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Expression of Recombinant G-Protein Coupled Receptors for StructuralBiology

Filippo Mancia and Wayne A. Hendrickson

First published as an Advance Article on the web 7th September 2007

DOI: 10.1039/B713558K

1 Introduction

1.1 Signal Transduction through G-protein Coupled Receptors

A cell perceives its environment through the receptor

molecules embedded in the plasma membrane and endowed

with a selective sensitivity toward various stimuli.

Conformational changes or associations that occur when a

receptor interacts with an external stimulus are transmitted to

the cell interior where responses are induced, often elicited

through a cascade of signal transduction events. The mechan-

ism of signal transduction depends on molecular character-

istics of the receptor, and there are several classes of receptors.

Besides those linked to the downstream elements by hetero-

trimeric G-proteins, our subject here, there are many receptors

linked to protein tyrosine kinases, ones which are linked to ion

channels, and diverse receptors coupled in other ways as in the

TGFb/Smad, Notch, and Wnt systems. The stimulus detected

by a receptor may be physical, e.g. light or an electrostatic

potential, but in most cases the stimulus is a chemical ligand.

Some ligands are macromolecules, others are small com-

pounds; some are diffusible, others are associated with another

cell or the extracellular matrix.

The most salient molecular characteristic of G-protein

coupled receptors (GPCRs) is a pattern of seven hydrophobic

segments that correspond to transmembrane a-helices; thereby

GPCRs are also known as seven transmembrane (7TM)

receptors. This 7TM pattern was first seen in sequences of

rhodopsins1–3 and a little later in the sequence of the hamster

b2-adrenergic receptor.4 The involvement of heterotrimeric

G-proteins in signaling through 7TM receptors was first

worked out for the b2-adrenergic receptor,5 where binding of

the hormone epinephrine activates Gas which in turn

stimulates adenylyl cyclase to produce the second messenger

cyclic AMP. The parallel role of the G protein transducin in

visual signaling, where photoactivation of rhodopsin stimu-

lates cyclic GMP phosphodiesterase and sodium-channel

closure, was discovered a little later.6 Taken in this context,

the evident homology between these two biologically disparate

7TM receptors prompted the realization that they were the

founding members of the GPCR family. A flood of GPCR

clonings ensued, including the first for serotonin receptors7,8

and the discovery of the huge subfamily of odorant receptors.9

A total of 948 GPCR genes were identified in a recent analysis

of the human genome sequence,10 up somewhat from the 616

found initially.11 These receptors include sensors of exogenous

stimuli including light and odors and others that respond to

endogenous ligands ranging from cationic amines such as

serotonin to peptides such as angiotensin and to proteins such

as chemokines and glycoprotein hormones.

Ligand binding (or photoisomerization of retinal in the case

of opsins) activates a GPCR to serve as a nucleotide exchange

factor for the cognate heterotrimeric G protein. Each

heterotrimer is a labile association of the GTPase component,

Ga, with a Gb:Gc heterodimer. Ga(GDP):Gb:Gc dissociates

to Ga(GTP) and Gb:Gc when stimulated by an activated

GPCR, and the trimer reassociates after GTP hydrolysis. Both

components are tethered to the membrane, by N-terminal

myristoylation of Ga and C-terminal prenylation of Gc, and

after activation they can diffuse away from the receptor to

effector sites on their membrane-associated targets. There are

at least 15 different Ga proteins, 5 Gbs, and 5 Gcs12 and

different combinations are selective for specific GPCRs and

for target effector molecules.13 In particular, Gas(GTP)

stimulates adenylyl cyclase whereas Gai(GTP) inhibits it, and

Gaq(GTP) stimulates phospholipase C-b. Crystal structures

have been determined for Ga proteins in various states, includ-

ing a complex between Gas(GTP) and the catalytic portion of

adenylyl cyclase, of Gb:Gc and of heterotrimers.14–19 GPCRs

are thought to exist in equilibrium between inactive and active

states, which naıvely correspond to empty and ligand-occupied

receptors. The ligand-binding site is known, from studies on

rhodopsin20 and b2-adrenergic receptor,21 to be located

between helices near the center of the membrane. How

activation occurs for GPCRs that bind peptide ligands, such

as substance P,22 or intact proteins, such as follicle stimulating

hormone,23 is not entirely clear; portions of such ligands might

insert between transmembrane helices or else bind to

interhelical loops to effect conformational changes (for a

review, see Ref. 24). Changes in 7TM conformation that

accompany ligand binding or activation are linked to the

receptor binding of the G protein; G protein association with a

receptor increases its ligand affinity. A model of G-protein

coupling in GPCR activation to effector targets is given in

Figure 1.

