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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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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 Ciências e Letras de Ribeirão Preto, Universidade de São Paulo,
Ribeirão 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 Systèmes de Signalisation, Institut National de la
Recherche Agronomique, UMR85, Unité Physiologie de la Reproduction et des
Comportements; CNRS, UMR7247, Nouzilly, France; LE STUDIUMÒ Loire Valley
Institute for Advanced Studies, Orléans, France
R. Bar-Shavit
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
xiii
xiv Contributors
Nicolas F. Berbari
Department of Biology, Indiana University-Purdue University Indianapolis,
Indianapolis, IN, USA
Hélène Bonin
Department of Biochemistry and Molecular Medicine, Institute for Research in
Immunology and Cancer, Université de Montréal, Montreal, QC, Canada
Michel Bouvier
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
Dominic Devost
Department of Pharmacology and Therapeutics, McGill University, Montréal, 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 Farmacobiologı́a, Centro de Investigación y de Estudios
Avanzados del IPN, México D.F., Mexico
Timothy N. Feinstein
Department of Developmental Biology, University of Pittsburgh School of
Medicine, Pittsburgh, PA, USA
Contributors xv
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 González-Espinosa
Departamento de Farmacobiologı́a, Centro de Investigación y de Estudios
Avanzados del IPN, México 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. Hébert
Canada
Mellisa M. Hege
Ilpo Huhtaniemi
London, UK
M. Jaber
Jerusalem, Israel
Kim C. Jonas
London, UK; Institute of Medical and Biomedical Education, St George’s
University of London, London, UK
Pedro A. Jose
Manali Joshi
Savitribai Phule Pune University, Pune, India
xvi Contributors
Agnieszka A. Kaczor
Medical Analytics, Medical University of Lublin, Lublin, Poland; School of
Pharmacy, University of Eastern Finland, Kuopio, Finland
A. Kancharla
Jerusalem, Israel
Rafik Karaman
Bioorganic Chemistry Department, Faculty of Pharmacy, Al-Quds University,
Jerusalem, Israel
Hiroyuki Kobayashi
Ajeet Kumar
Pradesh, India
Christian Le Gouill
Anat Levit
Department of Pharmaceutical Chemistry, University of California e San
Francisco, San Francisco, CA, USA
Bin Lu
Viktorya Lukashova
Marina Macı́as-Silva
Departamento de Biologı́a Celular y Desarrollo, Instituto de Fisiologı́a Celular,
Universidad Nacional Autónoma de México, México D.F., Mexico
M. Maoz
Jerusalem, Israel
Contributors xvii
Dariusz Matosiuk
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
Israel; Fritz Haber Center for Molecular Dynamics, The Hebrew University,
Jerusalem, Israel
Carlos Nogueras-Ortiz
San Juan, PR, USA
Melanie Philipp
Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany
Cristina Roman-Vendrell
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
Fabio Marques Simoes de Souza
Center for Mathematics, Computation and Cognition, Federal University of ABC,
São Bernardo do Campo, Brazil
xviii Contributors
Michal Slutzki
Israel
Chandan Sona
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
Jerusalem, Israel
Genaro Vázquez-Victorio
Departamento de Biologı́a Celular y Desarrollo, Instituto de Fisiologı́a Celular,
Universidad Nacional Autónoma de México, México 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
Richard Wargachuk
Canada
Kunhong Xiao
Laboratory for GPCR Biology, Department of Pharmacology & Chemical Biology,
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Prem N. Yadav
Pradesh, India
Guillermo A. Yudowski
Contributors xix
Yaping Zhang
Xiaoxu Zheng
Cynthia Zhou
Canada
Naiming Zhou
Preface
G proteinecoupled receptors (GPCRs) also referred as seven transmembrane

receptors (7TMRs) lie at the heart of almost every physiological and pathophysio-
logical 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 manage-
ment, 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 over-
view 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 high-
light 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 sen-
sitive cellular assays to investigate GPCR expression, signaling, and downregula-
tion. 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 GPCR-
beta-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 edito-
rial 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
xxi
xxii 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
Van Anthony M. Villar1, Santiago Cuevas, Xiaoxu Zheng, Pedro A. Jose1

1
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: vvillar@gwu.edu; pjose@mfa.gwu.edu
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 messen-
gers, 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, http://dx.doi.org/10.1016/bs.mcb.2015.11.008 3

