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Department of Physiology (D.A.R.), Cell and Molecular Biology Program (R.D.H.), and Department of Chemistry (H.M., B.G.B.), Colorado State University, Fort Collins, Colorado 80523
Address all correspondence and requests for reprints to: Dr. Deborah A. Roess, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523. E-mail: daroess{at}lamar.colostate.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonetheless, the question of whether liganded LH receptors are intimately self-associated, forming receptor dimers or oligomers after binding of hormone, has not been resolved. Such interactions have been suggested by electron microscopy studies of this receptor, which show grouping of hormone-conjugated ferritin molecules. However, the diameter of ferritin molecules used to image LH receptors is about 24 nm (1), approximately 3-fold greater than the diameter of the hormone itself (5). Thus, these studies fail to distinguish actual receptor oligomerization from simple concentration of receptors with small membrane microdomains. Similarly, light microscopy results showing fluorescent clusters containing LH receptors (2, 6) can arise either from receptor oligomers or from restriction of receptors to specific small domains.
Because there is evidence that at least dimeric structures may be
necessary for function of G protein-coupled receptors, such as the
-opioid receptor (7) and
ß2-adrenergic receptor (8), the
question of whether the functional LH receptor self-associates is of
interest. We have approached this question by first examining the
differences in the lateral dynamics of the wild-type rat LH receptor
and a nonfunctional LH receptor expressed in human embryonic kidney 293
cells. The nonfunctional receptor contains a single point mutation in
lysine 583 located in the third extracellular loop, a domain believed
to be involved in signal transduction (9, 10). In cells
stably expressing the mutant receptor, the cAMP response to human CG
(hCG) is either eliminated completely (9) or reduced by
over 75% (10). We have also examined receptor-receptor
interactions on individual cells, using fluorescence resonance energy
transfer techniques (11) to determine whether large,
slowly diffusing complexes contain self-associated LH receptors.
Fluorescence resonance energy transfer, whether via spectroscopic
methods or flow cytometric techniques (12), has proven
useful in detecting molecular associations in the plasma membrane.
Because the characteristic Förster distance
Ro for the fluorescein-rhodamine pair used in
these studies is approximately 56Å (13), energy transfer
between hormone-occupied LH receptors occurs under conditions where
receptors are within less than approximately 100Å of each another
(5).The results presented here suggest that functional,
but not nonfunctional, LH receptors are self-associated and present in
slowly diffusing complexes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Dr. Tae Ji, from the Department of Chemistry at the University
of Kentucky, kindly provided 293 cells stably transfected with the
wild-type LH receptor (LHR-wt) or with an LH receptor modified in
position 583 by substitution of lysine with arginine (LHR-K583R)
(9). Untransfected 293 cells were maintained in DMEM
containing 10% horse serum (Summit Biotechnology),
100 U penicillin, 1000 µg/ml streptomycin, and 10 mM
HEPES, pH 7.4. LHR-wt cells and LHR-K583R cells were maintained in the
same medium supplemented with 400 µg/ml geneticin.
Preparation of TrITC- and FITC-derivatized hormones
Hormones were derivatized with FITC or TrITC using a
modification of methods described by Brinkley et al.
(14) and described in detail elsewhere (11).
Briefly, hormones were dissolved in PBS (1.9 mM
NaH2PO4, 8.4
mM
Na2HPO4, 0.15
M NaCl, PBS) containing 50
mM sodium borate, pH 9.3. Protein concentrations
were determined spectrophotometrically at 280 nm. A 5-fold molar excess
of TrITC or FITC was added to the protein solutions, and the mixtures
were kept at 4 C for 18 h in the dark. After quenching with 1
M Tris, the fluorophore-derivatized hormones were
separated from the unreacted free dye on a Sephadex G-25 column. After
extraction of remaining free dye with n-butanol and extensive dialysis,
the molar ratios of dye to hormone were determined
spectrophotometrically. Hormone preparations used in these experiments
had 1.0–1.5 mol TrITC or FITC per mol oLH or hCG. It has been
previously shown that there is no effect of these fluorophore
conjugations on hormone activity (15). Before use, all
fluorophore-derivatized proteins were centrifuged at 130,000 x
g for 10 min in an Airfuge (Beckman Instruments, Inc., Palo Alto, CA) to remove any protein aggregates
formed during storage at 4 C.
