©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Recombinant Homo-oligomeric 5-Hydroxytryptamine Receptors Provides New Insights into Their Maturation and Structure (*)

(Received for publication, November 23, 1994)

Tim Green (1)(§) Kathrin A. Stauffer (2)(¶) Sarah C. R. Lummis (2)(**)

From the  (1)Centre for Protein Engineering and the (2)Laboratory of Molecular Biology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A recombinant baculovirus containing a mouse 5hydroxytryptamine(3) (5-HT(3)) receptor subunit cDNA under the control of the polyhedrin promoter was shown to direct the production of large amounts of functional 5-HT(3) receptor in insect cells, as assayed by Western blotting and ligand binding. After solubilization, the receptor was purified to homogeneity by affinity chromatography and characterized pharmacologically. The ligand binding characteristics of the recombinant receptor were essentially identical to those of the native receptor, both before and after purification. Only fully glycosylated receptors bound to the ligand affinity resin, although subsequent removal of the sugar did not affect ligand binding. Visualization of the purified receptor using electron microscopy showed that the receptor preparation contained a homogeneous population of pentameric doughnut-shaped particles. The general appearance of the recombinant homooligomeric channels was indistinguishable from that of native 5-HT(3) receptors. Yields of purified receptor were of the order of 200 µg/3 liters of original culture. The amount and homogeneity of the purified receptor are sufficient to begin preliminary crystallization trials.


INTRODUCTION

The elucidation of the three-dimensional structure of transmitter-gated ion channels, a superfamily of receptors that includes the nicotinic acetylcholine (nACh), (^1)glycine, -aminobutyric acid type A, and 5-hydroxytryptamine(3) (5-HT(3)) receptors, is a major goal in molecular neurobiology. The best characterized receptor of this type is the nACh receptor from Torpedo electric rays, which was shown as early as 1980 to be formed from five homologous subunits with the stoichiometry alpha(2)beta(1) . These subunits are arranged in the lipid bilayer as a pseudo pentamer, with an aqueous pore formed in the center of the complex(2) . This general arrangement (five subunits around a central pore) has now been confirmed, either directly or indirectly, for most other members of the channel family(3, 4, 5) . This accords with their similar amino acid sequences, which additionally suggest that the various receptors will share common elements of their tertiary structure.

The Torpedo nACh receptor three-dimensional structure is known to a resolution of 9 Å (6) , but it is likely that a structure at atomic resolution will have to await three-dimensional crystals suitable for x-ray diffraction. Despite the relative ease of purifying large amounts of the Torpedo receptor, no such crystals have been obtained. This could simply be due to a structural property of this particular receptor (such species-specific effects on crystallization are often seen with soluble proteins). Alternatively, the pseudo rather than true symmetry of a receptor with four different subunit types per molecule may make crystallization in the third dimension difficult. A channel composed of a single subunit type rather than several might therefore have a better chance of crystallizing, in addition to making overexpression more straightforward.

Several of the cloned subunits from the neurotransmitter-gated ion channel family form functional channels when expressed alone in heterologous systems(7, 8, 9, 10) . There is as yet no evidence that any of these homo-oligomers are present in vivo, and for certain subunits this is clearly not the case (for example, the glycine alpha subunit) (see (11) ). Such subunits have, however, proved useful in experiments designed to elucidate receptor structure and mechanism by mutagenesis (e.g.(12) and (13) ). The various subunits comprising native receptors are so similar in terms of sequence that structurally they are expected to be effectively interchangeable. Although this is obviously a simplification (otherwise all subunits would readily form homo-oligomeric channels), single subunits forming functional receptors must represent some sort of consensus structure. The determination of the three-dimensional structure of such a receptor should therefore provide a valid model both for the receptor concerned and for the other members of the superfamily.

