Large-scale functional expression of visual pigments

Expression and Purification of Membrane Proteins

Large-scale functional expression of visual pigments: towards high-resolution structural and mechanistic insight Willem J. DeGrip', Corne H. W. Klaassen and Petra H. M. Bovee-Geurts Department of Biochemistry, Institute of Cellular Signalling, I60-FMW, University of Nijmegen, P.O. Box 9 I0 I , 6500 HB Nijmegen, The Netherlands

Introduction

sufficient amounts of purified material. This is largely due to the facts that tissue expression of GPCRs is very low (0.01-0.2 pmollmg of membrane protein) and that purification is usually accompanied by heavy losses due to their low stability in detergent solution. Only the lightsensitive GPCR rhodopsin is expressed at relatively high levels in retinal photoreceptor membranes, which allows the isolation of reasonable amounts of purified protein. Consequently, structural studies with GPCRs have so far mainly been performed with rhodopsin. Recently, preparation of two-dimensional crystals of rhodopsin was further improved and analysis of images from cryo-microscopy produced a medium-resolution (7.5 A) 3D structure of the membrane domain, which agrees with seven transmembrane segments with a high degree of a-helical character [3]. Studies using Fourier-transform infrared (FTIR) spectroscopy support a large content of a-helical structure that extends into some helical loops, and indicate considerable p-structure in the intracellular domain [4].NMR analysis of soluble peptides representing intracellular loops also suggests a high proportion of p-structure and has generated a model for the intracellular domain of rhodopsin

In response to endogenous and exogenous stimuli, multicellular organisms develop highly co-ordinated responses. An essential element in this communication system involves G-protein-coupled receptors (GPCRs). This ubiquitous class of membrane proteins arose early during evolution and has developed into one of the largest protein superfamilies known in nature. GPCRs have been identified in a variety of organisms, varying from yeast and Dictostelium to mammals. Several hundreds of distinct GPCRs couple extracellular stimuli as diverse as light, odours, monoamines, glycohormones and ions to intracellular signals. As such, GPCRs are of fundamental importance for cell-to-cell communication. Consequently, these proteins are the target of approximately 60 yo of the currently available medication against human diseases. Moreover, a large number of additional GPCRs is still expected to be discovered in genome projects. As a consequence, the GPCR family is considered to be the most important target in the development of the next generation of drugs. The superfamily of GPCRs shares two basic structural and mechanistic features : seven transmembrane segments with strong a-helical characteristics, that connect at the intracellular side through loop structures, which in the activated state of the receptor generate a high-affinity binding domain for heterotrimeric GTP-binding proteins or G-proteins [1,2]. Following binding to GPCRs, G-proteins are able to modulate a variety of cellular functions via several distinct intracellular mechanisms [1,2]. A thorough understanding of ligand-GPCR interaction is still obstructed by a lack of detailed knowledge of the actual three-dimensional (3D) structure of these receptors. Structural studies of GPCRs have been seriously hampered by lack of

PI. Visual pigments: photon-counting receptors Visual pigments comprise a subfamily of GPCRs that accounts for the light sensitivity of the photoreceptor cells in the vertebrate (rods and cones) and invertebrate retina, which is essential for the process of vision [6]. Members of this family are probably also involved in the mechanisms regulating photoperiodic processes [7-91. In their evolution towards a photosensory function, the visual pigments have developed some unique features among the GPCRs [6]. They contain a photosensitive ligand (1 1-&-retinal), that is covalently bound to the protein moiety (opsin) through a protonated Schiff base linkage with a membrane-domain-embedded lysine side chain. The retinylidene ligand is responsible for the visible absorbance band of these receptors (e.g.

Abbreviations used: FTIR, Fourier-transform infrared: ss-MAS, solid-state magic-angle spinning: p i , post infection: GPCR Gprotein-coupled receptor: IMAC, immobilized-metal affinrty chromatography; DoM,8-I -dodecylmaltoside: 3D,three-dimensional: MOI, multiplicity of infection. 'To whom correspondence should be addressed.