© 2016 Elsevier Inc. All rights reserved.
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 environ-
ment. 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 phospho-
lipid 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, & Párraga, 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 5
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 protein-
coupled 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 transduc-

tion, 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 pro-
cesses 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 meaning-
ful 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 sub-
unit 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
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 (trans-
Golgi network) and then to the late endosomes for eventual lysosomal degradation.
Additional proteolytic mechanisms, such as proteasomes or cell-associated endo-
peptidases, 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 down-
regulation 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 phosphoryla-
tion, 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 activ-
ity 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 (Barnett-
Norris 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 7
fractionation procedures that break the cells apart and destroy cell morphology
before GPCR analysis using biochemical or immunological assays. A complemen-
tary 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 membrane-
associated 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 resis-
tance 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 experi-
mental controls (Figure 2). Cholesterol depletion to disrupt the lipid rafts is
commonly performed by pretreatment with b-MCD for 1 h at 37 C.
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 choles-
terol/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 concomi-
tantly 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 2000 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 180,000 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.
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 20 C, while the rest of the fractions without the 6X sample
buffer can be stored at 80 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) 30% Sucrose 20% Sucrose 10% Sucrose 5% Sucrose
50% Optiprep 3.0 2.0 1.0 0.5
(mL)
MBSTS (mL) 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 10.
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 ultra-
centrifuge 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 175,000 g
(42,000 rpm) at 4 C for 4 h. Other rotors may be used, such as the SW 55
(170,000 g for 4 h) or TLS55 (250,000 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 20 C, while the rest of the fractions without the 6X
sample buffer can be stored at 80 C.
1.1.3 Immunoblotting and data interpretation

Western blot is the most commonly used method to determine the lipid raft distribu-
tion 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 detergent-
free 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.
1.2 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 avail-
able 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.
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, coloc-
alization 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, be-
sides 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 (Ohno-
Iwashita 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 (Schütz, 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 deter-
mine 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
illumination microscopy, stimulated emission depletion (STED) microscopy, near-

field 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-2-
phenylindole (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 concom-
itant 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 2000 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 anti-
body 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 3 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 do-
mains. 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 3 with cold PBS. Spin cells and decant wash buffer.
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

Maximum Absorption and Emission
CTxB Fluor Conjugate (Catalog #) (nm)
Alexa FluorÒ 488 (V-34403) 495/519
Alexa FluorÒ 555 (V-34404) 555/565
Alexa FluorÒ 594 (V-34405) 590/617
The maximum absorption and emission for DAPI are 358/461 nm.
2. GPCR signaling in lipid rafts 15
2.5 Permeabilize the cells with 0.3 mL of 0.5% Triton X-100 in deionized water
for 10 min. Permeabilization provides access to intracellular antigens.
Triton X-100 can effectively solvate cellular membranes without disturb-
ing proteineprotein interactions. Other detergents such as saponin,
Tween-20, or sodium dodecyl sulfate may also be used.
2.6 Wash cells with PBS.
3. Immunostaining.
3.1 Add 0.3 mL of the primary antibody against the GPCR of interest dissolved
in 10% BSA (1:100e200 dilution) for 30e60 min.
3.2 Wash cells 3X with PBS.
3.3 Add 0.3 mL of the secondary antibody (against the host of the primary
antibody used in step 3.1) in 10% BSA. The secondary antibody should be
tagged with a Fluor other than the one used to label the CTxB. As coun-
terstain, 300 nM DAPI may also be added to this working solution.
3.4 Wash 2X with PBS and once with deionized water. The use of deionized
water washes away the residual NaCl crystals from PBS.
3.5 Mount cover slips using a mounting medium on glass slide. Gently remove
excess mounting medium by aspiration. Allow the mounting medium to
harden completely.
3.6 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.
2. GPCR SIGNALING IN LIPID RAFTS

There are many established protocols available that allow the study of GPCR activity
per se using commercially available kits or, less commonly, proprietary materials.
Studying the activity of GPCRs in the context of their residency in lipid rafts often
requires additional steps that would disrupt the integrity of the lipid raft microdo-
main or dissociate the protein of interest from the rafts. Most of the current strategies
to disrupt lipid raft involves either perturbation of the raft stability or modifying the
cholesterol content of the lipid rafts. Most of these treatments are performed on cells
prior to agonist/antagonist treatment and functional assays, such as cAMP produc-
tion, sodium transport, and NADPH oxidase activity (Gildea et al., 2009; Han et al.,
2008; Yu et al., 2004, 2014).
2.1 PERTURBATION OF RAFT STABILITY