In this study, we have overexpressed a functional, homo-oligomeric 5-HT(3) receptor. This receptor, one of a number for the neurotransmitter serotonin (the others couple to G-proteins), gates a cation-selective ion channel(14) . Small quantities of the 5-HT(3) receptor have been purified from cell lines by various groups(15, 16, 17) , and their analyses of the purified protein seem to indicate that the native receptor is a hetero-oligomer. Cloning, however, has led to the isolation of only one subunit cDNA, encoding a mature polypeptide of 53.6 kDa (9) and several slight variants (e.g.(18) ). All of these, when expressed on their own in mammalian cells or Xenopus oocytes, form functional channels that display many, if not all, of the characteristics of the native receptor (expression in oocytes, (9) ; expression in HEK cells,(^2)). If and until further subunits are cloned, the possibility remains that there is only one subunit and that the results from purification studies have been misleading. This study does not shed any further light on this, but any eventual structure would inevitably be more relevant if it corresponded to a truly native receptor.

We have used Spodoptera frugiperda Sf9 cells, infected with recombinant Autographa californica baculovirus, to express the mouse 5-HT(3) receptor subunit with the eventual aim of crystallization for structural study. The baculovirus system has been successfully used both for the expression of neurotransmitter-gated ion channels (19, 20, 21, 22, 23) and for the expression of other membrane channels and pores(24, 25) . The high cost of scale-up with insect cell culture means that expression levels in the milligram per liter range are required for the isolation of the quantities of pure protein required for structural studies. As some of the overexpression studies show, this is a realistic goal(20, 24, 25) .


EXPERIMENTAL PROCEDURES

Materials

TNM-FH media was purchased in powdered form from Sigma. [^3H]granisetron (80 Ci/mmol) was obtained from DuPont NEN. GR65630 and GR119566X were synthesized by the Chemistry Research Department (Glaxo Group Research). Quipazine dimaleate and m-chloro-phenylbiguanide were obtained from Cookson Chemicals, and MDL 72222 was from Research Biochemicals Inc. All other compounds were of the highest grade available.

Isolation of 5-HTR Recombinant Baculovirus

All baculoviral and insect cell manipulations were performed essentially as described by Summers and Smith(33) . The 5-HT(3)R cDNA open reading frame, contained in the vector pCDM8-5HT(3)aS, was removed as a BamHI-EcoRI fragment. This was inserted directly into the baculoviral transfer vector pVL1393 (Invitrogen), cut with the same enzymes, to form the vector pT147. Recombinant virus was obtained by homologous recombination in vivo, using 5 µg of pT147 and 1 µg of linearized wild-type A. californica nuclear polyhedrosis virus DNA (Invitrogen).

Cell Culture and Infection

Cells (Sf9) were grown in TNM-FH media supplemented with 10% fetal calf serum. Spinner flasks were used for cultures on the scale 50-500 ml, but for protein production culture was performed in a 50-liter fermentor (SGI). This allowed growth at a 32-liter scale, with control of pH, oxygen levels, and temperature. Cells were infected with virus at a multiplicity of infection of 5-10 and then harvested after 50 h by continuous centrifugation. Cells were stored at -70 °C.

Western Blots, Confocal Imaging, and Deglycosylation

Immunolabeling experiments were performed using polyclonal antisera (a gift from Dr. R. M. McKernan), raised against the M3-M4 loop of the murine 5-HT(3) receptor expressed in Escherichia coli(26) . Western blots were performed essentially following the method of Towbin et al.(27) . For the fluorescence-labeled confocal imaging, Sf9 cells were grown directly on 22-mm glass coverslips in 6-well dishes. 48 h after infection (multiplicity of infection, 5-10) with either the control or the 5-HT(3)R virus, the cells were washed with PBS and fixed for 5 min with glutaraldehyde (0.5%). After three further washes with PBS, the cells were incubated with the primary antisera (1:1000 dilution in PBS, 0.1% Triton X-100, overnight, 4 °C), biotinylated secondary antibody (1:200 dilution in PBS, 3 h, 22 °C), and fluorescein isothiocyanate-avidin (1 h, 22 °C). The final wash was with saline-free phosphate buffer. Images were obtained on an MRC-600 confocal microscope.

