Methods in cell biology, volume 132

Methods in Cell
Biology
G Protein-Coupled Receptors:
Signaling, Trafficking and
Regulation
Volume 132


Series Editors
Leslie Wilson
Department of Molecular, Cellular and Developmental Biology
University of California
Santa Barbara, California

Phong Tran
University of Pennsylvania
Philadelphia, USA &
Institut Curie, Paris, France


Methods in Cell
Biology
G Protein-Coupled Receptors:
Signaling, Trafficking and
Regulation
Volume 132

Edited by

Arun K. Shukla

Department of Biological Sciences and Bioengineering,
Indian Institute of Technology, Kanpur, India

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ISSN: 0091-679X
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Contributors
Agnes M. Acevedo Canabal
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,
San Juan, PR, USA; Department of Anatomy and Neurobiology, School of
Medicine, University of Puerto Rico, San Juan, PR, USA
D. Agranovich
Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel
Stefan Amisten
Diabetes Research Group, King’s College London, London, UK
Gabriela Antunes
Laboratory of Neural Systems (SisNE), Department of Physics, Faculdade de
Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo,
Ribeira˜o Preto, Brazil
Chaitanya A. Athale
Division of Biology, IISER Pune, Pune, India

Nicolas Audet
Department of Pharmacology and Therapeutics, McGill University, Montreal, QC,
Canada
Mohammed Akli Ayoub
Biologie et Bioinformatique des Syste`mes de Signalisation, Institut National de la
Recherche Agronomique, UMR85, Unite´ Physiologie de la Reproduction et des
Comportements; CNRS, UMR7247, Nouzilly, France; LE STUDIUMÒ Loire Valley
Institute for Advanced Studies, Orle´ans, France
R. Bar-Shavit
Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel
Damian Bartuzi
Department of Synthesis and Chemical Technology of Pharmaceutical
Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of
Medical Analytics, Medical University of Lublin, Lublin, Poland
Maik Behrens
Department of Molecular Genetics, German Institute of Human Nutrition
Potsdam-Rehbruecke, Nuthetal, Germany

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xiv

Contributors

Nicolas F. Berbari
Department of Biology, Indiana University-Purdue University Indianapolis,
Indianapolis, IN, USA
He´le`ne Bonin

Department of Biochemistry and Molecular Medicine, Institute for Research in
Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada
Michel Bouvier
Department of Biochemistry and Molecular Medicine, Institute for Research in
Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada
Amitabha Chattopadhyay
CSIR-Center of Cellular and Molecular Biology, Hyderabad, India
Linjie Chen
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang
University, Hangzhou, Zhejiang, China
Santiago Cuevas
Division of Renal Diseases & Hypertension, Department of Medicine, The George
Washington University School of Medicine and Health Sciences, WA, USA
Francheska Delgado-Peraza
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,
San Juan, PR, USA; Department of Anatomy and Neurobiology, School of
Medicine, University of Puerto Rico, San Juan, PR, USA
Dominic Devost
Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC,
Canada
Antonella Di Pizio
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith
Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot,
Israel
Shalini Dogra
Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar
Pradesh, India
Zyanya P. Espinosa-Riquer
Departamento de Farmacobiologı´a, Centro de Investigacio´n y de Estudios
Avanzados del IPN, Me´xico D.F., Mexico

Timothy N. Feinstein
Department of Developmental Biology, University of Pittsburgh School of
Medicine, Pittsburgh, PA, USA


Contributors

Colleen A. Flanagan
School of Physiology and Medical Research Council Receptor Biology Research
Unit, Faculty of Health Sciences, University of the Witwatersrand, Wits Parktown,
Johannesburg, South Africa
Alexandre Gidon
Molecular Mechanisms of Mycobacterial Infection, Center for Molecular
Inflammation Research, Norwegian University of Science and Technology,
Trondheim, Norway
Claudia Gonza´lez-Espinosa
Departamento de Farmacobiologı´a, Centro de Investigacio´n y de Estudios
Avanzados del IPN, Me´xico D.F., Mexico
S. Grisaru-Granovsky
Department of Obstetrics and Gynecology, Shaare Zedek, Jerusalem, Israel
Aylin C. Hanyaloglu
Institute of Reproductive and Developmental Biology, Imperial College London,
London, UK
Terence E. He´bert
Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC,
Canada
Mellisa M. Hege
Department of Biology, Indiana University-Purdue University Indianapolis,
Indianapolis, IN, USA
Ilpo Huhtaniemi

Institute of Reproductive and Developmental Biology, Imperial College London,
London, UK
M. Jaber
Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel
Kim C. Jonas
Institute of Reproductive and Developmental Biology, Imperial College London,
London, UK; Institute of Medical and Biomedical Education, St George’s
University of London, London, UK
Pedro A. Jose
Division of Renal Diseases & Hypertension, Department of Medicine, The George
Washington University School of Medicine and Health Sciences, WA, USA
Manali Joshi
Savitribai Phule Pune University, Pune, India

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Contributors

Agnieszka A. Kaczor
Department of Synthesis and Chemical Technology of Pharmaceutical
Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of
Medical Analytics, Medical University of Lublin, Lublin, Poland; School of
Pharmacy, University of Eastern Finland, Kuopio, Finland
A. Kancharla
Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel

Rafik Karaman
Bioorganic Chemistry Department, Faculty of Pharmacy, Al-Quds University,
Jerusalem, Israel
Hiroyuki Kobayashi
Department of Biochemistry and Molecular Medicine, Institute for Research in
Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada
Ajeet Kumar
Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar
Pradesh, India
Christian Le Gouill
Department of Biochemistry and Molecular Medicine, Institute for Research in
Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada
Anat Levit
Department of Pharmaceutical Chemistry, University of California e San
Francisco, San Francisco, CA, USA
Bin Lu
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang
University, Hangzhou, Zhejiang, China
Viktorya Lukashova
Department of Biochemistry and Molecular Medicine, Institute for Research in
Immunology and Cancer, Universite´ de Montre´al, Montreal, QC, Canada
Marina Macı´as-Silva
Departamento de Biologı´a Celular y Desarrollo, Instituto de Fisiologı´a Celular,
Universidad Nacional Auto´noma de Me´xico, Me´xico D.F., Mexico
M. Maoz
Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel


Contributors

Dariusz Matosiuk
Department of Synthesis and Chemical Technology of Pharmaceutical
Substances with Computer Modelling Lab, Faculty of Pharmacy with Division of
Medical Analytics, Medical University of Lublin, Lublin, Poland
Jeremy C. McIntyre
Department of Neuroscience, University of Florida, Gainesville, FL, USA; Center
for Smell and Taste, University of Florida, Gainesville, FL, USA
Masha Y. Niv
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith
Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot,
Israel; Fritz Haber Center for Molecular Dynamics, The Hebrew University,
Jerusalem, Israel
Carlos Nogueras-Ortiz
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,
San Juan, PR, USA
Melanie Philipp
Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany
Cristina Roman-Vendrell
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,
San Juan, PR, USA; Department of Physiology, School of Medicine, University of
Puerto Rico, San Juan, PR, USA
Ewelina Rutkowska
Department of Biopharmacy, Faculty of Pharmacy with Division of Medical
Analytics, Medical University of Lublin, Lublin, Poland
Jana Selent
Research Programme on Biomedical Informatics (GRIB), Universitat Pompeu
Fabra, IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain
Durba Sengupta
CSIR-National Chemical Laboratory, Pune, India

Ying Shi
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang
University, Hangzhou, Zhejiang, China
Fabio Marques Simoes de Souza
Center for Mathematics, Computation and Cognition, Federal University of ABC,
Sa˜o Bernardo do Campo, Brazil

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xviii

Contributors

Michal Slutzki
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith
Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot,
Israel
Chandan Sona
Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar
Pradesh, India
Katarzyna M. Targowska-Duda
Department of Biopharmacy, Faculty of Pharmacy with Division of Medical
Analytics, Medical University of Lublin, Lublin, Poland
Teresa Casar Tena
Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany
B. Uziely
Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel
Genaro Va´zquez-Victorio

Departamento de Biologı´a Celular y Desarrollo, Instituto de Fisiologı´a Celular,
Universidad Nacional Auto´noma de Me´xico, Me´xico D.F., Mexico
Jean-Pierre Vilardaga
Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology,
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Van Anthony M. Villar
Division of Renal Diseases & Hypertension, Department of Medicine, The George
Washington University School of Medicine and Health Sciences, WA, USA
Richard Wargachuk
Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC,
Canada
Kunhong Xiao
Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology,
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Prem N. Yadav
Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, Uttar
Pradesh, India
Guillermo A. Yudowski
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,
San Juan, PR, USA; Department of Anatomy and Neurobiology, School of
Medicine, University of Puerto Rico, San Juan, PR, USA


Contributors

Yaping Zhang
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang
University, Hangzhou, Zhejiang, China
Xiaoxu Zheng
Division of Renal Diseases & Hypertension, Department of Medicine, The George

Washington University School of Medicine and Health Sciences, WA, USA
Cynthia Zhou
Department of Pharmacology and Therapeutics, McGill University, Montre´al, QC,
Canada
Naiming Zhou
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang
University, Hangzhou, Zhejiang, China

xix


Preface
G proteinecoupled receptors (GPCRs) also referred as seven transmembrane
receptors (7TMRs) lie at the heart of almost every physiological and pathophysiological process in our body. These receptors bind to and get activated by a wide
range of ligands ranging from small molecules, hormones, peptides, proteins to
lipids. The overall activation and signal transduction mechanisms of GPCRs are
highly conserved where binding of an agonist results in a conformational change
in the receptor followed by activation of heterotrimeric G proteins and subsequent
generation of second messengers and downstream signaling. Downregulation of
GPCRs is also primarily a conserved process where activated receptors are
phosphorylated by GRKs (GPCR kinases) followed by binding of beta arrestins
which leads to receptor desensitization and internalization. GPCRs are targeted by
about one-third of the currently prescribed drugs which include angiotensin blockers
for hypertension, beta-blockers for heart failure, antihistamines for allergy management, and opioid agonists as analgesic medication.
In this volume of Methods in Cell Biology, we cover multiple aspects of GPCR
signaling, trafficking, regulation, and cellular assays in a form of either an overview or as step-by-step protocol. This is an effort to bring together different
domains of GPCR pharmacology and signaling on to a common platform and highlight the incredibly versatile nature and diverse functional manifestation of
GPCRs. Section I includes chapters on GPCR trafficking in lipid rafts and cilia,
imaging endogenous receptor in neurons, single molecule imaging of GPCRs,
and a comprehensive analysis of GPCRs in adipose tissue. In Section II, we cover