1.2 Structural and Functional Characteristics of GPCRs

The characteristic 7TM pattern of hydrophobic segments in

GPCRs provides powerful constraints on the possibilities for a

3D structure. Moreover, this pattern when seen in rhodopsin

was reminiscent of that in bacteriorhodopsin where the

structure from purple membranes had shown the disposition

of helices.25 Although the sequences showed no detectable

homology and these two photoreceptors have very different

biochemical actions they do both use a Schiff-base linked

retinal to detect light. Later, the topological connections in

bacteriorhodopsin were found at a high resolution,26 andHoward Hughes Medical Institute and Department of Biochemistry andMolecular Biophysics, Columbia University, New York, NY 10032, USA

PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems

724 | Mol. BioSyst., 2007, 3, 723–734 This journal is � The Royal Society of Chemistry 2007

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ultimately electron crystallography showed that the helices in

rhodopsin have the same topology as those in bacteriorho-

dopsin, although their orientation is somewhat different.

Meanwhile, as sequences accumulated, conserved features

were realized and comprehensive alignments were made.27

The vast majority of GPCR sequences are homologous with

rhodopsin, but there also are groups of the superfamily that

are atypical. These include the secretin and metabotropic

glutamate receptors.

GPCR sequence alignments reveal many features besides the

7TM pattern, and some of these are evident in the subset

shown in Figure 2. Most strikingly, there is substantial

conservation in the transmembrane segments. There is,

however, a great variation in size as well as sequence for the

N- and C-terminal segments and also for most loops. Some

N-termini, e.g. those of glycoprotein hormone receptors,

include very large domains (not shown in Figure 2). The

cytoplasmic 5–6 loop is extremely variable in size. By contrast,

the extracellular 2–3 and cytoplasmic 3–4 loops are relatively

constant in size. The positions of several functional sites are

also typically in common. These include a disulfide bridge

between the N-terminus of TM3 and the 4–5 extracellular

loop, N-linked glycosylation at 10–20 residues before TM1, a

palmitoylation site at the end of H8, and phophorylation sites

in loop 5–6 and the C-terminal segment.28

Structural models have also been predicted from GPCR

sequences.29,30 The best constrained model came from

combining the structure of frog rhodopsin, determined at

9 s resolution by electron microscopy of 2D crystals,31 with an

analysis of the sequences of some 500 rhodopsin-family

members.32 The result was an alpha-carbon template for the

transmembrane helices for the rhodopsin family of GPCRs.

Fig. 1 Coupling of ligand (L) binding to a GPCR (R) through catalysis of GTP for GDP exchange in Ga and dissociation of free Ga(GTP) to

interact with an effector target (T). Components here are approximately at scale, based on known structures of inactive rhodopsin, Ga:Gb:Gc, and

Ga (GTP):adenylyl cyclase

Fig. 2 Alignment based on the structure of rhodopsin of GPCR sequences. Sequences are from the following sources: Rhod, bovine rhodopsin;

rat 5HT2c; human 5HT1a; mouse 5HT7; B2AR, human b2-adrenergic receptor; human CCR5; LHR, human leuteinizing hormone; FSHR, human

follicle stimulating hormone; and SP1 mouse mOR28 olfactory receptor. Bars over the sequences represent transmembrane helices TM1–TM7 and

C-terminal helix H8, respectively. Highlighted residues designate identities or certain close similarities

This journal is � The Royal Society of Chemistry 2007 Mol. BioSyst., 2007, 3, 723–734 | 725

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Subsequently, in a landmark study, the structure of bovine

rhodopsin was reported at 2.8 s resolution,33 and later refined

to 2.6 s resolution34 (Figure 3). A second structure has also

been reported.35 These structures confirm predictions based on

the alpha-carbon template and add rich detail on rhodopsin in

the inactive, 11-cis retinal state.

1.3 Structure Determination of GPCRs

Integral membrane proteins present formidable, albeit not

insurmountable, problems for structural analysis. There have

been striking successes starting with the first result in 3Ds, by

electron crystallography at 7 s resolution, on bacteriorho-

dopsin25 and the first atomic-level structure, at 3 s resolution

by X-ray crystallography, on a photosynthetic reaction

center.36 Membrane-protein structures have been determined

at an accelerated pace in recent years, and many of these new

structures have had a dramatic impact as in the cases of

cytochrome c oxidases37,38 and of potassium39,40 and water

channels.41,42 Recently, the scope of successes has broadened

to include several transporters,43–47 pumps,48,49 and other

channels.50–54 Nevertheless, the structural output on mem-

brane proteins is a very small fraction of that for soluble

macromolecules. While membrane proteins comprise 20–30%

of all proteins in both prokaryotic and eukaryotic organisms55

they are but a fraction of a percent of those with a known

structure. It is, of course, the natural association of membrane

proteins with lipid bilayers that complicates their structural

analysis. Once membrane-protein crystals are obtained, for

example, the diffraction analysis is as straightforward as it is

for aqueous soluble macromolecules. Problems that arise in

the recombinant expression of membrane proteins are even

more limiting than difficulties in purification and crystal-

lization. There have been recent successes in producing

recombinant bacterial proteins for analysis, but eukaryotic

membrane proteins have been strikingly recalcitrant in

expression at the scale needed for a structural analysis.