Enzymatic deglycosylation was carried out using endoglycosidase H from Boehringer Mannheim. The reaction was carried out with 5 µl of sample in a final volume of 20 µl in 10 mM MES, pH 5.5. Enzyme (1 milliunit) was added to 1 aliquot, and another was left as the control. The reaction mixture was incubated at 24 °C for 3 h. Western blots and binding assays were carried out on each sample.

Radioligand Binding Assays

For radioligand binding experiments with either membranes or solubilized receptor, the samples were incubated in HEPES buffer (10 mM, pH 7.4) containing 0.1 nM [^3H]granisetron in a final volume of 0.5 ml for 1 h at 0 °C. Nonspecific binding was defined as that not displaced by 100 nM GR65630. For competition studies, 0.5 nM [^3H]granisetron was used; concentrations ranged from 0.01 to 18 nM in the saturation experiments. Bound and unbound radioligand were separated by vacuum filtration using a Brandel cell harvester onto GF/B filters (presoaked for 1 h in 0.1% polyethyleneimine), followed by 2 times 2-ml washes in ice-cold HEPES buffer. All assays were performed in triplicate. Experimental data were analyzed using the programs EBDA, LIGAND, and KINETIC(28, 29) .

Receptor Purification

All buffers contained leupeptin (5 µg/ml), pepstatin A (2.5 µg/ml), and phenylmethylsulfonyl fluoride (1 mM), and all operations were carried out at 4 °C unless otherwise stated. The cell pellet from 3 liters of the fermentor culture was resuspended in 40 ml of 10 mM HEPES, pH 7.0, 1 mM EDTA, and lysed by 20 strokes in a hand-held Potter homogenizer. The sample was centrifuged at 1000 times g for 15 min. This procedure was repeated three times with the pellet from the low-speed spin. The supernatants were pooled and re-centrifuged at 130,000 times g for 45 min to pellet the membranes. These were resuspended in a total volume of 15 ml using the HEPES buffer. For solubilization, the membranes were added to 10 volumes of the solubilization buffer (0.1% (w/v) Triton X-100, 10% (v/v) glycerol, 10 mM bis-Tris propane, pH 9.0), and incubated in this buffer for 1 h on ice with stirring. The sample was then centrifuged at 130,000 times g for 1 h to remove insoluble material.

After centrifugation, the Triton and NaCl concentrations and the pH level were adjusted to 0.5%, 300 mM, and 7.5, respectively. The supernatant was loaded at room temperature onto a column containing GR119566X-linked Affi-Gel 15 resin(16) , pre-equilibrated with 10 volumes of solubilization buffer adjusted as described. After application, the resin was washed with 30 bed volumes of medium salt buffer (0.25% Triton, 300 mM NaCl, 10 mM HEPES, pH 7.5), followed by 10 bed volumes of low salt buffer (as medium salt, containing 150 mM NaCl). Finally, the resin was washed with 10 bed volumes of low salt buffer containing 0.6% CHAPS. The receptor was eluted from the resin using 10 bed volumes of the medium salt buffer containing 0.6% CHAPS and 0.1 mM quipazine.

The eluted sample was concentrated to 0.5 ml using a 50-ml Amicon stirred cell and Microsep 100K concentrators (Filtron) and loaded onto a 10/30 Superose 6 column (Pharmacia Biotech Inc.), pre-equilibrated with 0.6% CHAPS, 500 mM NaCl, 1 mM EDTA, 10 mM HEPES, pH 7.5 (no protease inhibitors). Fractions containing pure, oligomeric 5-HT(3)R were pooled and further concentrated using Microsep 100K concentrators. Where detergent exchange from CHAPS was required, this was carried out on the Superose 6 column.

Electron Microscopy

Samples were adsorbed onto freshly glowdischarged, carbon-coated copper grids, washed in 100 mM cacodylate, pH 6.8, to remove detergent, and stained with 2% aqueous uranyl acetate. Micrographs were taken at nominal magnifications of 45,000-49,000times on Phillips 420 or CM12 microscopes using low dose units and Kodak SO163 film.


