(Received for publication, November 23, 1994)
From the
A recombinant baculovirus containing a mouse
5hydroxytryptamine (5-HT
) receptor subunit cDNA
under the control of the polyhedrin promoter was shown to direct the
production of large amounts of functional 5-HT
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
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.
The elucidation of the three-dimensional structure of
transmitter-gated ion channels, a superfamily of receptors that
includes the nicotinic acetylcholine (nACh), ()glycine,
-aminobutyric acid type A, and 5-hydroxytryptamine
(5-HT
) 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
(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 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 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
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,(
)). 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 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) .
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.
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-HTR 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.
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-HTR
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
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
[
H]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 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
receptor virus (panelA, rightside, and panelB). Cells
were prepared and labeled with the anti 5-HT
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 receptor affinity purification. Samples were separated by
SDS-polyacrylamide gel electrophoresis and transferred onto a
nitrocellulose membrane. Anti 5-HT
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.
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.
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)
for the ligand [
H]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
) 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 [
H]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).
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 [H]granisetron
on either membrane fractions of the baculovirus-infected insect cells (panelA) or the purified recombinant 5-HT
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.
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'').
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
subunit 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
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.
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. ()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 receptor
subunits and possibly also for the other subunits from the superfamily.
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 receptors are indistinguishable from native 5-HT
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. (
)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.
The
recombinant 5-HT 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.