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L,,, = 498 nm for bovine rhodopsin), and is called the chromophore. This ligand actually functions 'as an inverse agonist. It reduces the basal activity of the apoprotein opsin to such a low level that it allows the rod photoreceptor to operate under very dim light intensities, basically functioning as a photon counter [lo]. Absorption of a light photon triggers an ultrafast (within 200 fs [ll]) conformational change in the ligand (isomerization, 11-cis --* all-trans), which converts it into a full agonist, resulting in rapid high-level activation of the receptor (within several ms to a x106-fold increase in activity over the dark state [6]). A single light-pulse can lead to synchronous activation of a sufficient number of receptors to detect the accompanying conformational steps in the protein. Symbiosis between opsin and chromophore has pushed the efficiency of the photochemical reaction (cis + trans isomerization) close to the limit. A nearly complete stereospecificity, femtosecond kinetics and a quantum yield of 0.67 are far beyond the values presently achieved in model systems [121. Subsequent discrete conformational steps leading to the active receptor (metarhodopsin I I) have been identified by their individual spectral properties and can be isolated thermally by cryospectroscopy [ 131 or kinetically by picosecond spectroscopy [12]. The spectral intermediates (batho-, lumi-, metarhodopsin I and metarhodopsin 11) have been shown to indeed represent discrete structural intermediates by vibrational spectroscopy [11,141. It is obvious that visual pigments have become a paradigm for studies of the molecular mechanism of a GPCR. Ideally, a high-resolution 3D structure would serve as a basis for such studies. However, 3D crystals suitable for highresolution X-ray diffraction analysis have not yet been produced for any GPCR. The available twodimensional crystals of rhodopsin currently only suffice for a medium-resolution 3D structure of the membrane domain [3]. In spite of the progress in molecular modelling, the available models based on this medium-resolution structure still differ considerably (e.g. [15,16]) and reliable high-resolution structural information cannot be extracted from such structures, yet. Recently, alternative experimental approaches to extracting structural data from supramolecular membrane systems have been developed. FTIR spectroscopy in the difference mode can monitor changes at the molecular level (changes in absolute structure, in interaction 0 1999 Biochemical Society

pattern or in microenvironment) by the corresponding change in vibrational frequency and/or intensity [14]. In complex systems, identification or assignment to specific chemical groups usually requires site-specific mutagenesis or, preferentially, stable-isotope labelling [17,18]. Novel developments in the field of solid-state magic-angle spinning (ss-MAS) NMR spectroscopy now allow the derivation of highly precise structural constraints from supramolecular systems. Isotropic chemical shifts and corresponding tensor elements in combination with relaxation properties can provide detailed information on chemical and electronic characteristics of a specific atom [ 191. Distances between atoms of up to 1 nm can be measured with high accuracy (fO.O1 nm) using one-dimensional rotational resonance NMR [20,21]. Torsional angles in bonds between adjacent atoms can be addressed by double-quantum heteronuclear local-field NMR [22]. The latter approach is also very appropriate for measuring short distances (0.1-0.2 nm). Such structural studies require relatively large amounts of purified protein (FTIR, 0.20.5 mg; ss-MAS NMR, 1C30 mg). In addition, stable-isotope labelling is usually very helpful in FTIR studies for the purpose of identification ("C, 'H, 15N, '"0,34S), and absolutely essential for NMR studies ("C, 'H, "N). Consequently, we have invested considerable efforts in developing a functional expression system for visual pigments that would allow appropriate levels of stableisotope incorporation as well as scale-up to production levels of tens of milligrams of purified protein per batch.

Functional expression and scale-up With the exceptional progress in molecular biology it is now possible to overexpress various GPCRs in a variety of host systems. Expression levels that are 100-1000-fold higher than those available in native tissue can be obtained. As to visual pigments, early studies were primarily performed with the relatively stable bovine rod visual pigment rhodopsin. More 'simple ' systems like Escherichia coli or the yeast strains Saccharomyces or Pichia produced either not much recombinant protein at all ( E . coli) or reasonable amounts of total protein, but very low levels of functional receptor (4-8 yoin yeast) [23,24]. Mammalian cell lines (COS, HEK293) generate satisfactory yields ( > 10' copies/cell) of functional protein [25,26], but stable-isotope labelling and culture scale-up is not easily achieved, even if one