Lipid rafts are dynamic assemblies of phospholipids and glycosphingolipids that
contain mostly saturated hydrocarbon chains which allow cholesterol to intercalate
between the fatty acyl chains. The surrounding membrane has greater fluidity
because of the preponderance of phospholipids with unsaturated acyl groups. The
addition of exogenous gangliosides (Webb, Hermida-Matsumoto, & Resh, 2000)
and polyunsaturated fatty acids (Simons et al., 1999), such as docosahexaenoic acid
(Ravicci et al., 2013), in the growth medium results in a change in the lipid raft
composition and the dissociation of proteins from the lipid raft. Inhibition of the
biosynthesis of glycosphingolipids and sphingomyelins using the fungal metabolite
fumonisin B1 (Lipardi, Nitsch, & Zurzolo, 2000; Nakai & Kamiguchi, 2002) may
also perturb the integrity of lipid rafts. Supplementation with 7-ketocholesterol,
which differs from cholesterol by the additional ketone group that protrudes perpen-
dicularly to the cyclopentano-perhydro-phenanthrene ring, decreases lipid raft order,
and increases membrane polarity (Rentero et al., 2008; Schieffer, Naware, Bakun, &
Bamezai, 2014). Interestingly, the nonsteroidal, anti-inflammatory drug aspirin has a
high affinity for phospholipid membranes and partitions into the lipid head groups.
This interaction impairs the molecular organization brought about by cholesterol
and thus, leads to increased mobility in a lipid raft model (Alsop et al., 2015; Kyrikou,
Hadjikakou, Kovala-Demertzi, Viras, & Mavromoustakos, 2004). The use of short-
chain ceramides, i.e., C2-ceramide and C6-ceramide, decreases the plasma mem-
brane lipid order and disrupts the lipid rafts as indicated by a reduction in the extent
of FRET between lipid raft markers (Gidwani, Brown, Holowka, & Baird, 2003).
2.2 CHANGING THE CHOLESTEROL CONTENT

Cholesterol is an integral component of lipid rafts in mammalian cell membranes,
and membrane cholesterol levels are crucial in determining the stability and organi-
zation of lipid rafts (Silvius, 2003). Thus, modifying the content of cholesterol in the
plasma membrane is another option to disrupt the lipid raft and evaluate the function
of GPCRs. The antifungal polyene antibiotics filipin (Brown & London, 2000;
Drake & Braciale, 2001), nystatin (Oakley, Smith, & Engelhardt, 2009), and ampho-
tericin (Wysoczynski et al., 2005) disrupt lipid rafts by binding and sequestering
cholesterol within the plasma membrane. Pore-forming agents such as saponin
(Hering, Lin, & Sheng, 2003; Schroeder, Ahmed, Zhu, London, & Brown, 1998),
digitonin (Oliferenko et al., 1999), and streptolysin O (Fernandez-Lizarbe, Pascual,
Gascon, Blanco, & Guerri, 2008) may also be used. b-MCD is one of the most
frequently used agents to deplete the endogenous cholesterol content of lipid rafts
(Han et al., 2008; Yu et al., 2014, 2013). One advantage in using b-MCD is the avail-
ability of a control for its use, i.e., a-MCD (Vial & Evans, 2005). Inhibitors of the
rate-limiting enzyme for cholesterol synthesis, the HMG-CoA reductase, may be
used also to inhibit endogenous cholesterol biosynthesis. These include drugs
such as simvastatin (Drake & Braciale, 2001) and lovastatin (Meszaros, Klappe,
Hummel, Hoekstra, & Kok, 2011). A summary of protocols using these approaches
is found in Table 3.
2.3 FLUORESCENCE IMAGING

The advent of FRET and BRET (bioluminescence resonance energy transfer)
biosensors has allowed the study of GPCR activity in lipid rafts in living cells.
2. GPCR signaling in lipid rafts 17
Table 3 Common Strategies to Disrupt the Lipid Raft