RESULTS

5-HT Receptor Subunit Expression in Sf9 Cells

A cDNA clone encoding a subunit of the murine 5-HT(3) receptor was obtained from Dr E. Kawashima (Glaxo, Geneva). After introduction of the 5-HT(3) receptor subunit cDNA into the A. californica nuclear polyhedrosis virus genome by double homologous recombination, a single round of plaque purification was sufficient to obtain three occlusion-negative viral clones, all of which proved to be recombinant. Expression levels in the infected insect cells were defined using radioligand binding assays and Western blots. Initial Western blots indicated that all three viruses caused the production of 5-HT(3) receptor subunit polypeptides. Two of the three isolated viruses caused the appearance of specific binding sites for a radiolabeled 5-HT(3) receptor antagonist in the infected cells. Binding was originally assayed on whole cells. It was assumed that the ligand was sufficiently lipophilic to enter the cells, as addition of brefeldin A (25 µg/ml) to the culture medium did not affect the observed ligand binding (brefeldin A blocks outward transport from the endoplasmic reticulum). One of these two clones was selected for further work. Scatchard plots indicated expression levels of 0.4-2 mg of protein/liter of culture on a small scale (50-500 ml), with approximately half this for cells grown in a fermentor.

Immunofluorescence labeling studies were carried out on infected insect cells that had been fixed and permeabilized to determine the cellular distribution of the receptors. Labeling was to a cytoplasmic epitope using fluorescein-labeled anti 5-HT(3)R subunit antisera raised against the region M3-M4(26) . Confocal imaging of these cells revealed strong fluorescent labeling (Fig. 1), with a low background in the control cells (infected with a virus expressing connexin-32). Western blots of the insect cell membranes using the anti-5-HT(3)R antisera showed multiple bands (see Fig. 2, lane1), with three major species running at molecular masses of approximately 43, 49, and 56 kDa. Numerous other bands were visible both above and below these, the higher ones presumably due to incomplete dissociation of subunits in the SDS. The lower bands can be attributed either to proteolysis or incomplete translation products. Control Western blots showed no cross-reactivity of the antisera with insect proteins. The receptor could be solubilized using various detergents. Irrespective of the solubilization conditions used, the pattern of polypeptides seen in Western blots remained the same (Fig. 2, lanes1 and 2). Ligand binding assays using [^3H]granisetron were used to estimate the efficiency of different solubilization procedures, with the efficacy of a variety of buffers, pHs, salts, and detergents being examined before optimal conditions were determined. Solubilization in 0.1% Triton X-100, 10 mM bis-Tris propane, 10% glycerol, pH 9.0, proved most effective, typically solubilizing over 70% of the binding sites present.


Figure 1: Immunofluorescence detection of 5-HT(3) receptors in baculovirus-infected Sf9 insect cells. The cells were infected with either a control virus (producing connexin-32) (panelA, leftside) or the 5-HT(3) receptor virus (panelA, rightside, and panelB). Cells were prepared and labeled with the anti 5-HT(3) antisera as described under ``Experimental Procedures.'' Both images in A are shown at the same magnification and image gain. Scalebar in panelA, 40 µm; scalebar in panelB, 20 µm.




Figure 2: Western blot of samples from 5-HT(3) receptor affinity purification. Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Anti 5-HT(3)R antisera (1:1000 dilution) was used in the primary incubation, and biotinylated anti-rabbit IgG was used in the secondary incubation. Horseradish peroxidase coupled to avidin was added as the tertiary label. Samples loaded were as follows: lane1, membrane fraction from baculovirus-infected insect cells; lane2, protein solubilized from membranes in 0.1% Triton; lane3, flow-through from affinity column after loading solubilized sample; lane4, combined washes from affinity resin; lane5, eluent from column. Only the samples in lanes1 and 2 were from equivalent volumes. Molecular size markers were stained with Ponceau S, and their positions are shown on the left.