topics ranging from GPCR signaling from endosomes, olfactory receptor signal
transduction, studies of a specialized GPCR smoothened in zebra fish model,
and the outcome of GPCR signaling in cytoskeletal dynamics. In recent years, a
key focus area in GPCR biology has been the development of novel and more sensitive cellular assays to investigate GPCR expression, signaling, and downregulation. Section III of this volume is focused on GPCR assays which include classical
radioligand binding, label-free, biosensor and fluorescenceebased approaches to
study GPCR trafficking and signaling, and TANGO assay for measuring GPCRbeta-arrestin interaction. Finally, Section IV consists of chapters on structural
and computational aspects of protease-activated receptors, bitter taste receptors,
and GPCR dimerization.
I would like to thank all the authors who have contributed to this focused volume
despite their busy schedule. I also express my sincere gratitude to the journal editorial staff and production team for a wonderful job in putting this volume together in a
timely fashion. With this brief background, on behalf of the entire Methods in Cell

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Preface

Biology Team, I present to you this volume entitled “G ProteineCoupled Receptors:
Signaling, Trafficking, and Regulation.” I sincerely hope that you enjoy the topics
covered in this issue and please feel free to share your feedback with us.
Arun K. Shukla
Indian Institute of Technology, Kanpur, India


CHAPTER

Localization and signaling
of GPCRs in lipid rafts

1

Van Anthony M. Villar1, Santiago Cuevas, Xiaoxu Zheng, Pedro A. Jose1
Division of Renal Diseases & Hypertension, Department of Medicine, The George Washington
University School of Medicine and Health Sciences, WA, USA
1

Corresponding authors: E-mail: ;

CHAPTER OUTLINE
Introduction ................................................................................................................ 4
1. Localization of GPCRs in Lipid Rafts ........................................................................ 6
1.1 Isolation of Lipid Rafts ............................................................................ 7
1.1.1 Detergent-free method.......................................................................... 7
1.1.2 Detergent-based method ...................................................................... 9
1.1.3 Immunoblotting and data interpretation............................................... 10
1.2 Localization of GPCRs in Lipid Rafts....................................................... 11
1.2.1 Cells in suspension............................................................................. 13
1.2.2 Adherent cells .................................................................................... 14
2. GPCR Signaling in Lipid Rafts ............................................................................... 15
2.1 Perturbation of Raft Stability.................................................................. 15
2.2 Changing the Cholesterol Content ........................................................... 16
2.3 Fluorescence Imaging............................................................................ 16
References ............................................................................................................... 18

Abstract
The understanding of how biological membranes are organized and how they function
has evolved. Instead of just serving as a medium in which certain proteins are found,
portions of the lipid bilayer have been demonstrated to form specialized platforms that

foster the assembly of signaling complexes by providing a microenvironment that is
conducive for effective proteineprotein interactions. G protein-coupled receptors
(GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key
enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains,
called lipid rafts. As such, lipid rafts are crucial for the trafficking and signaling of
GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization
of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and
Methods in Cell Biology, Volume 132, ISSN 0091-679X, />© 2016 Elsevier Inc. All rights reserved.

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4

CHAPTER 1 GPCRs in lipid rafts

the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack
of standardized methodology to study lipid rafts, in general, and of the workings of
GPCRs in lipid rafts, in particular, and the inherent drawbacks of current methods have
hampered the complete understanding of the underlying molecular mechanisms. Newer
methodologies that allow the study of GPCRs in their native form are needed. The use of
complementary approaches that produce mutually supportive results appear to be the best
way for drawing conclusions with regards to the distribution and activity of GPCRs in
lipid rafts.

INTRODUCTION
Lipid Raft Microdomains. The plasma membrane is a semipermeable, biological
membrane that demarcates the intracellular milieu from the extracellular environment. Amphipathic lipids, such as phospholipids and sphingolipids, are the building
blocks of these bilipid membranes because of their aggregative properties, i.e., their
hydrophobic tails associate together, while their hydrophilic heads interact with both

extra- and intracellular aqueous environments (Sonnino & Prinetti, 2013). The
fluidity of the fatty acyl groups of phospholipids at 37  C enables the membranes
to act as a medium in which dissolved membrane proteins are afforded ample lateral
mobility, especially in response to environmental cues. Since the first description of
an “organization of the lipid components of membranes into domains” (Karnovsky
et al., 1982) and the elaboration of the “lipid raft hypothesis” by Simons and van
Meer (van Meer & Simons, 1988; Simons & Ikonen, 1997; Simons & van Meer
1998), the existence of lipid rafts is now established.
Lipid rafts are tightly packed, highly organized plasma membrane microdomains
that are enriched in phospholipids, glycosphingolipids, and cholesterol and serve as
a platform for the organization and dynamic interaction of biomolecules involved in
various biological processes (Figure 1). The cholesterol bestows a semblance of
rigidity and order by intertwining into the hydrophobic gaps between the phospholipid acyl chains. Certain structural proteins abound in lipid rafts to serve as scaffold
or anchor for other proteins, including caveolins (Head, Patel, & Insel, 2014; Quest,
Leyton, & Pa´rraga, 2004; Yu, Villar, & Jose, 2013; Yu et al., 2004), flotillins
(Rajendran, Le Lay, & Illges, 2007; Yu et al., 2004) and tetraspanins (Hemler,
2005), and glycosylphosphatidylinositol-linked (GPI-linked) proteins. The spatial
concentration and organization of specific sets of membrane proteins allow greater
efficiency and specificity of signal transduction by facilitating proteineprotein
interactions and by preventing crosstalk between competing pathways. The
nonhomogeneous lateral distribution of membrane components helps explain the
differences in composition between apical and basolateral membrane domains of
polarized epithelial cells (Sonnino & Prinetti, 2013).
The best characterized lipid raft microdomains are the caveolae, which were first
described by Palade and Yamada in the 1950s (Palade, 1953; Yamada, 1955). These
are small (60e80 nm) invaginations of the plasma membrane formed by the
polymerization of caveolins with cholesterol (Parton & del Pozo, 2013). Caveolae