Strategies Protocol
A. Disruption of raft stability
Addition of gangliosides 10e100 mM for 1 h (Simons et al., 1999)
Addition of PUFAs 50 mM, overnight (Webb et al., 2000)
Fumonisin B1 25 ug/mL, 48e72 h (Lipardi et al., 2000)
7-cholesterol 35e70 mM for 5 min to 2 h (Schieffer et al.,
2014)
Aspirin 10% For 30e60 min in noncellular
experiments (Alsop et al., 2015)
C2- and C6-ceramide 32 and 8 mM, respectively (Gidwani et al.,
2003)
B. Changing the cholesterol content
Filipin 2.5e5 mg/mL for 15 min (stock solution:
5 mg/mL in ethanol) (Drake & Braciale,
2001)
Nystatin 20e50 mg/mL for 1 h (Oakley et al., 2009)
Amphotericin 10 mg/mL for 1 h (Wysoczynski et al., 2005)
Saponin 5% in 20 mM phosphate buffer, pH 7.4, at
4 C for 10 min followed by extraction in
0.5% Triton X-100 at 4 C (Hering et al.,
2003)
Digitonin 0.003% for 30 min on ice (Oliferenko et al.,
1999)
Streptolysin O 500 ng/mL for 2 h (Fernandez-Lizarbe
et al., 2008)
b-MCD 2% for 1 h (Han et al., 2008; Yu et al., 2014,
2013)
10 mM for 1 h (Simons, 1999)
Lovastatin 1 mg/mL for 20 h (Meszaros, 2011)
Simvastatin Use 5 mg/mL for 12 h (Drake & Braciale,
2001)
PUFAs, Polyunsaturated Fatty Acids.
N-Way FRET microscopy can quantify interacting and noninteracting FRET pairs in
live cells (Hoppe, Scott, Welliver, Straight, & Swanson, 2013). The freely diffusible
FRET sensor Epac2-camps has been used to measure global cAMP responses of
lipid raft-associated receptors since it responds to changes in cAMP occurring
throughout the cytosolic compartment of cells (Agarwal et al., 2014). Moreover, ver-
sions of the Epac2-camps probe allow the selective targeting to lipid raft (Epac2-
MyrPalm) and nonraft (Epac2-CAAX) domains, which are useful in monitoring
local cAMP production near the plasma membrane (Agarwal et al., 2014). PALM,
as indicated above, and dSTORM have also been used to track the reorganization
of lipid rafts (Tobin et al., 2014; Wu et al., 2013). Movement of single molecules in
living cells could also be tracked (single molecule tracking) (Scarselli et al., 2015).
In addition, fluorescent nanosensors that measure sodium in real time are reversible
and completely selective over other cations (Dubach, Das, Rosenzweig, & Clark,
2009). Real-time monitoring of sodium transport in response to stimulation or inhi-
bition of GPCRs in intact or disrupted lipid rafts has become feasible.
Current biochemical and biophysical techniques for studying GPCRs in lipid
rafts, while helpful in many instances, are still rife with methodological drawbacks
and limitations. These include the requirement for cell membrane disruption, the
reliance on antibodies that are specific for the GPCR of interest, the inability to study
native proteins, and the use of exogenous, often tagged, proteins. Newer methodol-
ogies that allow the study of GPCRs in their native form in intact cells are needed,
such as the FRET biosensors for cAMP monitoring. Meanwhile, the use of comple-
mentary approaches that yield mutually supportive results may be the most judicious
way for drawing conclusions regarding the distribution and activity of GPCRs in
lipid rafts.
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CHAPTER
Imaging GPCRs
trafficking and signaling
with total internal
reflection fluorescence
2
microscopy in cultured
neurons
Francheska Delgado-Peraza*, x, Carlos Nogueras-Ortiz*,
Agnes M. Acevedo Canabal*,x,
Cristina Roman-Vendrell*, {, Guillermo A. Yudowski*, x, 1
*Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus,
San Juan, PR, USA
x
Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico,
San Juan, PR, USA
{
Department of Physiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA
1
Corresponding author: E-mail: Guillermo.yudowski@upr.edu
CHAPTER OUTLINE
1. Image Acquisition ................................................................................................ 27
1.1 Important Considerations before Imaging ................................................ 27
1.2 Materials .............................................................................................. 28
1.2.1 Cell culture......................................................................................... 28
1.2.2 TIRF microscopy equipment and settings............................................ 28
1.3 Imaging................................................................................................ 28
1.3.1 Cell culture......................................................................................... 28
1.3.2 Live cell imaging................................................................................. 29
1.4 Notes ................................................................................................... 30
2. Image Analysis .................................................................................................... 30
3. Final Comments.................................................................................................... 31
Acknowledgments ..................................................................................................... 31
References ............................................................................................................... 31

26 CHAPTER 2 Imaging GPCRs trafficking and signaling
Abstract
Total internal reflection fluorescence (TIRF) microscopy allows probing the cellular
events occurring close and at the plasma membrane. Over the last decade, we have seen a
significant increase in the number of publications applying TIRF microscopy to unravel
some of the fundamental biological questions regarding G protein-coupled receptors
(GPCRs) function such as the mechanisms controlling receptor trafficking, quaternary
structure, and signaling among others. Most of the published work has been performed in
heterologous systems such as HEK293 and CHO cells, where the imaging surface
available is higher and smoother when compared with the narrow processes or the smaller
cell bodies of neurons. However, some publications have expanded our understanding of
these events to primary cell cultures, mostly rat hippocampal and striatal neuronal cul-
tures. Results from these cells provide a bona fide model of the complex events con-
trolling GPCR function in living cells. We believe more work needs to be performed in
primary cultures and eventually in intact tissue to complement the knowledge obtained
from heterologous cell models. Here, we described a step-by-step protocol to investigate
the surface trafficking and signaling from GPCRs in rat hippocampal and striatal primary
cultures.
The unique ability of total internal reflection fluorescence (TIRF) microscopy to

