties of the membranes in which 
GPCRs reside, it has been demonstrated that a number of 
Abstract
Due to their membrane location, G protein-coupled 
receptors (GPCRs) are subject to regulation by soluble 
and integral membrane proteins as well as membrane 
components, including lipids and sterols. GPCRs also 
undergo a variety of post-translational modifications, 
including palmitoylation. A recent article by Zheng et 
al. in BMC Cell Biology demonstrates cooperative roles 
for receptor palmitoylation and cholesterol binding in 
GPCR dimerization and G protein coupling, underlining 
the complex regulation of these receptors.
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 
CO M M E N TA RY  Open Access
*Correspondence: anthony.watts@bioch.ox.ac.uk 
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, 
South Parks Rd, Oxford, OX1 3QU, UK
© 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 
http://www.biomedcentral.com/1741-7007/10/27
receptors interact directly with cholesterol (Figure  1), 
which has led to the discovery of consensus cholesterol 
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 
analogues can greatly increase GPCR stability upon 
purification and also improve crystal quality. e ability 
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 
should be noted that the effects of cholesterol on a 
particular receptor are unpredictable, and it has been 
demonstrated that certain GPCRs can function in 
Escherichia coli membranes, which lack cholesterol [6].
GPCR palmitoylation
GPCRs are post-translationally modified in a number of 
ways, including glycosylation, phosphorylation and pal-
mitoy lation (Figure  1). Palmitoylation is the addition of 
palmitic acid (a 16C saturated fatty acid) and occurs on 
one or more cysteines on the intracellular side of GPCRs. 
e thioester bond that links the palmitate to the cysteine 
is cleavable and thus the palmitoylation state of a receptor 
can be used to regulate its activity. It should be noted 
that, in rare cases, other lipids can be attached to GPCRs 
and that palmitoylation can occur on residues other than 
cysteine [7]. Again, the effects of palmitoylation are un-
predictable and GPCR-dependent.
Palmitoylation influences all aspects of GPCR 
signaling. e palmitoylation state of certain receptors 
can preferentially direct signaling through particular G 
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, 
which may select for certain G protein interactions [7]. 
Palmitoylation can influence the phosphorylation state of 
the receptor, modulating desensitization and internali-
zation, and can also control internalization independent 
of phosphorylation. It has been suggested that palmitate 
binding in the endoplasmic reticulum ensures correct 
processing and trafficking of receptors, and, once at the 
cell membrane, may target GPCRs to lipid rafts. However, 
not all palmitoylated receptors associate with rafts and 
not all raft-associated GPCRs are palmitoylated [7]. In 
the case of OPRM1, it appears that palmitoylation and 
cholesterol association (and presumably raft interactions) 
are intrinsically linked [4].
Packed with fat
An intriguing suggestion from the structure of β2AR was 
the ability of cholesterol and palmitate to act in concert 
to mediate GPCR dimer formation [3]. It is possible that 
this effect was largely observed due to crystal packing 
conditions but such an arrangement could influence 
GPCR activity in vivo.
e recent study by Zheng et al. [4] systematically 
investigates the effects of cholesterol and palmitoylation 
on OPRM1 dimerization and G protein coupling using a 
variety of colocalization techniques. Cholesterol can be 
depleted by statins and Zheng et al. used the synthetic 
statin simvastatin to probe the effects of cholesterol on 
OPRM1. Simvastatin reduced the amount of cholesterol 
associated with OPRM1 but also decreased receptor 
dimerization and receptor-G protein interactions. e 
authors also demonstrate that mutagenesis of the pal-
mitoy lated cysteine residue in OPRM1 has no effect on 
ligand binding but does decrease signaling efficiency, 
probably by impairing GPCR-G protein association. e 
same mutant had significantly reduced dimerization, and 
it was proposed that this was responsible for the reduced 
G protein coupling. Although it has been suggested that a 
Figure 1. Schematic of G protein-coupled receptor post-
translational modications and interactions. GPCRs have 
seven transmembrane domains (TMs) with an extracellular amino 
terminus and intracellular carboxyl terminus and alternating 
intracellular and extracellular loops. They undergo a variety of post-
translational modications and interact with various components 
of the membrane. (1) The amino terminus and extracellular loops of 
receptors are often glycosylated, which can be vital for correct cell-
surface localization. (2) The carboxy-terminal tail undergoes reversible 
phosphorylation, which is important in signal desensitization and 
receptor internalization. (3) Reversible palmitoylation of cysteine 
residues within intracellular loops and the carboxy-terminal tail 
results in dierent loop conformations as the palmitate groups 
penetrate into the lipid bilayer. (4) Cholesterol can interact at specic 
sites within the TM helices of GPCRs, in this case TM2 and TM4. 
(5) GPCRs also may also interact with specic lipid components of the 
membrane. These interactions and modications act in concert to 
modulate the activity of GPCRs.
P P P 
Extracellular 
Intracellular 
(1) 
(2) 
(3) 
(4) 
(5) 
Goddard and Watts BMC Biology 2012, 10:27 
http://www.biomedcentral.com/1741-7007/10/27
Page 2 of 3
G protein heterotrimer is too large to signal via a mono-
meric GPCR, monomeric receptors have been shown to 
signal efficiently [8], although this ability may be 
receptor-specific. Finally, Zheng et al. demonstrate that 
