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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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ISBN: 978-0-12-803595-5
ISSN: 0091-679X
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
Department of Biochemistry and Molecular Medicine, Institute for Research in
Immunology and Cancer, Université de Montréal, 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, 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
Department of Pharmacology and Therapeutics, McGill University, Montréal, 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
xvi 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, Université de Montréal, 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, Université de Montréal, 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, Université de Montréal, Montreal, QC, Canada
Marina Macı́as-Silva
Departamento de Biologı́a Celular y Desarrollo, Instituto de Fisiologı́a Celular,
Universidad Nacional Autónoma de México, México D.F., Mexico
M. Maoz
Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center,
Jerusalem, Israel
Contributors xvii
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,
São Bernardo do Campo, Brazil
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 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
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, Montréal, 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 xix
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, Montréal, QC,
Canada
Naiming Zhou
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang
University, Hangzhou, Zhejiang, China
Preface
xxi
xxii Preface
Arun K. Shukla
Indian Institute of Technology, Kanpur, India
CHAPTER
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
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
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.
REFERENCES
Agarwal, S. R., Yang, P. C., Rice, M., Singer, C. A., Nikolaev, V. O., Lohse, M. J., et al.
(2014). Role of membrane microdomains in compartmentation of cAMP signaling.
PLoS One, 9(4), e95835.
de Almeida, R. F., Loura, L. M., Fedorov, A., & Prieto, M. (2005). Lipid rafts have different
sizes depending on membrane composition: a time-resolved fluorescence resonance
energy transfer study. Journal of Molecular Biology, 346(4), 1109e1120.
Alsop, R. J., Toppozini, L., Marquardt, D., Kucerka, N., Harroun, T. A., & Rheinstädter, M. C.
(2015). Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes.
Biochimica et Biophysica Acta, 1848(3), 805e812.
Barnett-Norris, J., Lynch, D., & Reggio, P. H. (2005). Lipids, lipid rafts and caveolae: their
importance for GPCR signaling and their centrality to the endocannabinoid system.
Life Sciences, 77(14), 625e639.
Boesze-Battaglia, K. (2006). Isolation of membrane rafts and signaling complexes. Methods
in Molecular Biology, 332, 169e179.
Breton, S., Lisanti, M. P., Tyszkowski, R., McLaughlin, M., & Brown, D. (1998). Basolat-
eral distribution of caveolin-1 in the kidney. Absence from Hþ-ATPase-coated
endocytic vesicles in intercalated cells. Journal of Histochemistry and Cytochemistry,
46(2), 205e214.
Brown, D. A., & London, E. (2000). Structure and function of sphingolipid- and cholesterol-
rich membrane rafts. Journal of Biological Chemistry, 275(23), 17221e17224.
Chang, J. C., & Rosenthal, S. J. (2012). Visualization of lipid raft membrane compartmental-
ization in living RN46A neuronal cells using single quantum dot tracking. ACS Chemical
Neuroscience, 3(10), 737e743.
Drake, D. R., 3rd, & Braciale, T. J. (2001). Cutting edge: lipid raft integrity affects the
efficiency of MHC class I tetramer binding and cell surface TCR arrangement on
CD8þ T cells. Journal of Immunology, 166(12), 7009e7013.
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