Labeling cells with fluorophore-derivatized hormones
Before labeling with TrITC- or FITC-derivatized hormones, cells
were incubated in balanced salt solution containing 0.1%
NaN3, at 37 C for 30 min, to prevent hormone
internalization (15). Typically, 2–5 x
106 cells in 1 ml balanced salt solution were
labeled with 1 µM TrITC-derivatized oLH or hCG for each
fringe photobleaching recovery experiment. This hormone concentration
saturates available receptors and results in maximum cAMP
production by LHR-wt cells (data not shown). For fluorescence energy
transfer measurements, four groups, each containing 1 x
106 cells, were labeled and examined on a given
day. Thus, receptor number per cell was comparable for cells in each
group. One group of cells was not labeled. The remaining groups were
labeled with a total hormone concentration of 2.0 µM.
Cells labeled with fluorescent donor alone were incubated with
1.5 µM unlabeled hormone (either LH or hCG) and 0.5
µM of the same hormone was derivatized with FITC. Cells
labeled with fluorescence donor and acceptor were incubated with 1.5
µM TrITC- and 0.5 µM FITC-derivatized
hormone. Cells labeled with fluorescence acceptor were treated with 1.5
µM TrITC-derivatized hormone and 0.5 µM
unlabeled hormone. The 3:1 ratio of fluorescent acceptor to donor has
been shown previously to produce optimal signal (11).
After labeling for 1 h at 37 C, cells were then washed two times
by centrifugation at 300 x g for 3 min in balanced
salt solution to remove any unbound ligand. In some lateral diffusion
and fluorescence energy transfer experiments, cells were pretreated
with 40 µg/ml cytochalasin D for 30 min at 37 C before cell
labeling.
Fringe fluorescence photobleaching recovery measurements
The optical system for performing fringe fluorescence
photobleaching recovery measurements and the method used for data
analysis have been described previously (16). The
microscope objective used in these studies was a 40x objective
of NA 0.65 (Carl Zeiss, Inc., New York, NY). Cells were
examined under coverslip on well slides while temperature was
maintained by a thermoelectrically cooled/heated thermal stage with a
temperature range of 0–40 C. For fringe measurements, the region
illuminated at the sample has a 1/e2
radius of about 18 µm, and the photometer acceptance region is
large enough to encompass the entire cell. The fringe spacing used in
these experiments was 2.3 µm. Because of the large interrogated area,
1.3 W in the bleaching pulse and 3 mW in the probe beam were used.
Unadjusted raw data were represented directly in terms of the various
parameters associated with a given measurement, including the prebleach
and immediate postbleach fluorescence levels, the extent M of
fluorophore mobile on the timescale of the experiment, and a function
representing the recovery kinetics in terms of a decay half-time. These
parameters were evaluated directly by a nonlinear least-squares
procedure; and, from the measured time t1/2 at
which fluorescence recovery was half-complete and from the known
optical parameters, the desired diffusion coefficient D was
evaluated.
Single-cell fluorescence energy transfer
Fluorescence energy transfer between FITC- and TrITC-derivatized
LH or hCG was evaluated based on the reduced rate of irreversible
photobleaching of FITC fluorophores when TrITC fluorophores were
present (11). Slower rates of fluorescence decay for cells
labeled with the FITC fluorescence donor and TrITC fluorescence
acceptor, than for cells labeled with FITC only (D), were
indicative of energy transfer from fluorescence donor to acceptor and
occurred only when the donor and acceptor were separated by distances
less than Ro, a characteristic of the specific
donor/acceptor pair. For FITC and TrITC, this distance is 56Å
(13).
