2013

http://informahealthcare.com/rstISSN: 1079-9893 (print), 1532-4281 (electronic)

J Recept Signal Transduct Res, Early Online: 1–4! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2013.781625

MINI-REVIEW

Measurement of G protein-coupled receptor surface expression

Pieter Beerepoot, Vincent M. Lam, and Ali Salahpour

Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada

Abstract

The quantity of G protein-coupled receptors (GPCRs) expressed on the cell surface is animportant factor regulating receptor signaling. Maturation, internalization, recycling anddegradation together determine the net amount of receptor surface expression. Understandingevery aspect of the receptor lifecycle will facilitate the development of therapeutic applications.A number of assays for measuring the surface expression of GPCRs are currently available.This minireview summarizes the currently available assays and their suitability and usage formeasuring GPCR surface expression.

Keywords

ELISA, flow cytometry, GPCR, HTS,internalization, surface expression

History

Received 22 January 2013Revised 26 February 2013Accepted 27 February 2013Published online 4 April 2013

Introduction

The interaction of G protein-coupled receptors (GPCRs) with

ligands and subsequent alterations in signal transduction

depends on the presence of the receptor at the plasma

membrane. GPCR cell surface expression is highly dynamic

and is regulated at the stages of maturation, internalization,

recycling and degradation. Ultimately the level of receptor

maintained at the surface is one of the principle homeostatic

mechanisms to modulate signal transduction.

Insertion of nascent receptor into the plasma membrane is

a key point for regulating surface expression levels. GPCRs

mature in the endoplasmic reticulum (ER), after which they

traffic through the Golgi apparatus before arriving at the cell

surface (1). The ability of a GPCR to reach the surface as

functional protein is affected by folding rate, interaction

with ER-resident chaperones, and dimerization with other

proteins and GPCRs. These processes are in turn influenced

by environmental conditions and the application of exogenous

agents (2,3). For example, folding in the ER can be modulated

by altering temperature or by an application of chemical or

pharmacological chaperones, small molecules that selectively

bind and stabilize protein conformations (4). GPCR traffick-

ing to the plasma membrane can also be modulated by the

absence or presence of endogenous molecular chaperones as

well as retention sequences within the GPCR itself (5).

Once at the cell surface, GPCRs can undergo constitutive

internalization as well as ligand-mediated internalization (6).

In most cases, agonist-induced internalization is dependent

on the phosphorylation of the cytoplasmic tail and third

intracellular loop by G protein-coupled receptor kinases

(GRKs), allowing binding of b-arrestins, followed by intern-

alization in a clathrin-dependent mechanism (6,7).

Internalized receptors are then either sorted to recycling

endosomes to return to the cell surface or to lysosomes for

degradation (8).

The cumulative effect of these processes determines the

amount of functional receptors at the surface of the cell at any

given time. Since the ability of a receptor to signal depends on

its presence at the surface, accurately measuring surface

expression is important for the interpretation of signaling

data. Furthermore, multiple aspects of GPCR trafficking are

modulated by binding of ligands, which may be exploited

for therapeutic applications, an approach that has only

recently become appreciated (2,6,9). Ligands can differ in

their ability to activate separate signaling cascades through

the same receptor, and in their ability to alter GPCR

trafficking. This phenomenon is described as the compound’s

functional selectivity (9). For example, it was recently shown

that some ligands for the 5HT2A receptor have functional

selectivity in internalization and recycling that is independent

from their activity on canonical signaling pathways through

this receptor (10). Similarly, m-opioid ligands differ in their

ability to induce receptor internalization separately from

their activity on G protein-mediated pathways, which is

therapeutically relevant due to the relationship between

internalization and the development of opioid tolerance

(11,12). Therefore, assays that are able to accurately measure

cell surface expression in a high-throughput manner are

essential components of understanding GPCR/ligand inter-

action and will play an increasing role in continued drug

discovery. In this article, we will discuss current strategies to

measure surface expression of both wild type and recombin-

ant proteins.