Receptor Purification to Homogeneity

The solubilized receptor preparation was affinity purified using a 5-HT(3) receptor antagonist (GR119566X) coupled to Affi-Gel 15. The Western blot of the eluted fraction revealed a single band of 56 kDa (Fig. 2, lane5). Ligand binding assays conducted on the sample applied to the resin and on the column flow-through indicated that >90% of the active receptors were binding to the resin. Surprisingly, the flow-through from the column (lane3) appeared to be largely unchanged, with little depletion of the 56-kDa polypeptide apparent. Passage of the solubilized sample through the column reduced the intensity of the 56-kDa band by less than 20% (estimated from densitometer scans relative to the 49-kDa band). Therefore, only a small fraction of the polypeptides visible in Western blots (and therefore also the confocal images) were active in terms of ligand binding. It also means that the absolute level of protein expression (including inactive receptors), is much higher than the estimates obtained from Scatchard plots. During affinity chromatography, the detergent was exchanged from 0.1% Triton to 0.6% CHAPS to enable concentration of the eluted sample without concomitant concentration of the detergent.

The receptor preparation obtained from the affinity column was further purified on a Superose 6 gel filtration column. In addition to removing the few contaminating protein species, this step served both to remove the eluting ligand (necessary to allow binding assays) and any aggregated receptor particles. The peak corresponding to single channel molecules was identified by electron microscopy visualization of individual fractions. Exchange into a detergent more suited than CHAPS for crystallization could also be done at this point. The sample from this step was concentrated to a final concentration of 0.2-1.0 mg/ml by ultrafiltration. The overall yield of purified receptor was 100-200 µg of protein from 3 liters of infected Sf9 cells.

Characterization of Purified Receptor Preparation

The purified receptor appeared as a single band in silver-stained gels and Western blots (Fig. 3, A and B), with a faint band at a higher molecular weight corresponding to the subunit dimer. The precise nature of this 56-kDa purified polypeptide in relation to the other major species present in the membranes was investigated further. To determine if the 56-kDa polypeptide was glycosylated, the purified receptor was incubated with endoglycosidase H. There was a shift in apparent molecular mass from 56 to 49 kDa in the labeled band on a Western blot (Fig. 3B). The size of the deglycosylated receptor corresponded to the molecular weight of the major immunoreactive polypeptide in the membranes (Fig. 2, lane1). Single point binding assays on the deglycosylated protein and controls revealed that, contrary to expectations, there was no loss of binding sites upon removal of sugar residues. In contrast, when N-linked glycosylation in the infected insect cells was blocked by the addition of 5 µg/ml tunicamycin to the culture medium, no ligand binding was observed in crude membrane preparations (data not shown).


Figure 3: SDS-polyacrylamide gel electrophoresis analyses of purified receptor and receptor following enzymatic deglycosylation. The receptor sample obtained from the Superose 6 column was separated by SDS-polyacrylamide gel electrophoresis and silver stained (panelA). Purified receptor was also incubated with endoglycosidase H (EndoH) as described under ``Experimental Procedures,'' and the resulting samples were analyzed by Western blot (panelB). Molecular size markers are shown on the left of each gel.



The pharmacology of the receptor complex was also investigated both in the membrane fraction and after the final purification step. The dissociation constants (K(d)) for the ligand [^3H]granisetron were 0.37 ± 0.07 nM (n = 5) for the membrane fraction and 0.57 ± 0.06 nM (n = 3) for the purified receptor. These were comparable with the value of 0.3 ± 0.06 nM reported for the receptor in rat cortical membranes(30) . The values for maximal binding (B(max)) were 10.9 ± 1.1 pmol/mg protein for the membranes and 4.2 ± 2.3 nmol/mg for the purified receptor. This gave an overall purification factor of approximately 380-fold. All of the Scatchard plots could be fitted using a single site model. Displacement analyses for [^3H]granisetron with various unlabeled agonists and antagonists (Fig. 4, A and B) gave the expected order of potency and apparent affinities compared with native membranes (Table 1).^2