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growth. This contributes peptides from which the cells also derive free amino acids, thereby ' diluting ' added stable-isotope-labelled amino acids with unlabelled ones. Nevertheless, upon supplementing InsectXPress medium deficient in either lysine or tyrosine with 4 m M labelled lysine or 2 m M labelled tyrosine, label incorporation levels of 57&5 Yo for lysine (n = 4) and of 74 f4 % for tyrosine (n = 3) were achieved, amply sufficient for F T I R and N M R analyses [18,32]. Label incorporation was checked, after proteolytic or acid hydrolysis and proper derivatization, by GC-MS analysis of the mass distribution of all amino acids (C. H. W. Klaassen, unpublished work). Scrambling of label from lysine (15N,13C) or tyrosine ('H, 13C) into other amino acids was not detectable. This serves to show that this system can be used for protein labelling with essential amino acids as well as with non-essential amino acids with slow metabolic turnover. For amino acids with a high metabolic flux like Glu, Gln or Ala, alternative procedures are being investigated. For infection with recombinant baculovirus a high M O I is usually employed (5-10) with cells in logarithmic growth. Large volumes of high-titre virus suspension ( X 200 ml of x 10' p.f.u./ml) would then be required to infect a 10-litre bioreactor. Therefore we investigated the effect of lower MOIs. Earlier we observed quite similar expression levels for MOIs between 1 and 10 [30]. Further reduction of the M O I actually results in an increase in total expression level if longer incubation times can be tolerated (Figure 1, left panel). For the lowest M O I tested (0.01), detectable opsin expression starts with several days' delay, not surprisingly, but the total level surpasses that of M O I 0.1 and 1 by 5 days post infection (p.i.) and does not fully reach its maximal level at 6 days p.i. Since the expression level per cell only shows slight MOI-dependency [33], this net increase must be due to the higher final cell densities attained at very low M O I [33], where the cells can still go through another division before the infection really spreads. These conditions could be transferred to a 10-litre bioreactor culture with identical results (Figure 1, right panel). I n this way, we are able to produce 30-40mg of functional rhodopsin in a single 10-litre bioreactor run. Large differences in the percentage of functional protein (5-80 %) are reported in the literature upon expression of GPCRs with recombinant baculovirus, sometimes with quite discrepant fig-

turns to stably transformed cell lines [27,28]. We experienced good expression levels in the eukaryotic recombinant baculovirus expression system in combination with the insect Sf9 cell line [29]. This cell line is easily adapted to suspension culture as well as to serum-free culture conditions. We have therefore optimized the baculovirus system towards batch production of rhodopsin (20-40 mg of receptor/batch) with respect to the following parameters : (i) baculovirus promoter to drive opsin expression, (ii) serum-free culture medium, and (iii) multiplicity of infection (MOI) and incubation time. Suitable expression levels ( > 5 x lo6 copies/ cell) were only obtained with the very strong verylate-phase p l 0 and polyhedrin promoters [30], which are switched on between 12 and 18 h after infection of the cell. T h e earlier, much weaker basic protein promoter did not generate detectable levels of functional opsin. As most baculovirus expression vectors utilize the polyhedrin promoter, this has become our promoter of choice. Despite the 'late' activity of this promoter, we always observe a high percentage of functional protein (60-80 yo; total receptor protein assayed by competitive ELISA; functional fraction by ligand-binding [30]), even at very high expression levels of total receptor [(3WO) x lo6 copies/cell]. Functional receptor is properly modified (Nglycosylation at two sites ; thiopalmitoylation at two sites), while non-functional protein is incorrectly folded and/or not glycosylated [31]. Only at the occasional unusually high expression levels exceeding 40 x 10' copies/cell do relative functional levels begin to drop, suggesting that at those levels the translocation, folding and/or modification machinery of the cell becomes overloaded [30,31]. Stable-isotope labelling requires serum-free culture conditions. We screened several commercially available media in suspension culture (100ml spinner bottles) as to cell growth, maximal cell density and recombinant rhodopsin production. Best results were obtained with InsectXPress, a protein-free medium from BioWhittaker, Walkersville, M D , U.S.A. (doubling time x 22 h ; cell densities u p to 10 x 106/ml; 2&25 pmol of functional rhodopsin per mg of cellular protein ; x 15 x 10' copies/cell) that actually performed better in suspension culture than our serumsupplemented T N M F H medium [30] ( % 18 h ; up to 5 x 10'/ml; 15-20 pmol/mg of protein). All protein-free insect-cell media still require some yeast- or meat-derived supplement to sustain cell

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are rate-determining under these overexpression conditions, and that enhancing the endoplasmic reticulum capacity of the insect cell for protein folding and quality control may give significant improvement [35]. Indeed, we usually find higher levels of functional protein in bioreactor culture, where conditions are carefully monitored and cellular vitality is optimally preserved, as compared with spinner culture (Table 1).