Although there are structures of important eukaryotic

membrane proteins, these have all come from natural sources

except for the peripheral, single-leaflet associated cyclooxy-

genases56,57 and for the very recent rat voltage-gated potas-

sium channel.53

GPCRs are found only in eukaryotes, and the majority of

them are present only in mammals. Essentially all GPCRs are

scarce and cannot be prepared from membranes derived from

natural sources. Rhodopsin represents the most notable

exception as it can be isolated in large amounts and at close

to 100% purity directly from purified membranes of rod outer

segments.58 The abundance and relative ease with which pure

rhodopsin can be obtained from natural sources is widely

believed to be a dominant factor behind its successful structure

determination. High natural occurrence not only ensures an

abundant supply of material, a prerequisite for successful

structure determination, but it also bears two additional

advantages. The first is the ‘‘ideal,’’ naturally occurring, match

between the protein and the composition of the membrane in

which the protein resides. The composition of a membrane can

have a profound impact on the stability of a protein that

resides within it, especially, given the likely presence of specific,

high-affinity lipids that are able to remain attached to the

protein, once the lipid bilayer is destroyed by detergent. The

second, somewhat related, advantage of using abundant

natural sources is that the higher the expression level of a

given membrane protein the fewer are the purification steps in

a detergent-containing aqueous solution typically required to

obtain a homogeneous preparation. Detergents tend not to be

ideal substitutes for a lipid bilayer, and the greater the extent

that one has to use these during purification, the greater is the

likelihood for removal of lipids important for the stability of

the protein. Rhodopsin, for example, is present at such a high

concentration in rod outer segments that, with a careful

preparation of these membranes, the detergent-extracted

protein could be used directly for (successful) crystallization

experiments.58,59

Unfortunately, rhodopsin represents an exception.

Essentially every other GPCR is sufficiently inabundant as

to defy a rhodopsin-like approach for structural studies.

Moreover, GPCR systems do not exist in prokaryotes and thus

bacterial homologs, exploited so effectively in structural

studies of potassium channels39 and trans-porters,44,47 are

not an option in this case. Appropriate recombinant expres-

sion systems are therefore needed to support these structural

studies.

Fig. 3 Ribbon diagram of bovine rhodopsin, drawn with the

extracellular side facing up, and the intracellular side facing down.

The bound retinal is visible in the plane of the membrane. The figure

was drawn from coordinates deposited in the Protein Data Bank

(PDB) with accession number 1L9H34

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2 Expression of Recombinant GPCRs

2.1 Overview

GPCRs have been successfully expressed in a multitude of

hosts, ranging from bacteria to mammalian cells. The

expression systems currently available for the production of

GPCRs have been discussed extensively in several excellent

reviews.60–63 In particular, Sarramegna and colleagues62

provide a detailed comparison of all the expression systems

that have been used for the production of GPCRs. Moreover,

these authors compile a truly informative and up-to-date chart

that summarizes the published results (expression levels,

activity, etc.) of essentially every GPCR that has ever been

heterologously expressed. An analysis of these data suggests

that there is substantial variability (up to 100-fold) in the levels

of different GPCRs produced in the same expression system.

Similarly, expression levels for the same GPCR expressed in

different systems can vary dramatically. In summary, given the

so far scarce success of GPCR structure determination, a pre-

ferred expression system for these proteins has yet to emerge.

Regardless of the problematic nature of GPCRs as targets for

structural studies, an abundant supply of functional material is

a prerequisite for a successful structure determination.

GPCRs, like other membrane proteins, undergo a complex

and poorly understood folding and membrane-insertion

mechanism. Unique properties of a cell environment that

may facilitate homologous (as opposed to heterologous)

expression of a GPCR include the specific lipid composition

of the various membrane compartments, cell-type specific

chaperones, and unique post-translational modifications

including defined glycosylation, phosphorylation, sulfonation,

and other covalent modifications. Following this line of

thoughts, one should attempt to express the GPCR of choice

in a system that matches as closely as possible its native

environment. Not surprisingly, the highest expression level

reported62 for a GPCR is that of the Ste2 receptor from

Saccharomyces cerevisiae, homologously overexpressed in

S. cerevisiae.64 The majority of GPCRs are from mammalian

origin. Therefore, mammalian cell-based expression systems

represent a likely good choice for these proteins. Although it is

now routine to use mammalian protein expression systems in

functional studies, based on transient-transfection experi-

ments, their application to large-scale protein production has

often been deterred by difficulties in obtaining large amounts

of material, rapidly, and at a reasonable cost. A path toward

the solution of these deterrents lies in the generation of stable,

as opposed to transient, cell lines.

An alternative, in a way opposite, approach is to use a

robust, simple, rapid, and cheap albeit ‘‘primitive’’ system and

to optimize it to produce sufficient amounts of functional

proteins. Bacterial expression systems, primarily based on the

Gram-negative bacterium Escherichia coli, have been by far the

most successful for the production of recombinant proteins for

structural studies. E. coli-based expression of a number of

GPCRs has indeed worked remarkably well, producing

milligram amounts of functional receptors (for example, see

Ref. 65).

These two ‘‘opposing’’ strategies for the expression

of GPCRs, those of E. coli-based and stable mammalian

cell-based expression systems, will be discussed in detail,

analyzing their advantages and disadvantages. Other expres-

sion systems that are either an alternative to the generation of

stable cell lines, such as viral infection of mammalian cells, or

that fall conceptually in between these two extremes, such as

those based on yeast hosts and on viral infection of insect cells

will also be discussed briefly in separate paragraphs.