Figure 4: Representative examples of radioligand displacement analyses of membrane-bound and purified receptor. Two antagonists (GR65630 and MDL72222) and two agonists (m-chloro-phenylbiguanide and 5-HT) were used at the concentrations shown. The competition experiments were performed in triplicate using 0.5 nM [^3H]granisetron on either membrane fractions of the baculovirus-infected insect cells (panelA) or the purified recombinant 5-HT(3) receptor (panelB). Apparent affinities (K) are given in Table 1. Binding is shown as specific counts normalized to 100% at maximal binding. For clarity in panelB, further data points at 100% are not shown.





Electron Microscopy

Visualization of the negatively stained solubilized receptor by electron microscopy (Fig. 5, A and B) was used to assay the quality of receptor preparations and determine the gross structural features of the channel. The views of the particles were related to what is known of the structure of the nACh receptor. Many of the particles (some being circled in Fig. 5A) appear as toroidal rings (doughnut shaped) with a stain-filled center. These rings have an apparent diameter of 70 Å. This view corresponds to the receptor seen from above or below the bilayer (molecules in negative stain being seen in projection). Other particles (boxed in Fig. 5A) can be seen as two parallel strands separated by stain with dimensions of 70 times 140 Å. These can be interpreted as side views of the receptor, with the pore being visible along the whole length of the molecule. Other particles represent tilted (or distorted) views between these two extreme orientations. Fig. 5B shows the purified receptor at a higher concentration (estimated at 0.2 mg/ml), where it again appears homogenous and shows little or no aggregation. Storage of the receptor at this protein concentration in 0.6% CHAPS at 4 °C for over 3 months resulted in no changes in the appearance of the isolated receptor particles or in the level of aggregation.


Figure 5: Electron microscopy visualization of uranyl acetate-stained soluble receptor preparations. Receptor preparations after the gel filtration are shown both before (panelA) and after (panelB) concentration. Examples of receptors viewed from above are circled (see text). Examples of receptors on their sides are boxed. Views of receptors from fields similar to that in panelA are shown enlarged to show the channel's 5-fold symmetry more clearly (panelC). Scalebar in panelsA and B, 40 nm.



Closer analysis of the particles seen from above should reveal the rotational symmetry of the receptors and therefore the number of subunits, as has been done for the native -aminobutyric acid, type A receptor(5) . Fig. 5C shows particles similar to those seen in Fig. 5A, magnified photographically. These appear clearly 5-fold symmetric, although given the limitations imposed by negative stain, confirmation of this would require analysis of a larger number of particles (see ``Discussion'').


DISCUSSION

The 5-HT Receptors Appear to Remain in Internal Membranes

5-HT(3) receptors are normally located in the plasma membrane of neurons. The location of the recombinant receptor in insect cells was investigated using confocal imaging of permeabilized cells (Fig. 1). When expressed in insect cells it appears that the majority of the receptors fail to reach the cell surface. The pattern of labeling seen in confocal imaging is most consistent with a location in internal (cytoplasmic) membranes. Labeling of intact cells was not possible because the antisera used was raised against a cytoplasmic epitope. The granular nature of the labeling suggests that the receptor might be primarily located in vesicles of some sort. The low proportion of active receptors relative to the total amount of receptor detected by antibody labeling makes it impossible to discount the possibility that there are some receptors on the surface.

Localization of recombinant channels in internal membranes has been previously observed with proteins expressed at high level using the baculovirus system. The gap junction protein connexin-32 is found in the endoplasmic reticulum membranes(25) , and only 0.1% of the expressed glycine receptor alphasubunit can be detected on the surface as functional channels(20) . The internal buildup of recombinant protein, both in these cases and with the 5-HT(3) receptor, can be attributed either to overloading of the transport machinery or to a cellular response to the potential toxicity of the recombinant proteins. In the latter case, it is interesting to speculate that the lack of success in expressing such membrane channel proteins in prokaryotic organisms such as E. coli may be due to their lack of alternative membranes in which to store them.