ures for the same receptor [34]. We have tested several receptors under our optimized expression conditions. We found comparable total recombinant protein expression levels [(10-20) x 106 copies/cell], but again a large variety in functionality (10-80 yo),indicating that specific protein parameters determine this variability (Table 1). Receptors showing lower levels of functional expression tend to be retained in intracellular compartments, probably the endoplasmic reticulum in particular. This suggests that protein modification and folding in the endoplasmic reticulum

Purification and return to the roots A crucial element in receptor purification is the

Figure I Effect of MOI on production of functional recombinant rhodopsin in the baculovirus expression system Cultures were infected in their mid-loganthmic growth phase with a recombinant baculovirus expressing His-tagged rhodopsin [41] using the MOls indicated The volumetnc yield of rhodopsin (nmol/ltre of culture) was determined up to 6 days p i Results are given for representative expenments in I00 ml spinner (left panel) and I 0-litre bioreactor (nght panel) cultures Note the signifcantly earlier onset and higher level for MOI = 0 0 I in the bioreactor This probably reflects the supenor growth conditions of a carefully controlled environment

Id

100 mi spinner culture

-

1 1: 20-

07

a Days post lnfectlon

Days post infection

Table I Baculovirus expression of GPCR classes PM. plasma membrane; ER, endoplasmic reticulum.

Functional fraction of expressed protein (%)* Receptor

Spinner

Bioreactor

Rhodopsin Green cone pigment Red cone pigment Histamine H2

60-80 30-60 2MO

70 3 60

10-20

Not determined Not determined

*

lntracellular localization+

Detergent solubilizationf

Reference

Mainly PM PM+ER Mainly ER Mainly ER

CHAPS or DoM CHAPS+glycerol+lipids CHAPS+glycerol+lipids DoM+salts+antagonist

[30,331

Estimated by ELISA or immunoblot (total recombinant protein) and ligand binding (functional part). Determined by immunocytochemistry. f Optimal conditions for solubilization and stabilization.

t

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[48] [48] [43]

Expression and Purification of Membrane Proteins

His-tag has high affinity for transition metals like Zn2+, Ni2+ and Cu2+ and allows purification by immobilized-metal affinity chromatography (IMAC) [40]. Suitable IMAC matrices are commercially available and exhibit a high proteinbinding capacity (up to 70 nmol/ml of gel [33]). T h e selectivity of this approach is somewhat lower, since proteins with properly placed His residues as well as haem proteins exhibit medium affinity for such matrices. These can, however, usually be removed by proper washing procedures [33,41]. Elution is usually accomplished with high concentrations of imidazole (200-500 mM), but we prefer to use histidine that can be used at much lower concentrations (50-1 00 mM), which is better tolerated by solubilized receptors [36]. Depending on the stability of the receptor the purity of the eluted material varies between 40 and 90 yo. More detergent-resistant proteins like rhodopsin tolerate more extensive washing routines when bound to the matrix and purity levels of 80-90 yo can be easily achieved [33,41]. For quite unstable receptors like the cone visual pigments we restrict column residence time by minimizing washing procedures. This usually results in purity levels between 40 and 80 yo [36]. When evaluating solubilization conditions it is important to include binding tests to the IMAC matrix because we have observed quite receptorspecific phenomena in this respect. For instance, while CHAPS-solubilized His-tagged rhodopsin shows very high affinity for such a matrix, CHAPS-solubilized cone pigments have a very low affinity and DoM or a combination of CHAPS and DoM has to be used [33,36]. We surmise that size of the micelle, charge distribution and lipid affinity of the receptor exercise special effects. Indeed, excess lipid can seriously interfere with binding to the IMAC matrix. If lipids are required for stabilization of the receptor, they should be included after it has bound to the matrix. I n the final step, the purified receptor is reconstituted with appropriate lipids into proteoliposomes to generate a membrane matrix that is as close to native membranes as possible. This not only provides the most stable environment, but also allows proper analysis of structure and conformational changes induced by ligand binding or G-protein interaction. Since available methods for exchange of detergents for lipids are not generic and usually do not handle large batches very well, we have developed a special procedure for this reconstitution step, based on detergent inclusion by cyclodextrins, that tolerates large batches and is