All of the above-mentioned expression systems can be

expected to yield a functional protein. An alternative approach

is to generate abundant quantities of nonfunctional proteins

and to regain functionality by subsequent refolding, either in

lipid bilayers or in detergent micelles. Expression systems that

follow this approach and that have been used successfully for

the production of GPCRs will also be mentioned at the end of

this chapter.

2.2 Bacteria as Hosts for the Production of Functional GPCRs

High-resolution structural studies of proteins generally require

large amounts of pure, properly folded material. Indeed, the

advent of gene manipulation techniques for producing

recombinant protein in heterologous systems is arguably the

most important breakthrough of the past 30 years for

structural biology, exceeding even the wondrous developments

in synchrotron crystallography66 and NMR spectroscopy.67

Bacterial expression systems, primarily based on the Gram-

negative bacterium E. coli, have been by far the most

successful for the production of recombinant proteins for

structural studies. As of mid-2005, 20,504 of the 32,643 protein

structures deposited in the Protein Data Bank (PDB) had

‘‘Expression_System’’ records (www.rcsb.org). Of these, over

90% were produced using E. coli; y3.5% were produced in

yeast; y2.5% with insect cells, and y1.5% using mammalian

cells. The remaining y3% of these structures were determined

using proteins expressed in other systems, including bacterial

hosts such as Bacillus subtilis and cell-free expression systems.

The success of bacteria-based expression systems arises from

several factors including the ease with which such organisms

can be genetically manipulated; the thorough understanding of

their transcription and translation machinery, which has led to

the ability to achieve high levels of protein expression; the

rapidity of their growth; and the relatively low cost of their use.

Bacteria are unable to perform the post-translational

modifications that are typical of eukaryotic membrane

proteins. GPCRs undergo various modifications after protein

synthesis, with glycosylation, phosphorylation, and palmitoy-

lation being the most common. The essentiality of these

modifications for function of a given receptor cannot be

predicted a priori. For the most studied receptors, these data

are typically known. Glycosylation at Asn15 of bovine

rhodospin, for example, is critical for the stability of the

photoactivated metarhodopsin II state, and hence for coupling

to transducin.68 This information makes rhodopsin an

unsuitable candidate for bacterial expression. Many GPCRs,

however, are able to function without post-translational

modifications. If the protein can withstand it, the absence of

these modifications eliminates a source of heterogeneity. This

represents an important advantage of bacterial expression over

other, eukaryotic-cell based systems.

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GPCRs can be inserted in the inner bacterial membrane.

The lipid composition of the bacterial inner membrane is

rather different from that of eukaryotic cells.69 Most notable is

the absence of cholesterol (or any sterols for that matter) in

bacteria, a major component of membranes of higher

organisms, but there are other less notable but potentially as

important differences. Phosphatidylserine (PS), for example, is

an abundant phospholipid in mammalian cells. In E. coli, all

the PS is converted by the enzyme phosphatidylserine

decarboxylase to phosphatidylethanolamine (PE).70 PE accu-

mulates to become the predominant component of bacterial

membranes.69,71 In their recent review, Opekarova and

Tanner69 present conclusive examples showing that membrane

proteins depend on phospholipids and sterols for their

integrity and activity. Among GPCRs, the oxytocin receptor,72

the human m-opioid receptor,73 and the dopamine D-1

receptor74 have been shown to require specific lipid compo-

nents for optimal function.

G-proteins do not occur naturally in bacteria. This has two

effects on the expression of GPCRs in bacterial hosts. The first

is that, regardless of the fact that the receptor is properly

inserted in the membrane, most GPCRs will bind agonists only

in a low affinity state, unless exogenous G-proteins are

supplied. The second is that the system is not optimal for

functional studies of the signal transduction mechanism of

receptor in vivo. High affinity binding sites can be successfully

restored, however, either by exogenous addition of purified

G-proteins,75 by addition of membranes from cells expressing

G-proteins,76 or by constructing and expressing a GPCR

G-protein fusion.77,78 In contrast to the concerns about

bacteria lacking authenticity as hosts for GPCRs, the

observation that bacterially expressed GPCRs are able to shift

to a high-affinity state for agonists upon successful coupling to

G-protein heterotrimers provides compelling support for the

utility of this expression system.

Functional expression of a GPCR in E. coli was first shown

in the late 1980s: Marullo and colleagues79 fused the coding

sequence of the human b2-adrenergic receptor to an

N-terminal fragment of the b-galactosidase gene, and tested

cells expressing the fusion protein with b2-adrenergic receptor

specific ligands. Production of b2-adrenergic receptors was

shown by the presence, on intact bacteria, of binding sites for

catecholamine agonists and antagonists possessing a typical

b2-adrenergic pharmacological profile. Binding and photo-

affinity labeling studies performed on intact E. coli cells and on

membrane fractions showed that these binding sites are located

in the inner membrane of the bacteria. Expression levels

reported in this study were too low (0.4 pmol mg21 of

membrane protein) to enable the use of this system for

milligram-scale protein production. However, this study

showed that the use of a bacterial host for the expression of

GPCRs was a possibility. Protein levels improved considerably

with the expression of serotonin (5-HT) receptor subtype 1a

(5HT1a) as a fusion to the C-terminus of maltose-binding

protein (MBP).75 MBP, coded by the malE gene is a protein

that is targeted to the bacterial periplasm.