the palmitate-free mutant associated more weakly with 
cholesterol [4], suggesting a functional interaction 
between the palmitate group and cholesterol.
It is interesting to note that, in contrast to the β2AR 
crystal structure [3], the palmitate group on OPRM1 is 
not located on the carboxy-terminal tail but instead on 
the intracellular side of transmembrane domain (TM) 3 
[4]. In β2AR, the dimer interface involves TM1 and 
helix 8 (in the carboxy-terminal tail) whereas the OPRM1 
dimer interface is predicted to be between TM4 of each 
protomer, with the palmitate bound to the carboxy-
terminal side of TM3. This difference in receptor 
interface may be driven by the location of palmitoylation 
and it is possible that regulation of palmitoylation states 
could dynamically influence the dimerization interfaces 
of GPCRs. A model of the OPRM1 dimer in which 
cholesterol and palmitate pack together to facilitate 
receptor dimerization reveals that cholesterol inter actions 
contribute approximately 25% of the total interaction 
energy at the homodimer interface [4]. Dimerization of 
OPRM1 appears to be regulated by palmitate, which 
recruits cholesterol to stabilize the dimer. Most GPCR 
dimers are transient, and anything that affects the weak 
association between protomers may have dramatic 
effects on the population of dimers at equilibrium.
The complex interplay between receptor, cholesterol 
and palmitate illustrated by Zheng et al. [4] lends support 
to the model suggested by the crystal structure of β2AR 
[3] by demonstrating a role for sterols and lipids in GPCR 
dimerization in vivo. It remains to be seen if other GPCRs 
require such modulation to support dimerization. For 
example, neurotensin receptor 1 can dimerize in artificial 
lipid membranes even in the absence of palmitoylation 
[9] and also in low concentrations of detergent (no lipid 
bilayer) [10]. An interesting discussion remains - what 
truly constitutes a GPCR dimer? How close do the recep-
tors have to be to functionally interact? If a dimer is 
required solely to provide sufficient surface area for G 
protein coupling, then the proximity provided by such a 
lipid bridge seems sufficient, but if allosteric regulation 
by protomers within the dimer is to occur, more direct 
protein-protein interactions may be required.
These studies remind us that it is vital to consider 
membrane proteins in the context of their environment. 
They do not exist in isolation and are heavily influenced 
by lipids, sterols and other proteins. Cholesterol and 
palmitoylation can affect all aspects of GPCR activity and 
their precise roles are likely to be receptor-dependent, 
although only with more data can we hope to determine 
any consensus. Membrane environments are challenging 
to control and probe, but such studies are vital if we are 
to gain a complete understanding of GPCR function.
Acknowledgements
This work was supported by the Biotechnology and Biological Sciences 
Research Council (BBSRC; grant number BB/G019738/1).
Published: 19 March 2012
References
1. Hill SJ: G-protein-coupled receptors: past, present and future. Br J 
Pharmacol 2006, 147 Suppl 1:S27-37.
2. Maurice P, Kamal M, Jockers R: Asymmetry of GPCR oligomers supports 
their functional relevance. Trends Pharmacol Sci 2011, 32:514-520.
3. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, 
Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC: High-resolution crystal 
structure of an engineered human beta2-adrenergic G protein-coupled 
receptor. Science 2007, 318:1258-1265.
4. Zheng H, Pearsall EA, Hurst DP, Zhang Y, Chu J, Zhou Y, Reggio PH, Loh HH, 
Law P-Y: Palmitoylation and membrane cholesterol stabilize μ-Opioid 
receptor homodimerization and G protein coupling. BMC Cell Biol 2012, 
13:6.
5. Patel HH, Murray F, Insel PA: G-protein-coupled receptor-signaling 
components in membrane raft and caveolae microdomains. Handb Exp 
Pharmacol 2008:167-184.
6. Oates J, Watts A: Uncovering the intimate relationship between lipids, 
cholesterol and GPCR activation. Curr Opin Struct Biol 2011, 21:802-807.
7. Chini B, Parenti M: G-protein-coupled receptors, cholesterol and 
palmitoylation: facts about fats. J Mol Endocrinol 2009, 42:371-379.
8. Whorton MR, Bokoch MP, Rasmussen SG, Huang B, Zare RN, Kobilka B, 
Sunahara RK: A monomeric G protein-coupled receptor isolated in a high-
density lipoprotein particle efficiently activates its G protein. Proc Natl 
Acad Sci U S A 2007, 104:7682-7687.
9. Harding PJ, Attrill H, Boehringer J, Ross S, Wadhams GH, Smith E, Armitage JP, 
Watts A: Constitutive dimerization of the G-protein coupled receptor, 
neurotensin receptor 1, reconstituted into phospholipid bilayers. Biophys J 
2009, 96:964-973.
10. White JF, Grodnitzky J, Louis JM, Trinh LB, Shiloach J, Gutierrez J, Northup JK, 
Grisshammer R: Dimerization of the class A G protein-coupled neurotensin 
receptor NTS1 alters G protein interaction. Proc Natl Acad Sci U S A 2007, 
104:12199-12204.
doi:10.1186/1741-7007-10-27
Cite this article as: Goddard AD, Watts A: Regulation of G protein-coupled 
receptors by palmitoylation and cholesterol. BMC Biology 2012, 10:27.
Goddard and Watts BMC Biology 2012, 10:27 
http://www.biomedcentral.com/1741-7007/10/27
Page 3 of 3


A: Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

A: Uncovering the intimate relationship between lipids, cholesterol and GPCR activation. Curr Opin Struct Biol

Asymmetry of GPCR oligomers supports their functional relevance. Trends Pharmacol Sci

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction.

G-protein-coupled receptors, cholesterol and palmitoylation: facts about fats.

G-protein-coupled receptors: past, present and future.

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Regulation of G protein-coupled receptors by palmitoylation and cholesterol

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