choice of detergent and solubilization conditions. Generally, solubilization into detergent micelles strongly destabilizes membrane proteins. Functionality of the receptor needs to be sufficiently preserved to survive purification and reconstitution. In our experience these conditions can be quite receptor-specific. In the literature too a large variety of solubilization conditions are recommended for different GPCRs [34]. Nevertheless, our preliminary results with several receptors suggest that initial attempts are best made with CHAPS or p-1-dodecylmaltoside (DoM) in combination with specific additives to improve solubilization and/or stabilization of the protein (Table 1). For instance, solubilization of the histamine receptor required a ‘salty ’ environment. Cone visual pigments could only be sufficiently stabilized to survive purification by including glycerol as well as lipids [36]. Inclusion of a suitable ligand (antagonist or reverse agonist) can also make an important contribution to receptor stability. This is already obvious from the longer half-life of rhodopsin relative to the apoprotein opsin, by several orders of magnitude, in every detergent examined [37]. In view of this stability issue, a rapid, preferentially single-step, purification procedure should be selected involving column matrices offering a high binding capacity and high flow rates. This requires a highly selective interaction principle and in fact restricts the options to affinity procedures. For example, the use of immobilized ligand with eventual elution with (another) free ligand is very selective. A disadvantage is, however, that such affinity matrices almost invariably have to be specially designed, prepared and tested, therefore they are quite expensive and usually only suitable for one specific receptor isotype. Another very selective option is epitope tagging, e.g. extending the receptor with a peptide sequence containing the epitope of a suitable antibody. A quite popular option is the C-terminal octapeptide of rhodopsin, for which a monoclonal antibody has become freely available (1D4 [38]). This approach allows excellent purification in a single step [25,39]. T h e affinity matrix has to be prepared, however, and may vary in performance, and the free epitope-peptide is required for elution. Such elements also render epitope tagging rather expensive and less attractive for purification of large batches of receptor. For large-scale purification of receptors we prefer histidine tagging, i.e. extending the receptor with a sequence of 6-10 histidine residues. T h e

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compatible with any detergent type [42]. For reconstitution a suitable lipid mixture is used. For visual pigments we add a lipid extract from bovine retina [30]. Commercially available brain, egg or soy lipid extracts are often employed for other GPCRs. Detergents are selectively extracted from the mixed detergent/lipid/receptor micelles by addition of a suitable cyclodextrin [42], which evokes spontaneous formation of vesicles with receptor incorporated into the membrane. These proteoliposomes are separated from soluble components and non-incorporated protein by centrifugation through a sucrose gradient. This procedure affords another 2- to 3-fold purification, yielding receptor preparations with a purity always exceeding 80 yo,usually over 95 Yo, as long as the IMAC-purified receptor has a purity greater than 30 % [36,41,42].

been characterized extensively with respect to photochemical behaviour [30,36,41,42], to structural transitions accompanying receptor activation [18,33] and to G-protein activation [33,36] and have been shown to behave identically to their native counterparts. This demonstrates that the C-terminal His-tag does not interfere in any way with the structural and functional properties of these receptors. A similar conclusion has been drawn for other GPCRs [43-46]. The combination of large-scale production and stable-isotope labelling of recombinant rhodopsin has recently afforded the first F T I R [18] and solid-state NMR [28,32] analyses of a labelled eukaryotic membrane protein. The NMR data provide the first highly precise structural details for the protein-ligand interaction in the binding site. The FTIR data provide the first evidence for the involvement of tyrosine residues in the active state of the receptor. We recently also recorded FTIR difference spectra of labelled rhodopsin for the first step in the activation cascade (rhodopsin + batho). This clearly demonstrates that a tyro-

Functional properties and receptor mechanism The proteoliposome preparations of His-tagged visual pigments obtained as described above have

Analysis of the rhodopsin to batho transition by FTIR difference spectroscopy FTIR difference spectroscopy was performed on membrane films of unlabelled (bold, upper curve) or nng-*H,-Tyr-labelled (bottom curve) recombinant His-tagged rhodopsin, as descnbed [ 181. Note that both spectra are identical except for two bands at I252 and I244 cm-' (inset). In the labelled receptor the negative band at I252 cm-' has shifted t o lower frequency. thereby cancelling the positive I244 cm-' band. This identifies the I252 band as a tyrosine-nng vibration, and demonstrates that the interaction pattern of one tyrosine side chain is altered upon batho formation, the first step in receptor activation.