generate an evanescent field and excite fluorophores within a narrow optical section
(100 nm) provides an ideal tool to investigate the multiple and dynamic events
occurring close to and at the plasma membrane of living cells with reduced
phototoxicity and bleaching (Axelrod, 1981, 2003, 2008; Simon, 2009). This
characteristic resulted in the application of TIRF microscopy to multiple biological
areas ranging from single molecule analysis to cell migration. TIRF microscopy has
been particularly used to investigate protein translocation and trafficking to the
membrane, vesicular events, and single molecule analysis among others
(Mattheyses, Simon, & Rappoport, 2010; Reck-Peterson, Derr, & Stuurman,
2010; Roman-Vendrell & Yudowski, 2015; Steyer & Almers, 2001). Since astro-
cytes display a significant surface area, these cells have been used to visualize
and analyze various events such as the molecular mechanisms controlling vesicular
release (Li, Agulhon, Schmidt, Oheim, & Ropert, 2013). However, the application of
TIRF microscopy to investigate G protein-coupled receptors (GPCRs) in neurons is
still very limited. Our laboratory was among the first to use TIRF microscopy to
investigate GPCRs in neurons (Yudowski, Puthenveedu, & von Zastrow, 2006).
Our data showed that receptor recycling to the cell surface can be mediated by
two different vesicular events, a rapid event without diffusion barriers and a slow
event with a kinetic similar to kiss and run exocytosis (Roman-Vendrell et al.,
2014; Yudowski et al., 2006). These kiss and run events were not observed with
the Mu opioid receptor and were dependent on the carboxy terminal sequence of
the B2 adrenergic receptor (Roman-Vendrell & Yudowski, 2015; Yu, Dhavan,
Chevalier, Yudowski, & Zastrow, 2010). TIRF microscopy was also utilized to
demonstrate that GPCR recycling after ligand-induced internalization is actively
controlled by the activation of receptors still at the cell surface. Interestingly,
1. Image acquisition 27
identical results were observed in primary cell cultures and in heterologous systems
supporting the idea that heterologous systems can provide valuable information
(Bowman & Puthenveedu, 2015; Bowman et al., 2015; Roman-Vendrell, Yu, &
Yudowski, 2012; Yudowski, Puthenveedu, Henry, & Von Zastrow, 2009). Others
have utilized TIRF in neuronal cultures to investigate the pathways by which recep-
tors are recycled to the cell surface (Li et al., 2012). Our laboratory also utilized
TIRF microscopy to investigate endogenous GPCR signaling in hippocampal
neurons by loading neurons with calcium sensitive dyes and pharmacologically acti-
vating specific adrenergic receptors (Tzingounis, von Zastrow, & Yudowski, 2010).
More recently, we have investigated how cannabinoid receptor 1 interacts with beta-
arrestins at the cell surface of hippocampal neurons to regulate beta-arrestin
signaling (Flores-Otero et al., 2014). These are some of the applications of TIRF mi-
croscopy to investigate GPCRs. The application of TIRF and superresolution micro-
scopy in combination with novel fluorescent tags and nanobodies should only
expand the toolbox available to further probe the biology of these highly relevant
receptors.
1. IMAGE ACQUISITION
1.1 IMPORTANT CONSIDERATIONS BEFORE IMAGING
To visualize GPCRs at the cell surface, receptors must be tagged with fluorophores
that are ideally resistant to quenching/photobleaching and with a significant quantum
yield (Shaner, Steinbach, & Tsien, 2005). One of the fluorescent tags widely utilized
in TIRF microscopy to investigate GPCRs is the pH-sensitive eGFP-variant super
ecliptic pHluorin (SEP) (Miesenbock, De Angelis, & Rothman, 1998). By attaching
the SEP molecule to the extracellular domain of GPCRs, receptors are highly visible
when they are located at the cell surface (neutral pH) and their fluorescence is
rapidly quenched in intracellular compartments such as endosomes (pH acidic).
The use of GPCRs tagged with SEP at the extracellular domain in TIRF microscopy
is an ideal approach to investigate events at the cell surface minimizing fluorescence
from receptors in intracellular compartments while reducing phototoxicity. Other
available probes such as antibodies conjugated with quantum dots or SNAP tag
fusion proteins have been also utilized to investigate GPCRs surface trafficking
(Calebiro et al., 2013; Maurel et al., 2008; Mikasova, Groc, Choquet, & Manzoni,
2008; Reck-Peterson et al., 2010). However, their application to TIRF microscopy
is not as frequent as conventional genetically coded fluorophores.
It is important to note that regardless of the florescent tag utilized, we strongly
recommend tagging receptors at their extracellular domain and compare their
function with wild-type receptors before any further analysis. Only tagged receptors
with identical pharmacological and functional properties to wild-type receptors must
be used. In our experience, tags in intracellular domains of GPCRs generally disrupt
their signaling and or trafficking, rendering nonphysiological behavior. Finally, TIRF
microscopy is not exempt from the general rules and pitfalls from live cell imaging,
image acquisition, and analysis (Frigault, Lacoste, Swift, & Brown, 2009; Jaiswal &
Simon, 2004; North, 2006; Schnell, Dijk, Sjollema, & Giepmans, 2012).
1.2 MATERIALS
1.2.1 Cell culture
1. Striatal primary cultures obtained from embryonic day 17e18 Sprague-Dawley
rat embryos. Alternatively, brain tissue can be purchased from BrainBits LLC
(Springfield, IL).
2. Neuron culture media: Neurobasal medium supplemented with B27 (according
to manufacturer protocol) and 0.5 mM glutaMAXÔ (Life Technologies).
3. Imaging media such as Neurobasal nimus phenol red without serum and
supplemented with 20 mM HEPES (Life Technologies) (see Notes).
4. 30 mm coverslips #1.5 thickness. Coverslips must be acid washed and coated
with fresh poly-D-Lysine (Sigma). Glass bottom dishes (MatTek) can be also
utilized. They must be coated with PDL and the glass thickness must be 1.5.
5. Transfection reagents, Lipofectamine 3000 (Life Technologies).
6. Hippocampal cultures were >90% pure as calculated by MAP2 and GFAP
staining as previously described (Yudowski et al., 2006, Yudowski, Olsen,
Adesnik, Marek, & Bredt, 2013).
1.2.2 TIRF microscopy equipment and settings