Introduction

FIGURE 1 A Lipid Raft Membrane Microdomain.
Lipid rafts are highly organized plasma membrane microdomains enriched in phospholipids,
glycosphingolipids, and cholesterol, and serve as matrix for receptors, such as G proteincoupled receptors (GPCRs), and other signaling molecules. (See color plate)
Van Anthony M. Villar, MD, PhD.

have been implicated in a variety of cellular processes, including signal transduction, endocytosis, transcytosis, and cholesterol trafficking (Barnett-Norris, Lynch, &
Reggio, 2005). Lipid rafts accumulate in the apical plasma membrane in polarized
epithelial cells and in axonal membranes in neurons. Basolateral and dendritic
membranes contain lipid rafts but in more limited quantities (Simons & Ikonen,
1997). Interestingly, caveolae are found mostly at the basolateral membrane that
faces the blood supply and is more active during signal transduction (Simons &
Toomre, 2000). Lipid rafts are mostly found at the plasma membrane; however,
they may also be found in intracellular membranes involved in the biosynthetic
and endocytic pathways. Lipid raft microdomains play a crucial role in cellular processes such as membrane sorting, receptor trafficking, signal transduction, and cell
adhesion.
GPCR Signaling and Trafficking. G protein-coupled receptors (GPCRs)
constitute the largest superfamily of seven transmembrane proteins that respond
to a myriad of environmental stimuli that are transduced intracellularly as meaningful signals through secondary messengers. Agonist stimulation of a GPCR leads to a
conformational change that promotes the exchange of GDP for GTP on the Ga subunit of the G protein, resulting in the uncoupling of the G protein from the GPCR and
the dissociation of Ga and Gbg subunits. The Ga subunit either activates or inhibits
intracellular signaling pathways depending on the receptor subtype, while the Gbg
subunit recruits G protein-coupled receptor kinases which selectively phosphorylate
serine and threonine residues localized within the third intracellular loop and
carboxyl-terminal tail domains of the receptor to promote the binding of cytosolic
cofactor proteins called arrestins (Lefkowitz, 1998). The b-arrestins play a pivotal
role in the uncoupling process and in the sequestration and internalization of GPCRs

5

6

CHAPTER 1 GPCRs in lipid rafts

through a dynamin-dependent, clathrin-mediated endocytosis. Once internalized,
the GPCRs, in vesicles termed as early endosomes, are sorted by sorting nexins
and follow divergent pathways (Worby & Dixon, 2002). The receptors are sorted
into recycling endosomes for their return to the cell membrane (recycling and
resensitization), accumulate in late endosomes which target the lysosomes for their
subsequent degradation, or transported initially to the perinuclear endosomes (transGolgi network) and then to the late endosomes for eventual lysosomal degradation.
Additional proteolytic mechanisms, such as proteasomes or cell-associated endopeptidases, are also implicated in mediating the downregulation of certain GPCRs
(von Zastrow, 2003).
The signal transduction that follows ligand occupation of the GPCR is highly
regulated to ensure the specificity of the cellular response, both temporally and
spatially. The signal transduction can be attenuated with relatively fast kinetics
through a process called desensitization or through a much slower process of downregulation following prolonged or repeated exposure to an agonist. Desensitization,
or the waning of a receptor’s responsiveness to agonist with time, is an inherent
molecular “feedback” mechanism that prevents receptor overstimulation and helps
in creating an integrated and meaningful signal by filtering out information from
weaker GPCR-mediated signals (Ferguson, 2001).
It is accomplished through two complementary mechanisms, i.e., the functional
uncoupling of GPCRs from their cognate G proteins, which occurs without any
detectable change in the number of cell surface receptors, and GPCR phosphorylation, sequestration, and internalization/endocytosis. GPCR resensitization protects
the cells from prolonged desensitization and is carried out via dephosphorylation
by phosphatases as the GPCR traffics through the endosomal pathway. GPCR activity is the net result of a coordinated balance between receptor desensitization and
resensitization.
It is now established that lipid rafts serve as dynamic platforms for GPCRs and
pertinent signaling molecules such as G proteins, enzymes, and adaptors (BarnettNorris et al., 2005; Lingwood & Simons, 2010). However, understanding the
molecular mechanisms involved has been hampered by the lack of standardized

methodology to study lipid rafts, in general, and of the workings of GPCRs in lipid
rafts, in particular. Moreover, the minute size of lipid rafts has made lipid rafts
difficult to resolve by standard light microscopy, unless the lipid raft components
are cross-linked with antibodies or lectins (Simons & Toomre, 2000). Studying
how GPCR works in lipid rafts may be accomplished by determining if the
GPCR of interest localizes to the lipid rafts and by evaluating if GPCR signaling
and activity are lost when lipid rafts are disrupted.