Soon after the work on 5HT1a, Grisshammer and collea-

gues80 showed that neurotensin-binding could be detected in

E. coli membranes isolated from cells expressing the rat

neurotensin receptor (NTR). The authors compared expres-

sion levels of wild-type NTR, NTR N-terminally fused to a

bacterial signal peptide sequence (of enterotoxin B), and NTR

N-terminally fused to the C-terminus of MBP. Expression

levels, measured by radioligand-binding assay, showed a 40-

fold increase with the MBP–NTR fusion (to approximately

450 binding sites per cell, equivalent to 15 pmol mg21 of

membrane protein), unequivocally indicating that the fusion

with the malE gene was the preferred way to proceed. One of

the greatest benefits of working with a bacterial system is the

ease and rapidity with which an expression construct can be

engineered, modified, and evaluated. Grisshammer and

colleagues took advantage of this in the most thorough of

ways. They systematically varied the expression construct for

NTR, each time evaluating protein production levels, purifica-

tion yield, and efficiency.65,81 Following this approach,

expression levels increased to a maximum of approximately

1000 copies/cell. 3 mg of functional NTR can currently be

purified to homogeneity from a 20 L (100 g of cells) bacterial

culture.82 A similar approach has been applied to the

expression, for example, of the rat neurokinin A receptor,83

the human adenosine A2a receptor,84 and the M185 and M286

muscarinic acetylcholine receptors. The strategy followed for

the optimization of expression constructs for the above-

mentioned examples is similar. However, the results appear

to vary between different receptors, even within the same

family (for example, see Ref. 87). This implies that optimiza-

tion will be required for every receptor expressed in E. coli, and

that being able to increase expression to acceptable levels

cannot be guaranteed a priori. Analysis of the amino acid

sequence of a given receptor can only provide an (not too

reliable) indication of whether it will or will not express.88

Are there some aspects that can be generalized, and that can

serve as starting guidelines for someone trying to express a

GPCR in E. coli? Although GPCRs have been expressed

without N-terminal fusion, the presence of a fusion partner

seems to be advantageous, and in some cases even essential.

For example, the M2 muscarinic receptor could only be

expressed in an active form when fused to MBP.86 Given its

success, MBP would be the preferred choice. Other molecules

used successfully include the outer membrane protein

LamB,87,89 the periplasmic protein alkaline phosphatase90

and, surprisingly, the cytoplasmic protein b-galactosidase.91

The role of the fusion partner is unclear. In the case of MBP,

its translocation to the periplasm could help drive the

topologically correct insertion of the fused GPCR into the

inner membrane (Figure 4).

MBP can be removed easily from the GPCR by genetically

engineering a protease recognition sequence in the linker

region between the two proteins. Tobacco etch virus (TEV)

protease efficiently cleaves its hepta-residue recognition

sequence, engineered between the two proteins82 (Figure 4).

A stable mutant of TEV protease with enhanced catalytic

activity can be produced in abundance from E. coli.92 Fusion

of a small and stable bacterial protein such as a truncated form

of thioredoxin A (TRX) to the C-terminus of NTR leads to a

triple-protein fusion construct (MBP–NTR–TRX) that

appears to further stabilize the receptor and improve expres-

sion and purification yields.65,82 A similar stabilizing effect of

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TRX has been observed in the bacterial expression of the rat

serotonin receptor subtype 2c (5HT2c) (Mancia and

Hendrickson, unpublished results). The reasons for the

beneficial effects of TRX on bacterially expressed GPCRs

are unknown.

The slower the cell growth and the more attenuated the

induction, the better. Therefore, growing the bacteria at low

temperatures (18–25 uC) after having induced expression and

using an expression plasmid bearing a weak promoter element

(such as the lac wild-type promoter), are typically a good

starting point and choice, respectively, for an initial experi-

ment on a previously untested target. Finally, the need to

optimize codon usage for E. coli expression is questionable,

especially given the amount of time and effort required.

Regardless, there appears to be no disadvantage in using E. coli

strains that have been transformed to supplement the

bacterium with tRNAs for rare codons. Different strains

might need to be tested, as there is evidence for considerable

interstrain variability in the expression levels, with DH5a and

BL21 (and strains derived from this, such as Rosetta)

performing the best (Mancia and Hendrickson, unpublished

results).

2.3 Production of GPCRs in Stably Transfected Mammalian

Cells

Despite the advantages discussed in the preceding section,

bacterial systems often fail in their application to the expres-

sion of eukaryotic proteins.93–95 Failure to achieve acceptable

expression often arises from toxicity of the foreign protein or

its inability to fold or be targeted properly in the bacterial cell.