I

1700

1800

lmo

1

I

I

1400

1 m

1200

1100

Wavenumber (cm-1) 0 I999 Biochemical Society

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I

I

lo00

900

800

Expression and Purification of Membrane Proteins

sine residue already participates at this early stage in the activation mechanism (Figure 2). As this step only involves chromophore and binding-site residues, Tyr-268 is the only candidate. Such an early role for this residue agrees with site-specific mutagenesis studies showing that mutation of this residue seriously affects receptor function [47].

contrbution to the development of the next generation of medication. This research was supported by grants t o W. DeG. from the European Union (Bl02-CT93-0467), the Netherlands Organization for Scientific Research through its Council for Chemical Sciences (NWO-CW 328-050) and the Space Research Organization of the Netherlands (NWO-SRON MG-031). W e acknowledge technical contributions andlor fruitful discussions with Jacques Janssen, Just Vlak Lieveke DeCaluwe, Frank DeLange, Jenny VanOostrum, Ken Rothschild, Rob Leun, Herman Swarts, Johan Lugtenburg and Huub DeGroot.

Conclusion Cost-effective drug design requires detailed information on ligand-receptor interaction, preferably at atomic resolution. T o acquire such a high-resolution insight into the structure and mechanism of GPCRs, hundreds of milligrams of purified receptor are required. These amounts will not only allow extensive crystallization attempts to produce high-resolution 3D crystals, but they will also allow access to alternative powerful biophysical techniques like F T I R and solid-state NMR spectroscopy that are able to extract highresolution data from supramolecular systems. T h e large-scale expression and purification protocol that we have developed for the bovine visual pigment rhodopsin is able to generate the required amounts of purified protein. Recombinant baculovirus-based expression of 30-40 mg of functional receptor in a single 1 0-litre bioreactor batch of Sf9 cells has been reproducibly achieved and there is no limitation to further scale-up to 50or 100-litre batches. T h e single-step His-taggingbased purification procedure and rapid cyclodextrin-based reconstitution approach tolerate batches containing at least 10 mg of receptor and generate membrane-bound receptor of high purity ( 95%) and with a very satisfactory overall recovery (40-70 %). First results with other GPCRs indicate that, with some receptor-specific modifications in the solubilization step, this protocol can serve our purpose to develop a generic procedure for functional overexpression and efficient purification of these complex membrane proteins on a large scale. This development will finally pave the way to structural and mechanistic studies on a variety of GPCRs. Such structural knowledge will be of major scientific and medical importance. I t will expose the generic basis of the mechanism of receptor activation in the superfamily of GPCR proteins, as well as the individual variations in this common theme for ligand binding and G-protein activation, necessary to acquire subclass and isoform specificity. This will generate novel concepts in the field of drug design and make an important

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23 Mollaaghababa, R, Davidson, F. F., Kaiser, C. and Khorana, H. G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, I 1482-1 1486 24 Abdulaev, N. G., Popp, M. P., Smith, W. C. and Ridge, K. D. ( 1997) Protein Expression Purif 10, 6 1-69 25 Oprian, D. D., Molday, R S., Kaufman, R J. and Khorana, H. G. ( I 987) Proc. Natl. Acad. Sci. U. S. A. 84,8874-8878 26 Nathans, 1.. Weitz, C. J,, Agarwal, N., Nir, I. and Papermaster, D. S. ( I 989) Vision Res. 29, 907-9 I 4 27 Reeves, P. J., Thumond, R L and Khorana. H. G. ( I 996) Proc. Natl. Acad. Sci. U.S.A. 93, I 1487-1 1492 28 Eilers, M., Reeves, P. J., Ying, W. W., Khorana, H. G. and Smith, S. 0. ( I 999) Proc. Natl. Acad. Sci. U.S.A. 96, 487492 29 Janssen,J. J. M., VanDeVen. W. J. M., VanGroningen-Luyben, W. A. H. M., Roosien, J., Vlak, J. M. and DeGrip, W. J. ( I 988) Mol. Biol. Rep. 13, 65-7 I 30 DeCaluwe, G. L J., VanOostrum,J., Janssen,J.J.M. and DeGrip, W. J. (I 993) Methods Neurosci. IS, 307-32 I 31 DeCaluwe, G. L. J. and DeGrip, W. J. ( I 996) Biochern. J. 320,807-8 I5 32 Creemers, A. F. L., Klaassen, C. H. W., Bovee-Geurts, P. H. M., Kelle, R, Kragl, U., Raap, J., DeGrip, W. J., Lugtenburg, J. and DeGroot, H. J. M. ( I 999) Biochemistry 38,7 195-7 I99 33 Klaassen, C. H. W.. Bovee-Geurts, P. H. M., DeCaluwe. G. L. J. and DeGrip, W. J. ( I 999) Biochern. J. 342, 293-300 34 Grissharnmer, R and Tate, C. G. ( I 995) Q. Rev. Biophys. 28,3 I 5 4 2 2 35 Lenhard, T. and Reilander, H. ( I 997) Biochem. Biophys. Res. Comrnun. 238, 823-830