1. LOCALIZATION OF GPCRs IN LIPID RAFTS
Several techniques are available for the detection and localization of GPCRs in lipid
raft microdomains in cells. The most commonly employed approach utilizes cell


1. Localization of GPCRs in lipid rafts

fractionation procedures that break the cells apart and destroy cell morphology
before GPCR analysis using biochemical or immunological assays. A complementary biophysical approach involves the visualization of GPCRs in intact cell
membranes.

1.1 ISOLATION OF LIPID RAFTS
Lipid rafts are characterized by their relative insolubility in nonionic detergents at
4  C and light buoyant density on sucrose gradient (Schnitzer, McIntosh, Dvorak,
Liu, & Oh, 1995). The isolation of lipid rafts can be performed using either
detergent-based or detergent-free methods (Yu et al., 2013), with the latter generating
a greater fraction of inner leaflet membrane rafts and producing more replicable
results (Pike, 2004). Schnitzer et al. (1995) employed a detergent-free method to
isolate lipid rafts using cationic colloidal silica particles, which is appropriate for
non-cell culture studies. Lipid rafts may be extracted from total cell membranes
(Song et al., 1996) or just from surface plasma membranes (Smart, Ying, Mineo, &
Anderson, 1995). Detergent insolubility results from the segregation of membraneassociated proteins into the lipid rafts, which are abundant in cholesterol and

glycosphingolipids. Nonionic detergents, such as Triton X-100, b-octyl glucoside,
CHAPS, deoxycholate, Lubrol WX, Lubrol PX, Brij 58, Brij 96, and Brij 98, have
been used to prepare lipid raft fractions (Macdonald & Pike, 2005), resulting in
varying yields of proteins. Samples obtained by detergent-based methods are termed
detergent-resistant membranes or detergent-insoluble fractions. Different detergents
may yield different lipid raft components because of the varying degrees of resistance by the proteins to extraction using different reagents. The methods detailed
below are based on Yu et al. (2013).

1.1.1 Detergent-free method
Materials
2-N-morpholino ethanesulfonic acid (Mes), 250 mM, pH ¼ 6.8
Mes-buffered solution (MBS), 25 mM Mes þ 150 mM NaCl
Sodium citrate, 500 mM, pH w 11 (add protease inhibitors)
Sucrose, 5%, 35%, and 80% in MBS solution (add protease inhibitors)
Methyl-b-cyclodextrin (b-MCD), 2% dissolved in cell culture media
Cholesterol þ b-MCD (Sigma catalog #C4951), dissolved in cell culture
media
1X PBS, for washing
1. Cell culture and cell pellet collection. To obtain sufficient amounts of lipid raft
fraction, cells should be grown in 150-mm dishes until almost confluent using
the appropriate media at 37  C with 95% air and 5% CO2. Separate dishes of
cells should also be treated for cholesterol depletion and repletion as experimental controls (Figure 2). Cholesterol depletion to disrupt the lipid rafts is
commonly performed by pretreatment with b-MCD for 1 h at 37  C.

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CHAPTER 1 GPCRs in lipid rafts

FIGURE 2 Comparison groups for GPCR localization in lipid rafts.

Methyl-a-cyclodextrin (a-MCD) may be used as control for b-MCD (Vial &
Evans, 2005). Cholesterol repletion is performed by pretreating with cholesterol/b-MCD solution for 1 h at 37  C. Cholestane-3,5,6-triol, an inactive
analog of cholesterol, may be used as control for the use of exogenous
cholesterol (Murtazina, Kovbasnjuk, Donowitz, & Li, 2006). To determine the
effect of agonist or antagonist treatment, cells should be serum-starved for at
least 1 h prior to treatment to achieve “basal” conditions prior to treatment.
Additional controls, such as the use of the drug vehicle, should be concomitantly performed.
1.1 Wash cells with cold PBS and scrape the cells using a rubber-tipped cell
scraper.
1.2 Transfer cell suspension into 15-mL tube and spin at 2000Â g for 5 min.
1.3 Decant the supernatant to obtain the cell pellet.
2. Cell homogenate preparation. All steps are carried out at 4  C.
2.1 To the cell pellet, add 1.5 mL 500 mM sodium carbonate and vortex.
2.2 Homogenize the cell suspension by sonication using five 20-s bursts on ice.
2.3 Add 1.5 mL of 80% sucrose and mix by vortex and sonication (three 20-s
bursts) on ice. Protein concentration may be determined at this time using a
BCA kit.
3. Sucrose gradient ultracentrifugation. Prepare 5%, 35%, and 80% sucrose
solutions in MBS solution. The use of MBS solution with pH close to 7.0 may
be advantageous for most proteins.
3.1 Place 3 mL of cell homogenates into the bottom of precooled 12-mL
ultracentrifuge tubes.
3.2 Overlay sequentially 4.5 mL of 35% sucrose and 4.5 mL of 5% sucrose to
each tube.
3.3 With the tubes securely balanced in an SW-41 bucket, spin at 180,000Â g
(38,000 rpm) for 16 h at 4  C in a Beckman SW-41 centrifuge.
4. Lipid raft fraction preparation. A light-scattering band that is enriched with

caveolae/lipid rafts can be observed between the 5% and 35% sucrose gradients
and corresponds to the fourth fraction.
4.1 Carefully aspirate 12 1-mL fractions from the top of the tube and transfer
into prelabeled 1.5 microcentrifuge tubes.