1. Motorized Nikon (Melville, NY) Ti-E inverted microscope with a 100 apo-
chromat oil immersion TIRF objective lens (CFI Apo TIRF 100; Nikon), color
correction and a motorized stage with perfect focus (see Notes).
2. Light source: 488 and 561 nm Coherent sapphire lasers (Coherent Inc. Santa
Clara, CA) 50 and 100 mW lasers, respectively.
3. Temperature control is utilized to keep cells at 37 C with a stable Z stage and
objective warmer (Bioptechs, Butler, PA)
4. Interchangeable Coverslip Dish (Bioptechs) (http://www.bioptechs.com/
Products/ICD/coverslipdish.html)
5. Camera: iXonEM þ DU897 back-illuminated electron multiplying charged
coupled device sensor camera (Andor, Belfast, UK).
6. Readout speed: 10 Hz, exposure time: continuous 50e100 ms exposure for
receptor recycling, electron multiplying gain of 300, no binning, bit depth ¼ 14
bits, camera temperature set to minimum and laser power to 10% for 488 nm at
50 mW.
1.3 IMAGING
1.3.1 Cell culture
1. Acid clean coverslips or glass bottom dishes by incubation in 1 M HCl shaking
overnight. Rinse two times with abundant ddH2O and once with 70% ethanol.
1. Image acquisition 29
Leave in 95% ethanol until ready to use. Before coating, UV and dry in the
culture hood no less than 1 h.
2. Coat acid clean coverslips or glass bottom dishes with freshly prepared
100 mg/mL poly-D-lysine (PDL) for 2e4 h at 37 C. Wash 3 times with sterile
water and air dry in sterile environment. It is important to prepare PDL fresh for
every use. Old PDL solutions result in less than ideal cell attachment.
3. Plate 250,000e300,000 neurons per 35 mm dish. Neurons are transfected at 4e5
days in vitro (DIV) and imaged at 15 DIVor later. Expression levels for receptor
trafficking is ideal between 14 and 25 DIV.
4. Transfect cells with DNA constructs using Lipofectamine 3000 or Effectene
according to the manufacturer’s instructions. We perform imaging after DIV 15.
(High expression levels will impair observation of individual events. They can
also result in trafficking artifacts such as reduced internalization). Lentivirus
can be also used to infect targeted cells. In our experience, infected cells do not
look as healthy as transfected cells.
5. Replace conditioned media with 2 mL of freshly prepared Opti-MEM with
HEPES 15e30 min before imaging sessions. Important: Do not to allow
neurons to dry during this process. A small amount of media must be left at the
dish covering all cells during the process.
6. Incubate cells at 37 C >10 min to allow acclimatization to the new media.
7. Transfer neurons to the microscope.
1.3.2 Live cell imaging