Such problems inevitably result in low levels of expression or

protein misfolding.93–95 Thus, despite drawbacks in efficiency,

alternative expression systems based on eukaryotic hosts, have

been developed for large-scale protein production. These

include expression in yeast, insect cells, and mammalian cells,

all of which have been used successfully in producing proteins

for structure determination. In the particular case of mamma-

lian proteins, although heterologous eukaryotic cell systems or

bacteria can be effective, optimal expression of some such

proteins may require mammalian host cells.

Mammalian proteins evolved in a mammalian cellular

milieu, and it is understandable that both proper folding and

stability may depend on this environment. Unique properties

of the mammalian cell environment that may facilitate

homologous expression include specific lipid compositions of

the various membrane compartments, cell-type specific cha-

perones, and unique post-translational modifications including

defined glycosylation, phosphorylation, palmitoylation, and

sulfonation. Production of recombinant protein in mammalian

cells can be accomplished either through transient transfection,

viral infection, or through integration of expression constructs

into the host genome. Each of these methods has advantages

and disadvantages.

A very large number of GPCRs has been expressed in

mammalian cells, predominantly for functional studies,

following transient transfection protocols. Mammalian cells

offer the ideal host for functional studies, as these typically

require an active protein but not in large amounts. Expression

levels are highly variable from one receptor to another (for a

list of some examples, see Ref. 62), but functional analyses are

effective nevertheless. Rapid and efficient techniques for

transient transfection and site-directed mutagenesis facilitate

the experiment, and the presence of a complete signaling

machinery in mammalian cells provides an authentic func-

tional readout. Thereby, numerous successes are reported in

the tests of GPCR function in ligand binding, receptor

activation, oligomerization, desensitization, internalization,

and interaction with G-proteins. Moreover, given the presence

of a complete and functional receptor–effector signaling

pathway, mammalian cell-based expression of GPCRs is used

routinely in assays for drug discovery directed against these

important pharmaceutical targets. Although it is now routine

to use mammalian protein expression systems in functional

studies, their application to large-scale protein production has

often been deterred by difficulty in obtaining large amounts of

material, rapidly, and at a reasonable cost. When this is

Fig. 4 A schematic drawing of MBP–GPCR fusions. MBP is shown in blue, with its signal peptide in green. The GPCR is in red. The bacterial

inner membrane is drawn in yellow. SP is where the natural signal peptidase cleaves to produce the mature fusion protein. TEV is the TEV protease

cleavage site utilized to separate the two proteins after expression or purification

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achieved, however, advantages ensue both for the structural

work and also for functional tests of structure-inspired

hypotheses. Not surprising, given the above, is the scarcity

of reports in which a GPCR has been ectopically expressed in a

mammalian host and then purified for structural studies.

Bovine rhodopsin represents one of the very few exceptions,

having been expressed to high levels (up to 10 mg L21) in

human embryonic kidney 293 (HEK-293) cells, and purified to

homogeneity in a single step using an immunoaffinity

column.96

Stably transfected cells are desirable for their ability to

provide a constant source of recombinant protein, but the

production of stable transfectants is time consuming. To

generate a cell line, the coding sequence for the gene of interest

is placed under the control of a strong constitutive promoter

such as the promoter element derived from cytomegalovirus

(CMV).97 The promoter can be inducible, if protein-induced

toxicity to the cell should become an issue.98 The plasmid is

transfected into the host cells using standard techniques.

Generation of stably producing cell lines requires integration

of the expression construct into the genome of the host cell.

The selection of stable integrants is typically accomplished

with the use of antibiotic markers. The antibiotic marker can

be present in the same or in a separate plasmid as that carrying

the gene of interest. Integration is a rare event, and antibiotic-

resistant cells represent a minority of the total number of cells.

Expression levels will vary dramatically between these resistant

cells. Heterogeneity in expression levels arises from differences

in the number of integrants, and their sites of integration.

Thus, a key step in harnessing such cells for protein production

is the selection of those single cells that achieve the highest

expression levels. Colonies grow out of single antibiotic-

resistant cells. Traditionally, to screen for highly expressing

cells, individual colonies are handpicked and assayed for their

levels of protein production, usually involving immunological

detection. This method is time consuming and labor intensive.

A quicker and more efficient method is based on detection

of the fluorescence intensity of a co-expressed marker, such as

the green fluorescent protein (GFP).99,100 Downstream of the

promoter and of the coding sequence for the gene of interest,

an internal ribosome entry site (IRES)101 is followed by the

coding sequence for GFP (Figure 5A). A single bicistronic

messenger RNA encoding both genes is produced. The two

separate proteins are then translated from the same message,

and their expression levels are thereby coupled. Efficient

selection of highly expressing cells can be easily achieved by

monitoring fluorescence intensity of cells expressing variable

amounts of GFP. The process can be accelerated further by

using fluorescence-activated-cellsorting (FACS, see Refs. 102

and 103) technology for the rapid selection of either clonal or

non-clonal populations of high expressors (Figures 5B and

5C).

The use of IRES–GFP as a binary marker for successfully

transfected cells is well established. However, its use as a

monitor for target protein expression levels has seen far fewer

reports (for example, see Refs. 104 and 105). Stable HEK

293 cell lines expressing a considerable amount (140–

160 pmol mg21 of membrane protein) of the rat 5HT2c

receptor have been generated using this technique.106 The

authors show clear correlation between expression levels of the

GPCR and of GFP.