1. Localization of GPCRs in lipid rafts

4.2 Prepare 0.5 mL of each fraction by adding 0.1 mL 6X sample buffer, vortex,
and boil for 5 min before use for immunoblotting. These samples can be
stored at À20  C, while the rest of the fractions without the 6X sample
buffer can be stored at À80  C.

1.1.2 Detergent-based method
Materials
50% Optiprep Stock solution (45 mL of 60% Optiprep þ 9 mL of Optiprep
diluent)
MBSTS buffer (MBS þ 0.5% Triton X-100 þ protease inhibitors in 10% sucrose)
Sucrose solutions (Table 1):
Table 1 Preparation of Optiprep Gradient Solutions
Solution
(5 mL total
volume)
50% Optiprep
(mL)
MBSTS (mL)

30% Sucrose

20% Sucrose

10% Sucrose

5% Sucrose

3.0

2.0

1.0

0.5

2.0

3.0

4.0

4.5

1. Cell culture and cell pellet preparation. The same as with the detergent-free
method.
2. Cell extract preparation.
2.1 Add 0.3 mL ice-cold MBSTS to cell pellet and push through a 25G
needle 10Â.
2.2 Adjust cell extract (w0.4 mL; cell pellet volume is w0.1 mL) to 40%
Optiprep by adding 0.8 mL of cold 60% Optiprep and vortex. Determine
protein concentration using a BCA kit.
3. Optiprep gradient ultracentrifugation.

3.1 Place 1 mL of the cell extract into the bottom of precooled 5-mL ultracentrifuge tubes.
3.2 Overlay with 1 mL each of 30%, 25%, 20%, and 0% Optiprep solutions in
MBSTS buffer.
3.3 Secure each tube in a Beckman SW 50.1 bucket and spin at 175,000Â g
(42,000 rpm) at 4  C for 4 h. Other rotors may be used, such as the SW 55
(170,000Â g for 4 h) or TLS55 (250,000Â g for 2.5 h).
4. Lipid raft fraction preparation.
4.1 Carefully aspirate ten 0.5-mL fractions from the top of the tube and transfer
into prelabeled 1.5 microcentrifuge tubes.
4.2 Prepare 0.25 mL of each fraction by adding 0.5 mL 6X sample buffer,
vortex, and boil for 5 min before use for immunoblotting. These samples
can be stored at À20  C, while the rest of the fractions without the 6X
sample buffer can be stored at À80  C.

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CHAPTER 1 GPCRs in lipid rafts

1.1.3 Immunoblotting and data interpretation
Western blot is the most commonly used method to determine the lipid raft distribution of proteins, such as GPCRs. Antibody specificity is crucial for the identification
of the GPCR of interest. The lipid raft proteins are found in the more buoyant
fractions (top 5e6 fractions); however, their distribution among these fractions is
not uniform. Immunoblotting for lipid raft markers may help in determining the
fractions where the lipid rafts are most abundant. Caveolin-1 is the most commonly
used protein marker for lipid rafts, specifically for caveolae (Insel et al., 2005;
Lingwood & Simons, 2010). There are several other markers for lipid rafts, such
as flotillin-1, CD55, alkaline phosphatase, and pore-forming toxins, such as cholera

toxin subunit B (CTxB), equinatoxin II, perfringolysin (Foster, De Hoog, & Mann,
2003; Salzer & Prohaska, 2001; Skocaj et al., 2013). Flotillin-1 has been used as a
lipid raft marker protein in cells that do not contain caveolae, i.e., blood cells
(Salzer & Prohaska, 2001), neural cells (Huang et al., 2007), and rat renal proximal
tubule cells (Breton, Lisanti, Tyszkowski, McLaughlin, & Brown, 1998; Riquier, Lee,
& McDonough, 2009) and human embryonic kidney (HEK)-293 cells (Yu et al.,
2004). There is species specificity because human renal proximal tubule cells
express caveolin-1 (Gildea et al., 2009), while HEK-293 cells express caveolin-2.
These markers may also be used to indicate the integrity of lipid rafts in cholesterol
depletion or repletion experiments. In general, these markers should be distributed in
the more buoyant fractions and should redistribute into the less buoyant fractions
(fractions 7e12) after cholesterol depletion with b-MCD (Figure 3). Cholesterol
repletion reconstitutes the lipid rafts and thus, these markers should be observed in
the more buoyant fractions.

FIGURE 3 Lipid Raft Distribution of Caveolin-1 and D1R.
Lipid raft and non-lipid raft fractions from human renal proximal tubule cells treated with
b-MCD, a cholesterol-depleting and lipid raft-disrupting agent, were prepared by detergentfree method and sucrose gradient ultracentrifugation. The distribution of caveolin-1, a lipid
raft marker, and the dopamine D1 receptor (D1R), a GPCR, is shown in the immunoblots.
Images are courtesy of Peiying Yu, MD.