1. At least 30 min before any acquisition, turn on the microscope and the tem-
perature controllers. Turn on the laser key and let the laser warm up. Important:
Objective and imaging chamber temperature must be controlled before and
during experiments. Temperature must be 37 C at the glass.
2. Select TIRF objective, add a drop of immersion oil (type LDF, RI: w1.515) and
carefully place cells on the stage (see Notes).
3. To reduce the effects of photobleaching, it is important to find and focus the cells
using transmission light first. Then, find cells expressing tagged receptors using
epifluorescence and then switch to TIRF illumination (see Notes).
4. Add agonist diluted in warm imaging media by automated perfusion system or
manually outside the imaging area to minimize artifacts (see Notes).
5. Acquisition settings for endocytosis: 100e300 msec exposures every 2e3 s.
Total time: 10e30 min.
6. Acquisition settings for recycling: Continuous illumination and acquisition at
50e100 ms exposures for 1e2 min (see Notes).
7. Depolarization with 25 mM KCl is performed to test viability at the end of ex-
periments with Fluo-4.
8. Imaging sessions will generate large amounts of data. Careful data management
must be implemented in advance. Standardized electronic notebooks or
spreadsheets are recommended.
1.4 NOTES
1. Healthy primary cultures are essential to obtain reliable and valid results.
Neurons must conserve their integrity, without blebbing or detachments.
2. HEPES is used to maintain the pH constant for up to 45e60 min outside a CO2
incubator.
3. High quality cDNA is highly desired for neuronal transfection. Multiple
transfection agents are commercially available. We utilize lipofectamine 2000
on DIV 4e5 and perform imaging on DIV > 15. This delay results in optimal
expression levels for TIRF imaging.
4. Focal plane must be kept constant during imaging sessions.
5. It is very important that the cells grow in monolayer and are not more than
80e90% confluent on the day of imaging.
6. It is very important that the bottom of the imaging dish is completely dry and
clean. Any liquid or dirt will interfere during TIRF imaging.
7. The most critical step is to find the exact angle for TIRF. To align the laser
properly, focus on the plasma membrane. You can find the cell sharp edges and
use them as reference.
8. If ligands are added manually, extreme care is needed to prevent disturbing the
cells within the imaging area. Controls should be performed to test the effects of
dimethyl sulfoxide and other solvents on surface fluorescence and basal cell
activity.
9. Endocytosis should be visible within 1 or 2 min of agonist addition. Agonist-
induced recycling can be observed 2e3 min after initial exposure. A constant
rate of vesicular fusion is generally observed at w10 min.
2. IMAGE ANALYSIS
Exocytotic events are easily identified by direct visualization of the abrupt increase
(w1 s) in intensity at discrete points in the cell surface. Image sequences can be
analyzed using the acquisition software available from the microscope or by using
the public domain NIH Image program ImageJ/FIJI software, which is freely avail-
able at http://fiji.sc/Fiji. Orthogonal views (kymographs) can be used to distinguish
these events from other vesicular event due to their characteristic kinetics and to
quantify their frequency, decay kinetics, and location. Maximum intensity fluores-
cence can be extracted and plotted to demonstrate lateral diffusion and number of
exocytic molecules among others. More recently, software specifically designed
to identify exocytic events has been developed such as the exocytosis detection
recipe from SVCell (https://www.svcell.com/recipes/exocytosis-detection). Further
development of this and other open source codes should provide better tools to
extract and analyze exocytic events. We recommend performing all the analysis
blindly to reduce bias in the quantification.
Endocytic events are intrinsically more challenging to analyze due to their lower
signal to noise ration. Manual detection was initially utilized following specific
References 31
predetermined clathrin-coated pits behaviors including: (1) events must appear and
disappear within the time series (<250 s); (2) events must display limited movement
(no more than 4 by 4 pixels through their lifetime); and (3) events must not fuse or
collide with each other (Saffarian, Cocucci, & Kirchhausen, 2009). More recently,
particle-tracking algorithms have been developed to follow these events such as
the imageJ 2D-spot tracker plug-in (Sage, Hediger, Gasser, & Unser, 2005) or the
Matlab code developed by the groups of Danuser and Schmid (Loerke et al.,
2009, 2011). These codes were developed to analyze movies with ideal signal to
noise ratio such as those obtained by imaging fluorescently tagged clathrin and
dynamin. Analysis of GPCR endocytosis utilizing these tools has been more chal-
lenging with limited success. Nevertheless, these are fundamental steps toward a
more rigorous and unbiased tool to investigate endocytosis. Finally, to achieve sta-
tistical data, we define individual experiments by the number of cells analyzed from
the same transfection (n ¼ 1 experiment, x number of cells). To achieve statistical
significance, we analyze no less than 10 different cells from more than four different
experiments (different transfection). A minimum of 20 endocytic events are ex-
pected from every cell to secure results from healthy cells.
3. FINAL COMMENTS
The availability of turnkey TIRF microscopes and the development of multiple flu-
orophores have allowed the use of TIRF microscopy to peer into the complex lives of
GPCRs at the cell surface. We have learned that their surface trafficking, signaling,
and general function are more complex and exquisite than previously thought. Novel
layers of regulatory mechanisms and trafficking pathways have been discovered
along with complex associations between receptors and other proteins. This is
only the beginning of a new era of GPCR research in which novel imaging tools
such as TIRF and superresolution microscopy are key to unravel some of the funda-
mental questions in GPCRs biology.
ACKNOWLEDGMENTS
This work was supported by research grants from NIH DA023444 and DA037924 to GY, FDP,
and CNO. MBRS program RISE R25 GM061838 to CRV and AAC. We thank Dr Nevin
Lambert (Medical College of Georgia, Georgia Health Sciences University, Georgia Regents
University) for his feedback.
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CHAPTER
Trafficking of ciliary
G protein-coupled
receptors 3
Jeremy C. McIntyre*, x, 1, Mellisa M. Hege{, Nicolas F. Berbari{, 1
*Department of Neuroscience, University of Florida, Gainesville, FL, USA
x
Center for Smell and Taste, University of Florida, Gainesville, FL, USA
{
1
Corresponding authors: E-mail: jmcin@ufl.edu; nberbari@iupui.edu
CHAPTER OUTLINE
Introduction .............................................................................................................. 36
1. Rhodopsin as a Ciliary GPCR................................................................................. 38
2. Ciliary GPCRs in Other Neurons ............................................................................. 39
3. Olfactory Sensory Neuron Ciliary GPCRs ................................................................ 41
4. Dynamic Trafficking of Ciliary GPCRs..................................................................... 45
Conclusions and Future Directions ............................................................................. 47
References ............................................................................................................... 48
Abstract
In the last decade highly conserved cellular appendages called cilia have enjoyed a
renewed interest from basic, biomedical scientists, and clinicians alike. This interest has
grown upon the elucidation that cilia throughout the body serve as important sensory and
signaling centers in both development and adult homeostasis. Furthermore, the identifi-
cation of several rare genetic disorders associated with cilia dysfunction has broadened
the field. However, even though their potential role in human health and disease is now
recognized many basic questions about their functions remain. This chapter seeks to
explore the trafficking of cilia-specific G protein-coupled receptors (GPCRs) and
discusses several model systems in which this has been explored. We open the chapter by
briefly discussing cilia and GPCRs then begin discussing some aspects of rhodopsin
trafficking, arguably the most well studied of cilia GPCRs. We continue with sections on
neuronal cilia and olfactory cilia receptor trafficking. Finally, we conclude with the
emerging area of dynamic ciliary GPCR trafficking and speculate about future directions
and some of the questions that remain for ciliary GPCRs.