Glycosylation Is Central to Receptor Maturation

The isolation of a single protein band from the affinity resin was unexpected, given the number of immunoreactive bands in the crude extract. The receptor oligomers formed from such a heterogeneous mixture might be expected to be heterogeneous themselves. Even if the full-length glycosylated subunit is the only form with binding activity, an oligomer containing at least one such subunit might still be expected to attach to the resin. This obviously does not occur, as no shorter (unglycosylated) forms appear in the affinity-purified sample. Even after purification, there still appears to be significant amounts of a polypeptide corresponding to the full-length glycosylated subunit in the flow-through (Fig. 2, lane3), whereas 90% of the active receptor binds to the resin. This suggests that mixed receptors do occur but that they do not bind either to the affinity resin or to free ligand.

Deglycosylation of active receptors did not reduce their ability to bind ligand, demonstrating that sugars play no direct role in ligand binding. The problem is therefore one of why only the fully glycosylated oligomer binds ligand while partially glycosylated oligomers do not. Three possibilities present themselves. The first is that glycosylation is required to reach the final structure but is not necessary to maintain it. For example, the presence of sugar residues might alter the course of the folding pathway but not be necessary for the thermodynamic stability of the final structure. In this case, folding could also include oligomerization. The second possibility is that glycosylation is simply a marker indicating the completeness of other processing events required for activity. In other words, the crucial event is one that occurs only after the receptor has been completely glycosylated. If such an explanation is invoked, the change must be one that occurs in the endoplasmic reticulum, as brefeldin A has no detectable effect. A third possibility is that the partially glycosylated receptors are all incorrectly folded, but this is unlikely given the similar solubility of the various forms in detergents (see Fig. 2, lane2).

Support for the first explanation comes from analogous results reported for the epidermal growth factor receptor and the insulin receptor. For both of these proteins, it was reported that glycosylation was required to attain activity but not to maintain it(31) . This was attributed to a direct effect of glycosylation on the basis that tunicamycin blocked receptor activation in vivo. The basic cause of the activation was not investigated further. Tunicamycin added to the insect cell culture medium also prevented the formation of active receptors in our system. The simplest interpretation is again that glycosylation is affecting activation directly.

An indirect effect cannot be discounted, however. Evidence in support of such an indirect effect comes from experiments where the isolated insect cell membranes were incubated with canine pancreatic microsomes. There was a reproducible 17% increase in ligand binding seen in the treated membranes compared with controls. (^3)This microsome-mediated increase suggests the involvement of endoplasmic reticulum-linked enzyme systems other than those for N-linked glycosylation. This is because N-linked glycosylation is normally thought of as an entirely cotranslational event. Any post-translational increase in receptor activity must presumably therefore be caused by other enzymes present in the microsome preparation.

A more complete investigation of the possible explanations will be required before a definitive answer is known. Whether glycosylation turns out to have a direct or indirect affect on receptor activation, it is probable that processing pathways found only in eukaryotes are involved. One important consequence of this is that it precludes the use of E. coli expression systems for the expression of functional 5-HT(3) receptor subunits and possibly also for the other subunits from the superfamily.

Recombinant Homo-oligomeric Receptors Resemble Their Native Counterparts

The recombinant receptor, both in membranes and after purification, showed similar ligand binding characteristics to the native receptor (see Fig. 4and Table 1). One piece of information not accessible from studies of native membranes is the B(max), and thereby the stoichiometry, of the purified molecule. If our preparation is assumed to be completely homogenous, then a figure of 4.2 nmol/mg protein corresponds to a binding species of 238 kDa. This is very close to the figure of 280 kDa that would be expected if the receptor was pentameric and there was one binding site per molecule. It is tempting to speculate that this might explain why only fully glycosylated receptors were purified, with five correctly modified and assembled subunits required before binding is seen. It should be noted that the error in the B(max) (due primarily to the errors in protein estimation) is sufficient to allow for target sizes down to 90 kDa (at 3 s).