1. Localization of GPCRs in lipid rafts

1.2 LOCALIZATION OF GPCRs IN LIPID RAFTS
Another way to demonstrate the distribution of GPCRs in lipid rafts is by visualizing
them in intact cells, living or fixed, and tissues. There are now commercially available kits that have been developed for labeling the lipid rafts using the CTxB that is
tagged with fluorophores (Figure 4). CTxB binds to the pentasaccharide chain of
ganglioside GM1, which selectively partitions into lipid rafts. For visualizing lipid
rafts, cells are labeled with CTxB tagged with Alexa FluorÒ 488, Alexa FluorÒ

FIGURE 4 Colocalization of the D1 dopamine receptor (D1R) in Lipid Rafts of Human Renal
Proximal Tubule Cells.
Human renal proximal tubule cells were grown on a poly-L-Lysine-coated cover slip to 50%
confluence and serum-starved for 1 h to determine the basal distribution of D1R prior to
fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100. The lipid
rafts were labeled using cholera toxin subunit B (CTxB) tagged with Alexa FluorÒ 555
(Molecular Probes), while the endogenous D1R was immunostained using a proprietary
rabbit-anti-D1R antibody and a donkey anti-rabbit secondary antibody tagged with Alexa
FluorÒ 488 (Molecular Probes). DAPI was used to visualize the nucleus. At the basal state,
most of the D1R were found intracellularly, just below the inner leaflet of the plasma
membrane, although some colocalized with the lipid rafts (yellow areas pointed at by arrows).
The raw images were captured via laser scanning confocal microscope using separate
channels and the composite image was obtained using Zen 2011 software. 630X
magnification, scale bar ¼ 10 mm. (See color plate)
Van Anthony M. Villar, MD, PhD.

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CHAPTER 1 GPCRs in lipid rafts

555, or Alexa FluorÒ 647 before cross-linking with an anti-CTxB to maintain the
in situ protein distribution. To demonstrate the lipid raft distribution of GPCRs, colocalization experiments may be performed via laser scanning confocal microscopy by
labeling the lipid rafts using CTxB and immunostaining the GPCR of interest using
specific antibodies on the same cell. CTxB labeling may also be used to demonstrate
lipid raft endocytosis upon agonist stimulation in live cells (Qi, Mullen, Baker, &
Holl, 2010) and cultured explants (Hansen et al., 2005). The c-subunit of cytolethal

distending toxin (cdt) may also be utilized for lipid raft colocalization experiments
(the protocol is detailed in Boesze-Battaglia, 2006). Other pore-forming toxins, besides CTxB, used to visualize lipid rafts include equinatoxin II which binds dispersed
sphingomyelin, lysenin which binds clustered sphingomyelin, perfringolysin O which
binds to cholesterol, and ostreolysin which binds to the combination of sphingomyelin
and cholesterol (Makino et al., 2015; Skocaj et al., 2013).
An alternative to using CTxB, cdt, and other pore-forming toxins is to use
antibodies that specifically target the lipid raft protein markers, such as
caveolin-1, caveolin-3, and flotillin-1. Conversely, transferrin receptors, CD71,
and geranylated proteins are non-lipid raft markers (Boesze-Battaglia, 2006;
Magee, Adler, & Parmryd, 2005). The ganglioside GM1 may be labeled with single
quantum dots to measure the lateral mobility and extent of movement of the lipid
rafts (Chang & Rosenthal, 2012). Recently, GPI-anchored proteins that segregate
into lipid rafts have been visualized using a novel method called enzyme-mediated
activation of radical sources (Miyagawa-Yamaguchi, Kotani, & Honke, 2015).
Probes that target the lipid content of lipid rafts have also been used to visualize
these membrane microdomains. Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) and C-laurdan (6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)
amino]-naphthalene), which are membrane probes that are sensitive to membrane
polarity, allow the observation of lipid rafts via two-photon microscopy (Gaus,
Zech, & Harder, 2006; Kim et al., 2007, 2008). A fluorophore-tagged domain
D4 of perfringolysin O, a cholesterol-binding cytolysin produced by Clostridium
perfringens, has been used as probe to study membrane cholesterol (OhnoIwashita et al., 2004).
Aside from confocal microscopy, other biophysical approaches may also be
employed to study labeled GPCRs and/or lipid rafts. Single fluorophore tracking
microscopy (Schu¨tz, Kada, Pastushenko, & Schindler, 2000) and fluorescence
recovery after photobleaching (Kenworthy, 2007) may be used to monitor lateral
diffusion of lipid raft-anchored GPCRs, while fluorescence lifetime imaging
microscopyefluorescence resonance energy transfer (FLIM-FRET) (Kenworthy,
Petranova & Edidin, 2000; Thaa, Herrmann, & Veit, 2010) may be used to determine the proximity of GPCRs with other proteins of interest, or of lipid raft sizes
depending on membrane composition (de Almeida, Loura, Fedorov, & Prieto,
2005). Atomic force microscopy may be used to visualize the effects of detergent

solubilization of membranes during lipid raft studies (Garner, Smith, & Hooper,
2008). Lipid rafts can now be visualized using superresolution imaging below
the 200 nm limit of conventional microscopes, e.g., including structured