Address for correspondence: Ali Salahpour, PhD, Department ofPharmacology and Toxicology, University of Toronto, 1 King’s CollegeCircle, M5S 1A8, Toronto, Ontario, Canada. Tel: 416-978-2046. E-mail:ali.salahpour@utoronto.ca

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Measurement of surface expression of wild-typereceptors

Traditionally, assays measuring wild-type receptor surface

expression in heterologous cells have used either hydrophilic

ligands or antibodies, that do not cross the plasma membrane,

preventing labeling of intracellular stores of receptors. The

first assays to quantify surface expression of GPCRs utilized

hydrophilic cell-impermeable radioligands, or measured bind-

ing after isolation of plasma membrane receptors from the

whole cell lysate by sucrose-gradient centrifugation (13,14).

However, the limited availability of suitable cell-impermeable

ligands and the time required for cellular fractionation

reduced the utility of this approach.

Currently, surface expression measurements are mainly

performed by cell surface labeling either with antibodies

against an epitope present on the extracellular portion of the

receptor, or by surface biotinylation. When labeling receptors

with antibodies, secondary antibodies conjugated to enzymes,

fluorophores, or quantum dots can be used to allow for

visualization and quantification of receptors (15–17). The

most common assays used to measure antibody-labeled

receptors are enzyme-linked immunosorbent assays (ELISA)

and flow cytometry. Fluorescence microscopy can also be

used to monitor surface expression of antibody-labeled

GPCRs, while providing additional information about local-

ization of the protein. However, each of these approaches

necessitates the use of an antibody toward an extracellular

epitope of the receptor, which is often not available (18).

Additionally, these assays are time-consuming and have a low

throughput. Therefore, they are not suitable for drug discov-

ery, particularly in comparison to assays using recombinant

receptors (see below).

Another method to measure levels of native receptor is the

surface biotinylation assay. This assay does not depend on the

use of an antibody against extracellular epitopes, because cell

surface proteins are non-specifically labeled by chemical

conjugation of biotin (19). After labeling, total cell lysates

are prepared and biotin-labeled proteins are purified using

streptavidin-coated beads or resin (20). The assay derives its

specificity for a given GPCR by western blot with an antibody

against the receptor to compare the level in the total lysate

and the streptavidin-bound fraction. The major advantage of

this approach is that the intracellular and surface pools of

the receptors can be differentiated. However, lysates must be

analyzed by western blotting, making this method labor

intensive and only suitable for small-scale experiments.

Use of recombinant receptors for surfaceexpression assays

Native receptors are often expressed at low levels, and the

commercial availability of selective antibodies for any given

GPCR is limited. Therefore, the creation of recombinant

receptors by an insertion of epitope tags, fluorescent proteins,

or small reporter enzymes, in conjunction with heterologous

expression, is a common technique employed to facilitate

surface measurement of GPCRs (21). It is important to note

that inserting an additional sequence or epitopes to receptors

could potentially alter receptor function. With increasing

understanding of the functional domains of GPCRs, the

careful placement of an extracellular epitope generally does

not alter receptor biology.

The insertion of epitopes, such as HA, Myc or Flag, for

which commercially available high-affinity antibodies are

available, makes development of antibody-dependent surface

expression assays possible for essentially any membrane

protein (21). Although the use of antibodies against affinity

tags is cheaper and the choice of secondary antibodies is

much greater, they still require extended incubation and

wash steps, decreasing suitability for high-throughput screens.

A more direct approach is the use of fluorescent proteins,

such as green fluorescent protein (GFP), for tagging of

GPCRs, which allows for convenient tracking of protein

expression. However, GFP is much larger than classical

epitopes (27 kDa versus 8–12 amino acids) and is therefore

more likely to affect protein function (21,22). Moreover, since

fluorescence is constant regardless of the subcellular local-

ization of the receptor, surface expression can only be

measured by a microscopic analysis of fluorescence distribu-

tion (23). The image acquisition time and image analysis

that needs to be performed in these studies is substantial and

therefore decreases the throughput of this approach (24,25).

Recently, methods have been developed to circumvent

some of the limitations associated with GFP labeling. For

example, Takeda et al. developed an assay using a protein

tag and a peptide probe that form fluorescent heterodimeric

coiled-coil structures that are only 5–6 kDa in size (26). Cell

surface labeling takes place quickly (within 1 min) after an

addition of the probe, avoiding the incubation time needed

for antibodies. However, for this assay, quantification is

still performed by measuring fluorescence distribution with

a microscope, once again limiting throughput. A different

approach was taken by Fisher et al. by labeling a GPCR with a

fluorogen-activated protein (FAP) (20). Using cell-imperme-

able fluorogens, only populations of receptors on the plasma

membrane are fluorescently labeled. Flow cytometry can then

be used to quantify surface expression without extended wash

steps and incubations; this is in contrast to antibody-based

flow cytometry experiments that require multiple wash and

incubation steps. A FAP-based assay has recently been

adapted for high-throughput screening of ligands that induce

internalization of the b2-adrenoreceptor (27).