Many different mammalian cell lines have been used

successfully for the expression of GPCRs (for a list, see

Ref. 62). Baby hamster kidney (BHK) cells and African

monkey (COS) cells appear to be the best suited for viral

infection. HEK293 cells and Chinese hamster ovary (CHO)

cells have yielded the highest levels of GPCRs from stable

lines.

GPCRs expressed in cell lines often show a highly

heterogeneous pattern of glycosylation. This can constitute a

severe problem for structural studies. Mutated cell lines that

exhibit a reduced and homogenous glycosylation have been

generated and used successfully for the overexpression and

purification of recombinant bovine rhodopsin.107 These

experiments will be presented in detail in a subsequent chapter

of this book.

2.4 Production of GPCRs via Transient Transfection or Viral

Infection of Mammalian Cells

Transient expression is performed under non-selective condi-

tions. In transiently transfected mammalian cells, protein

expression levels peak around 48–72 h after transfection, and

Fig. 5 Schematic representation of the GFP-selection method. (A)

Scheme of the expression vector. (B) Diagram of the enrichment

procedure, through successive rounds of cell sorting, and expansion of

the most GFP-fluorescent cells. (C) Progression of the enrichment

procedure monitored by fluorescent microscopy. The cells depicted

here are HEK 293 cells expressing 5HT2c. Three stages are shown:

after antibiotic selection, after the first round of sorting, and after the

final round (Adapted from Ref. 106 with permission).

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inevitably decline thereafter. Transfection on a large scale is

not the most practical of solutions, because of the fact that

mammalian cells tend to transfect more efficiently in mono-

layers as opposed to suspension, making scale up arduous, and

because of reagent expenses. As a commonly accepted general

trend, expression levels of transiently transfected cells tend to

be higher than that of stable cell lines. This seems not to be the

case for GPCRs.106 However, constructs can be screened easily

and rapidly by transient expression, making this an excellent

screening tool to interplay with the more time-consuming

process of generating stable cell lines (Mancia and

Hendrickson, unpublished results).

Infection of cells with a recombinant virus such as the

Semliki Forest virus can be extremely efficient, leading to high

expression levels in the 48–72 h after infection (for a review of

this system and its application to the expression of GPCRs, see

Ref. 108). Excellent results have been obtained with this

system for the expression of GPCRs. The negative aspects are

that recombinant protein expression is again transient, as the

infected cells die after a finite number of days. Moreover, the

fact that viral infection typically requires the propagation of

recombinant virus in a ‘‘packaging’’ cell line, isolation of the

virus, and determination of viral titer prior to infection of the

host cells, makes this approach rather time consuming,

probably in the same time frame as generating a stable cell line.

2.5 Production of GPCRs in Yeast

The structure of a rat voltage-gated potassium channel has

recently been solved.53 The mammalian protein was expressed

in the yeast Pichia pastoris.109 Thus, a yeast-based expression

system has paved the way for what is, to the best of our

knowledge, the first crystal structure of a mammalian

membrane protein from a recombinant source. This success

should not be underestimated when choosing a system for the

expression of a GPCR.

Yeasts are, in principle, excellent organisms for the

expression of GPCRs. Yeasts are easy, quick, and inexpensive

to grow, and they can be cultured to high density for efficient

scaling up. Expression techniques are well established and

offer numerous different possibilities. Moreover, these uni-

cellular eukaryotes can potentially perform all the post-

translational modifications of mammalian cells. G-proteins

are also expressed in yeast.

Glycosylation is substantially different in yeast and mam-

malian cells. There are differences in both the amount and the

type of glycans. This can constitute a problem for those

GPCRs that are sensitive to glycosylation for proper function.

Moreover, there are examples of GPCRs that are not

glycosylated when expressed in yeast.110 The lipid composition

of the membranes is also different in yeast and mammalian

cells. Most notably, there is a reduced level of sterols in yeast.