1. Localization of GPCRs in lipid rafts

illumination microscopy, stimulated emission depletion (STED) microscopy, nearfield scanning optical microscopy, photoactivated localization microscopy
(PALM), and stochastic optical reconstruction microscopy (dSTORM) (Owen &
Gaus, 2013; Tobin et al., 2014; Wu et al., 2013).
Materials
VybrantÒ Lipid Raft Labeling Kits (Catalog #V-34403, V-34404, or V-34405)
prepare fresh working solutions according to manufacturer’s instructions
Primary antibody against the GPCR of interest
Secondary antibody against the host of the primary antibody
10% bovine serum albumin (BSA) solution
4% Paraformaldehyde in PBS
Mounting medium (EMS catalog #17985) without 40 ,6-diamidino-2phenylindole (DAPI)
DAPI, a nuclear stain, 10 mM stock solution
Triton X-100, 20% stock solution in deionized water
1X PBS for washing

1.2.1 Cells in suspension
Colocalization of GPCRs with lipid rafts can now be accomplished with the concomitant use of CTxB and an antibody against the GPCR of interest on cells. The cells can
be labeled in suspension and then mounted on glass slides for imaging, or the cells can
be grown and labeled on cover slips or in TranswellsÒ cell culture inserts when cell
polarity is important to distinguish between apical versus basolateral membranes.
1. Fluorescent labeling of cells.
1.1 Spin cells at 2000Â g for 5 min and decant the medium.
1.2 Resuspend the cells in cold medium, spin, and decant the medium.

1.3 Resuspend the cells in 2 mL of CTxBeAlexa FluorÒ working solution at
4  C for 10 min. The primary antibody against the GPCR of interest may
be added to this working solution at 1:100 dilution. The primary antibody
against the GPCR should be raised in mouse, goat, rat, or chicken but not in
rabbit when using the VybrantÒ Lipid Raft Labeling Kits. Alternatively,
the primary antibody against the GPCR (especially if only a rabbit antibody is available) may be prelabeled with a Fluor other than the one used
for CTxB. Directly labeling the primary antibody precludes the use of a
secondary antibody (in step 1.5).
1.4 Gently wash cells 3Â with cold PBS. Spin cells and decant wash buffer.
1.5 Resuspend in 2 mL of the rabbit CTxB antibody working solution at 4  C
for 30 min. The rabbit CTxB antibody cross-links it to the lipid raft domains. The secondary antibody against the primary antibody may be added
to this working solution at 1:100 dilution. The secondary antibody should
be tagged with a Fluor other than the one used to label the CTxB.
As counterstain, 300 nM DAPI may also be added to this working solution.
1.6 Gently wash cells 3Â with cold PBS. Spin cells and decant wash buffer.

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CHAPTER 1 GPCRs in lipid rafts

2. Mounting and imaging.
2.1 (Optional) Fix cells with 4% paraformaldehyde at room temperature for
15 min. Paraformaldehyde is a cross-linker fixative that preserves the
architecture of the cell but may reduce the antigenicity of some cell
components and thus, requires an additional permeabilization step if
additional intracellular proteins are needed to be visualized. Fixation may
also be achieved using organic solvents, such as alcohols and acetone, but

these remove lipids and precipitate the proteins and often disrupt the cell
structure.
2.2 Mount live cells in cold PBS or fixed cells in mounting medium on glass
slide and cover with cover slip.
2.3 Image the cells using a laser scanning confocal microscope. The appropriate
filters should be used depending on the Alexa FluorÒ dye that was used and
whether DAPI was used as a nuclear stain or not (Table 2).

1.2.2 Adherent cells
1. Cell culture on cover slips.
1.1 Grow cells on 12-mm cover slips placed in a 24-well tissue culture plate to
w50% confluence using complete cell culture medium at 37  C in 95% air
and 5% CO2. Cover slips coated with lysine, laminin, or collagen may
improve cell attachment for cells that easily detach, such as HEK-293 cells.
To determine the effect of agonist/antagonist treatment on GPCR
trafficking, cells should be serum-starved for at least 1 h prior to treatment
to achieve “basal” conditions prior to treatment. Additional controls, such
as vehicle treatment, should be performed.
1.2 Draw off the medium and wash cells with cold PBS. Place the cell culture
plate on ice to stop further receptor endocytosis and endosomal trafficking.
2. Fluorescent labeling, fixation, and permeabilization.
2.1 Add 0.3 mL of CTxBeAlexa FluorÒ working solution at 4  C for 10 min.
2.2 Draw off the solution and wash cells with cold PBS.
2.3 Fix cells with 0.3 mL of 4% paraformaldehyde at room temperature for
15 min.
2.4 Wash cells with PBS. Subsequent steps can be performed at room
temperature.
Table 2 Fluorescence Spectra of CTxB Conjugates
CTxB Fluor Conjugate (Catalog #)

Maximum Absorption and Emission
(nm)

Alexa FluorÒ 488 (V-34403)
Alexa FluorÒ 555 (V-34404)
Alexa FluorÒ 594 (V-34405)

495/519
555/565
590/617

The maximum absorption and emission for DAPI are 358/461 nm.