Expression of Recombinant G-Protein Coupled Receptors for Structural Biology

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 mechanism of signal transduction depends on molecular characteristics of the receptor, and there are several classes of receptors. Besides those linked to the downstream elements by heterotrimeric 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 TGFβ/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 compounds; 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 rhodopsins^1–3 and a little later in the sequence of the hamster β2-adrenergic receptor.⁴ The involvement of heterotrimeric G-proteins in signaling through 7TM receptors was first worked out for the β2-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 stimulates 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 receptors^7,8 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, Gα, with a Gβ:Gγ heterodimer. Gα(GDP):Gβ:Gγ dissociates to Gα(GTP) and Gβ:Gγ 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 Gγ, and after activation they can diffuse away from the receptor to effector sites on their membrane-associated targets. There are at least 15 different Gα proteins, 5 Gβs, and 5 Gγs¹² and different combinations are selective for specific GPCRs and for target effector molecules.¹³ In particular, Gαs(GTP) stimulates adenylyl cyclase whereas Gαi(GTP) inhibits it, and Gαq(GTP) stimulates phospholipase C-β. Crystal structures have been determined for Gα proteins in various states, including a complex between Gαs(GTP) and the catalytic portion of adenylyl cyclase, of Gβ:Gγ and of heterotrimers.^14–19 GPCRs are thought to exist in equilibrium between inactive and active states, which naïvely correspond to empty and ligand-occupied receptors. The ligand-binding site is known, from studies on rhodopsin²⁰ and β2-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.

Fig. 1 Coupling of ligand (L) binding to a GPCR (R) through catalysis of GTP for GDP exchange in Gα and dissociation of free Gα(GTP) to interact with an effector target (T). Components here are approximately at scale, based on known structures of inactive rhodopsin, Gα:Gβ:Gγ, and Gα (GTP):adenylyl cyclase

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 and ultimately electron crystallography showed that the helices in rhodopsin have the same topology as those in bacteriorhodopsin, 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

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 β2-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

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 Å 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. Subsequently, in a landmark study, the structure of bovine rhodopsin was reported at 2.8 Å resolution,³³ and later refined to 2.6 Å 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.

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

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 Å resolution, on bacteriorhodopsin²⁵ and the first atomic-level structure, at 3 Å 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 oxidases^37,38 and of potassium^39,40 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 membrane 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 crystallization. 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 cyclooxygenases^56,57 and for the very recent rat voltage-gated potassium 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 expression systems are therefore needed to support these structural studies.

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 preferred 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 experiments, 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 expression 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 (http://www.rcsb.org). Of these, over 90% were produced using E. coli; ∼3.5% were produced in yeast; ∼2.5% with insect cells, and ∼1.5% using mammalian cells. The remaining ∼3% 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 palmitoylation 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.

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 accumulates to become the predominant component of bacterial membranes.^69,71 In their recent review, Opekarová 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 μ-opioid receptor,⁷³ and the dopamine D-1 receptor⁷⁴ have been shown to require specific lipid components 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 β2-adrenergic receptor to an N-terminal fragment of the β-galactosidase gene, and tested cells expressing the fusion protein with β2-adrenergic receptor specific ligands. Production of β2-adrenergic receptors was shown by the presence, on intact bacteria, of binding sites for catecholamine agonists and antagonists possessing a typical β2-adrenergic pharmacological profile. Binding and photoaffinity 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 mg⁻¹ 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 colleagues⁸⁰ showed that neurotensin-binding could be detected in E. coli membranes isolated from cells expressing the rat neurotensin receptor (NTR). The authors compared expression 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 mg⁻¹ 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, purification 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 optimization 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 β-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).

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

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 expression and purification yields.^65,82 A similar stabilizing effect of 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 °C) 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 experiment 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 DH5α 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 expression 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 mammalian 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 chaperones, 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 functional 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 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 L⁻¹) 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).

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).

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 mg⁻¹ 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 conditions. In transiently transfected mammalian cells, protein expression levels peak around 48–72 h after transfection, and 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 monolayers 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 unicellular 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 mammalian 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 μg g⁻¹ 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 (hET_BR), fused with the C-terminal 10 residues of rhodopsin, in rod cells. Somewhat disappointingly, hET_BR protein levels were in the order of a 1000-fold less than 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 manipulations 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 currently 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 economically, 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 evaluation 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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