36 CHAPTER 3 Trafficking of ciliary G protein-coupled receptors
INTRODUCTION
The cilium is a highly conserved, near ubiquitous, small microtubule-based cell
appendage. Historically classified as either motile or immotile (primary), it is clear
that all cilia have the capacity to coordinate specific signaling pathways. Most
mammalian cells possess a singular immotile cilium or “primary” cilium which
is thought to play a crucial role as a complex sensory and signaling center (for a
review on cilia signaling associated pathways, see Berbari, O’Connor, Haycraft,
& Yoder, 2009) (Figure 1). An emerging class of relatively rare genetic diseases,
termed ciliopathies, are now associated with cilia dysfunction. Interestingly, the
clinical features of ciliopathies are broad and diverse. Phenotypes range from
FIGURE 1
Primary cilium schematic. Several of the structural elements are labeled and indicated with
arrows including the basal body, axoneme. The green and red arrows along the axoneme
indicate anterograde and retrograde intraflagellar transport, respectively. Other proteins and
complexes involved in general ciliary trafficking are indicated as well as generic molecules
found in the ciliary membrane such at ciliary-specific GPCRs. (See color plate)
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G protein-coupled receptors : signaling,

trafficking and regulation First Edition

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

Academic Press is an imprint of Elsevier

For information on all Academic Press publications

Agnes M. Acevedo Canabal

G proteinecoupled receptors (GPCRs) also referred as seven transmembrane

Biology Team, I present to you this volume entitled “G ProteineCoupled Receptors:

Localization and signaling

Van Anthony M. Villar1, Santiago Cuevas, Xiaoxu Zheng, Pedro A. Jose1

Methods in Cell Biology, Volume 132, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.11.008 3

FIGURE 1 A Lipid Raft Membrane Microdomain.

have been implicated in a variety of cellular processes, including signal transduc-

through a dynamin-dependent, clathrin-mediated endocytosis. Once internalized,

1. LOCALIZATION OF GPCRs IN LIPID RAFTS

1.1 ISOLATION OF LIPID RAFTS

1.1.1 Detergent-free method

FIGURE 2 Comparison groups for GPCR localization in lipid rafts.

Methyl-a-cyclodextrin (a-MCD) may be used as control for b-MCD (Vial &

1.1.2 Detergent-based method

1.1.3 Immunoblotting and data interpretation

FIGURE 3 Lipid Raft Distribution of Caveolin-1 and D1R.

1.2 LOCALIZATION OF GPCRs IN LIPID RAFTS

illumination microscopy, stimulated emission depletion (STED) microscopy, near-

1.2.1 Cells in suspension

2. Mounting and imaging.

1.2.2 Adherent cells

Table 2 Fluorescence Spectra of CTxB Conjugates

2. GPCR SIGNALING IN LIPID RAFTS

2.1 PERTURBATION OF RAFT STABILITY

2.2 CHANGING THE CHOLESTEROL CONTENT

2.3 FLUORESCENCE IMAGING

Table 3 Common Strategies to Disrupt the Lipid Raft

Kenworthy, A. K. (2007). Fluorescence recovery after photobleaching studies of lipid rafts.

Methods in Cell Biology, Volume 132, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.10.002 25

The unique ability of total internal reflection fluorescence (TIRF) microscopy to

1.2.2 TIRF microscopy equipment and settings

1.3.2 Live cell imaging

Axelrod, D. (2008). Total internal reflection fluorescence microscopy. Methods in Cell

Methods in Cell Biology, Volume 132, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.11.009 35

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