These differences can lead to the expression of GPCRs with

altered ligand-binding properties.111 Proteolysis can also be an

issue that should be taken into account, although protease-

deficient yeast strains can be used for expression.110

GPCRs have been expressed in a functional form in

S. cerevisiae, P. pastoris, and Schizosaccharomyces pombe.

Inducible as well as constitutive promoters have been used in

plasmids that are either maintained in an episomal form or

integrated into the host’s genome. Use of a yeast leader

sequence appears to be important for proper targeting to the

plasma membrane. Similarly to other systems, expression

levels of GPCRs in yeast are extremely variable (for a list of

examples, see Refs. 62 and 112). Not surprisingly, yeast

GPCRs express the best in yeast, as is the case for the Ste2

receptor in S. cerevisiae.64 For a more in depth understanding

of this expression system, the reader should consult some of

the excellent reviews that have been written on the expression

of GPCRs in yeast, and on the use of this system for functional

and structural studies on these proteins.62,112–116

2.6 Production of GPCRs in Insect Cells

Following E. coli and yeast, baculovirus-based expression

systems have had the greatest success in the number of

structures successfully determined. In particular, expression of

eukaryotic genes through the use of baculovirus-infected insect

cells117 has a long history of success (for a review of the

technique, see Ref. 118). Briefly, the gene of interest is placed

under the control of a strong constitutive promoter and the

plasmid is inserted into the viral genome by homologous

recombination. The commonly used virus is the Autographa

californica multiply embedded nuclear polyhedrosis virus

(AcMNPV). Insect cells are infected with the recombinant

virus, and the expression of heterologous proteins typically

peaks 48–72 h post-infection, with cell death occurring 4–5

days post-infection. Insect cells can be easily adapted to grow

in suspension. Insect cells are able to perform the same post-

translational modifications as those of mammalian cells,

although the type and amount of glycosylation is substantially

different.119 The lipid environments are quite different in insect

and mammalian cells (cholesterol levels, for example, are low

in insect cells).

Insect cells have been widely used for the expression of

GPCRs, yielding, once again, mixed results in terms of

expression levels. There are reports in which levels are among

the highest ever achieved. For an exhaustive review on the use

of this system for the expression of GPCRs, the reader should

consult a recent review written by Massotte.120

2.7 ‘‘In vivo’’ Expression in the Eye

The amount of rhodopsin that accumulates in rod cells is

extremely large. Researchers have thus begun to explore the

idea of expressing other GPCRs in these compartments

through the generation of appropriate transgenic animals.

Eroglu and colleagues121 were successful in expressing a

Drosophila melanogaster metabotropic glutamate receptor in

the Drosophila photoreceptor cells at levels (170 mg g21 of fly

heads, equivalent to approximately 3000 flies [Luisa

Vasconcelos, personal communication]) at least 3-fold higher

than those achieved with conventional baculovirus systems.

However, baculovirus-infected insect cells are undoubtedly

easier to scale up. Kodama and co-workers122 have instead

generated transgenic mice expressing the human endothelin

receptor subtype B (hETBR), fused with the C-terminal 10

residues of rhodopsin, in rod cells. Somewhat disappointingly,

hETBR protein levels were in the order of a 1000-fold less than

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rhodopsin in heterozygous animals. This approach is likely to

require a better understanding of how rhodopsin is efficiently

translated, folded, and transported before it can be used

successfully. Moreover, more examples are necessary before

the potential of this approach can be adequately assessed.

2.8 Extra-Membranous Expression Systems

All of the above-mentioned expression systems yield functional

proteins. A completely different approach is to generate

abundant quantities of protein outside of a membrane

environment, and then to use biochemical refolding manipula-

tions to reconstitute a properly folded and functional protein

from non-functional, aggregated material. Extra-membranous

expression may be possible in the bacterial cytoplasm or in

cell-free systems;123 reconstitution may be achieved either in

lipid bilayers or in detergent micelles. A human leukotriene

receptor has been expressed in the form of inclusion bodies in

E. coli and successfully refolded in detergent.124 The authors124

showed that the refolded protein was able to bind ligand as

well as interact with G-proteins.125 An olfactory receptor fused

to GST could also be accumulated in large amounts in the

form of inclusion bodies when expressed in E. coli and refolded

in detergent.126 Recently, Ishihara and colleagues127 were able

to express several GPCRs in a cell-free expression system as

fusions to thioredoxin (TRX). The insoluble proteins could be

solubilized by detergents and ligand binding restored by

incorporation into liposomes.127 These approaches are cur-

rently at an early stage of development, but could potentially

turn out to be extremely powerful for the field of structural

biology of GPCRs.

3 Conclusions

Several viable options exist for the recombinant expression of

GPCRs in sufficient abundance for structural studies. We have

emphasized two alternative expression systems; one in bacteria

where multiple constructs can be tested quickly and economic-

ally, and another in stable mammalian cell lines where natural

modifications and functional tests can happen. What we have

not stressed, but do find advantageous, is the synergy to be

found in pursuing the two simultaneously. A productive

interplay is then possible whereby feasibility tests are done at

relatively high throughput in bacteria and functional evalua-

tion of prime candidates can be made in a relevant mammalian

setting. We find that adequate yields are possible both from

MBP fusions in E. coli and from GFP-selected strains of HEK-

293 cells. Alternative pairs of systems might also provide the

respective advantages of these expression systems.

Although GPCRs have been expressed successfully in

various systems and by several investigators, none of these

studies has yet produced structure-quality crystals. Often, this

is despite the compelling evidence of natural ligand-binding

affinities in membranes or even in detergent micelles. It may be

that further structural stabilization is required, which might

come through the retention or reintroduction of certain lipids,

through provision of physiological interactions as in dimers or

in complexes with G proteins, or through complexation with

engineered binding partners such as antibodies. Methods for

implementing such approaches are beyond the scope of this

review, but the expression methods described here surely also

have relevance in these elaborated contexts.

Acknowledgments

We thank Richard Axel, Paul J. Lee, Yonghua Sun, and Risa

Siegel for their precious contributions to this work and for

helpful discussions. This work has been supported in part by

the NIH (GM68671).

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