Other approaches have used small enzymes as reporters

that can be used to tag proteins both intra- and extracellularly,

notably the SNAP-tag and CLIP-tag constructs (28,29).

The SNAP and CLIP tags use a mutant form of the 20 kDa

alkylguanine-DNA alkyltransferase protein with specificity

for benzylguanine and benzylcytosine derivative substrates

respectively. Cell-permeable and cell-impermeable fluores-

cent substrates for these tags are available that can covalently

bind and label the tagged protein. Using a combination

of cell-permeable and impermeable fluorophores of different

colors, both intracellular and surface fractions of GPCRs can

be quantified simultaneously in live cells. This approach

could be adapted to high-throughput applications and can

also provide information about protein–protein interaction

by using the fluorophore-labeled SNAP-tag GPCRs as FRET

donor and acceptors (30,31).

Assays specifically monitoring the entry of GPCRs into

endosomes, such as the enzyme complementation approach

2 P. Beerepoot et al. J Recept Signal Transduct Res, Early Online: 1–4

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taken by Hammer et al. are also available (32). In this assay,

b-galactosidase fragments are fused to the GPCR and an

endosomal marker, respectively. These fragments can revers-

ibly complement each other to recover enzymatic activity,

once the GPCR is localized to the endosomes. This concept is

applied by DiscoverRx (Fremont, CA) in their commercial

Pathhunter GPCR internalization assay. Additionally, there

also exist internalization assays that take advantage of the

acidic environment of endosomes by tagging receptors with

pH-sensitive fluorescent proteins (26,33,34). In these assays,

fluorescence is quenched when GPCRs enter endosomes,

which can be measured using a microscope. The key

difference in these endosomal-based assays is the measure-

ment of internalization rather than quantification of receptors

at the plasma membrane.

In sum, there are a number of assays available to monitor

GPCR surface expression depending on the particular

needs of an experiment (Table 1). New FAP-based assays

are versatile, homogenous and are particularly suited for high-

throughput screening applications. Assays employing enzyme

tags, such as the SNAP or CLIP-tag carry the promise to be

applied similarly in high-throughput fashion, but such an

application has thus far not been reported. Drug screens

monitoring multiple aspects of ligand activity in addition to

G-protein activation, including pharmacological chaperoning,

dimerization, b-arrestin-mediated signaling and internaliza-

tion, will provide a much more sophisticated and tailored

modulation of GPCR activity that could consequently lead to

improvements in therapeutic applications.

Acknowledgements

We thank Dr. Amy Ramsey for critical reading of the manuscript.

Declaration of interest

The authors declare no conflicts of interest. This work wassupported by the CIHR operating grant number (210296 to AS).

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Table 1. GPCR surface expression and internalization assays.

Measurement HTS suitabilityAssay Surface expression Internalization Signal to noise Throughput

Binding-cell impermeable ligand þ þ þ þBinding-surface receptor isolation þ þ � �Whole-cell ELISA þ þ þ þFlow cytometry – antibody þ þ þ þþ (25)Immunofluorescence þ þ þ þSurface biotinylation þ þ � �GFP imaging þ þ þþþ (35,36) þþ (25)Flow cytometry – FAP þ þ þþþ (27) þþ (25)Coiled-coiled tag/probe þ þ þþ (26) þþ (26)SNAP/CLIP þ þ þþþ (30) þþþ (30)Enzyme complementation � þ þþþ (32) þþþ (32)

þ yes, � no. For signal to noise: � not applicable, þ no tests reported, þþ moderate (Signal to noise ratio (SNR)below 5, Z0 below 0.5), þþþ high (SNR of 5 and above and Z0 0.5 and above). For throughput: � not applicable,þ no tests reported þþ5100 000 samples/day, þþþ4100 000 samples/day.

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