While the above results and other published studies of recombinant receptors have shown that these receptors behave in much the same way as native channels, there has been until now no direct evidence that they assemble into similar oligomers. The images of the homo-oligomeric 5-HT3 receptor presented here (Fig. 5) clearly show the channel to have the same basic quaternary structure as the well characterized Torpedo nACh receptor, with the subunits surrounding a central pore. In addition, these recombinant 5-HT(3) receptors are indistinguishable from native 5-HT(3) receptors purified from the neuroblastoma cell line NG108-15 and viewed by electron microscopy. These channels have been analyzed and shown to be 5-fold symmetric. (^4)This confirms that the recombinant protein assembles in the same way as the native channel. It is possible that small differences between the hetero- and homo-oligomeric forms might be seen at higher resolution. Any such differences should not affect the utility of recombinant homo-oligomers as structural models for the native channel.

Crystallization Trials as the Next Step

The eventual aim of this work is the crystallization and structure determination of the 5-HT(3) receptor. There are several main requirements before crystallization of any protein can reasonably be considered. First, there must be enough purified protein to produce a solution with a final protein concentration in the milligram per milliliter range. Second, the preparation must be homogeneous. Overall purity is often crucial for the success of crystallization experiments, but more subtle variations in the protein preparation can have an impact. For example, glycosylation can be a potential source of heterogeneity, and it is possible that the sugar residues will have to be removed before suitable crystals are obtained, as has been observed with lactoferrin(32) . The final consideration is one of stability. This is perhaps most important with an oligomeric integral membrane protein, where there are the dual problems of oligomer stability and nonspecific aggregation.

The recombinant 5-HT(3) receptor prepared here satisfies all of the above requirements. The purified receptor had an upper solubility limit of 1 mg/ml in the conditions described, and preparation of reasonable (200 µl) quantities of such a solution was possible from 3-liter preparations at the expression levels achieved. Working with homo-oligomeric rather than hetero-oligomeric channels removes one potential source of heterogeneity, but there are still others that will need considering, such as glycosylation. If it proves necessary to remove the sugar moieties, then enzymatic deglycosylation of the purified receptor will be the only option, as in vivo inhibitors of glycosylation prevent the formation of active receptor. As for stability, there was no visible deterioration in the sample over a period of months. The receptor preparation described here therefore demonstrates the main characteristics required for eventual crystallization.


FOOTNOTES

*
This work was supported in part by a Zeneca/Glaxo/DTi/MRC LINK grant (to T. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-1223-402163; Fax: 44-1223-402140.

Present address: Cambridge University Dept. of Pathology, Tennis Court Rd., Cambridge, United Kingdom.

**
A Royal Society University Research Fellow.

(^1)
The abbreviations used are: nACh, nicotinic acetylcholine; 5-HT(3), 5-hydroxytryptamine(3); 5-HT(3)R, 5-hydroxytryptamine(3) receptor; MES, 2-(N-morpholino)ethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline.

(^2)
S. C. R. Lummis, unpublished observations.

(^3)
T. Green, unpublished observations.

(^4)
R. Beroukhim, personal communication.


ACKNOWLEDGEMENTS

We thank Eric Kawashima (Glaxo, Geneva) for supplying the pCDM8-5HT3aS clone, Gavin Kilpatrick (Glaxo, Stevenage) for the ligand GR119566X, and Ruth McKernan (Merck, Sharp, and Dohme, Harlow) for providing the anti-5HT(3) antisera. We are also very grateful to Stephen Hunt for help with the confocal microscopy and Jill Chirnside for much invaluable advice on running the fermentor. Finally, we thank Chris Staples for keeping lab work going while the manuscript was written, Rameen, Reinhard, Jane, and Olga for useful comments on its content, and Nigel Unwin, whose suggestions throughout the project have been crucial to its success.


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