36 Vissers, P. M. A. M., Bovee-Geurts, P. H. M., Pottier, M. D., Klaassen, C. H. W. and DeGrip, W. J. ( I 998) Biochem. J. 330, 1201-1208 37 DeGrip, W. J. ( I 982) Methods Enzymol. 81, 256-265 38 Molday, R S. (I 989) Prog. Retinal Res. 8, 173-209 39 Oprian, D.D., Asenjo, A. B.. Lee, N. and Pelletier, S. L. ( I99 I) Biochemistry 30, I 1367- I I 372 40 Schrnitt, J., Hess, H. and Stunnenberg, H. G. ( I 993) Mol. Biol. Rep. 18, 223-230 41 Janssen,J. J. M., Bovee-Geurts, P. H. M., Merkx, M. and DeGrip, W. J. ( 1995) J. Biol. Chern. 270, I 1222- I I229 42 DeGrip, W. J., VanOostrum, J. and Bovee-Geurts, P. H. M. ( I 998) Biochem. J. 330,667-674 43 Beukers. M. W., Klaassen, C. H. W., DeGrip, W. J., Vemijl, D., Timrnerman, H. and Leurs, R ( I 997) Br. J. Pharmacol. 122, 867-874 44 Gimpl, G., Anders, J., Thiele, C. and Fahrenholz, F. (I 996) Eur. J. Biochem. 237,768-777 45 Hayashi, M. K. and Haga, T. ( I 996) J. Biochem. (Tokyo) 120, 1232-1238 46 Nekrasova, E. N., Sosinskaya, A,, Natochin, M. Y., Lancet, D. and Gat, U. (I 996) Eur. J. Biochern. 238,28-37 47 Nakayarna, T. A. and Khorana, H. G. (I 99 I) J. Biol. Chern. 266,42694275 48 Vissers, P. M. A. M. and DeGrip. W. J. ( I 996) FEBS Lett. 396,26-30

Received 2 I June I999

Heterologous expression of mammalian and insect neuronal nicotinic acetylcholine receptors in cultured cell lines Neil S. Millar Department of Pharmacology, University College London, Gower Street, London WC I E 6BT, U.K.

into Xenopus oocytes [5] and, subsequently, by stable transfection of cultured cell lines [ S ] ) . Since the molecular cloning of the Torpedo electric organ nAChR, a family of nAChR subunits have been identified in both vertebrate and invertebrate species. The best-characterized mammalian nicotinic receptor is the ' muscle-type ' nAChR, expressed at the neuromuscular junction, which has a subunit composition of either a141 yd or a14186 (two copies of the ligand-binding a1 subunits, co-assembled with three other ' structural' subunits). In addition to the muscle nAChR, a family of pharmacologically distinct nAChRs are expressed in the mammalian nervous system (' neuronal' nAChRs). The precise subunit composition of the various subtypes of neuronal nAChR is still being established, but strong evidence indicates that they are also pentamers, like the muscle and electric organ nAChRs. T o date, eleven vertebrate neuronal nAChR subunits have been identified (a2-a9 and /?2-/?4). In insects

Introduction Nicotinic acetylcholine receptors (nAChRs) are members of an extensive family of neurotransmitter-gated ion channels which also includes receptors for y-aminobutyric acid (GABA), glycine, glutamate and 5-hydroxytryptamine (serotonin). For many years nAChRs have been the best-characterized member of this family, largely as a consequence of there being an abundant and readily available source of nAChR (the electric organs of fish such as the marine ray, Torpedo, and the freshwater eel, Electrophorus). Indeed, the Torpedo electric organ nAChR was the first neurotransmitter receptor to be cloned (independently by four research groups in 1982 [l-41). It was also the first cloned neurotransmitter receptor to be expressed functionally in a heterologous expression system (in 1984, by micro-injection of cRNA Abbreviations used: nAChR nicotinic acetylcholine receptor; GABA, y-aminobutyric acid.

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