To perform these experiments, we used a fluorescence microscope photometer based on the inverted-configuration Carl Zeiss Axiomat microscope and associated components used for fringe fluorescence photobleaching recovery measurements at room temperature. Fluorescence excitation was provided by an Innova 100 argon ion laser (Coherent Inc., Santa Clara, CA) operating under light control at 488 nm. The intensity of the laser radiation focused on the cell was 15–20 mW, and this was held constant between measurements on cells labeled with FITC-derivatized LH or hCG only or on cells labeled with FITC- plus TrITC-derivatized hormone. The 1/e2 Gaussian spot diameter was 18 µm. Donor fluorescence from FITC was isolated with a standard fluorescein filter set together with a short-pass fluorescein-selective filter to remove red tetramethylrhodamine fluorescence. This combination was highly effective in rejecting TrITC fluorescence: TrITC-LH- or -hCG-labeled cells gave very low fluorescence signals using the fluorescein-selective filter set that were indistinguishable from those of unlabeled cells. Signals from cells labeled with either FITC-LH or hCG only or with FITC- and TrITC-LH or hCG were approximately 4-fold higher than background levels. In individual experiments, cells were identified and centered in the microscope field. At time zero, an electronically controlled shutter was opened to allow laser radiation to impinge on the cell. Simultaneously, a computer program was activated to record the output of the photomultiplier measuring membrane fluorescence. Data were collected at 0.01-sec intervals for 10 sec. Typically, about 20 cells in each sample were photobleached in this manner. The data traces were analyzed to give the energy transfer efficiency (%E), as has been described in detail previously (11).
Statistical analysis of data
In photobleaching recovery and fluorescence energy transfer
experiments, diffusion coefficients and energy transfer efficiencies
were obtained through curve fitting appropriate mathematical models to
experimental data sets. These data sets contained hundreds of points,
and fitting is accomplished using the Marquardt algorithm
(17). Because each of the many observations in a single
measurement provides independent information on the parameter of
interest, the SE of the parameter was calculated at the
same time as the fitted parameter itself. However, because any real
data set has some systematic deviation from a model representing the
parent experiment, these standard errors calculated during the
curve-fitting procedure almost certainly overestimate the reliability
of parameters. We thus present the uncertainties of a fitted parameter
x as <x>± 2s where s is the SEM of a set of three to
four complete, independent determinations of x. Uncertainties in
quantities, such as percent efficiency of energy transfer, which
involve parameters obtained in at least three separate experiments,
were calculated by standard propagation of errors methods. Decisions as
to whether parameters differ significantly between
(18) multiple treatment groups were made using
single classification ANOVA methods (SigmaStat, Jandel Scientific, San
Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Using this protocol,
lateral diffusion coefficients D for LH- and hCG-occupied receptors on
the various cell types ranged from 2.1–4.5 x
10-10cm2sec-1
at 37 C (Table 1). There was a
significant difference between the diffusion coefficients for LH- and
hCG-occupied receptors on LHR-wt cells: oLH-occupied receptors on
LHR-wt cells had a diffusion coefficient of 3.2 ± 1.0 x
10-10cm2sec-1,
whereas that of hCG-occupied receptors was 4.5 ± 1.5
x
10-10cm2sec-1.
There was no difference between the diffusion coefficients for
hormone-occupied receptors on LHR-K583R cells.
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Differences in the diffusion characteristics for LH receptors on LHR-wt and LHR-K583R cells were not the result of differences in receptor number. Before initiating each fluorescence photobleaching recovery experiment on an individual cell, we measured fluorescence counts per second (cps) from fluorophores bound to LH receptors in the area illuminated by the attenuated argon ion laser. The means and SD for counts from 20–40 experiments on 10 separate days were 2934 ± 1650 cps/cell on LHR-wt cells, compared with 3055 ± 1726 cps/cell on LHR-K583R cells. These values do not differ significantly.
Disruption of microfilaments increased the fraction of mobile
receptors for hCG but not LH-occupied receptors on LHR-wt cells at 37
C
Cytoskeletal components can affect the motions of LH receptors in
some cell systems. The most pronounced effects on protein motions have
been observed with cytochalasin D-treated ovine luteal
cells, where disruption of microfilaments increased the rate of LH
receptor lateral diffusion (19) and the fraction of mobile
receptors (19). On MA-10 cells, cytochalasin D treatment
resulted in faster rotational diffusion of the receptor
(20). To determine whether lower values for the mobile
fraction on LHR-wt cells were caused by restriction of receptor lateral
diffusion by microfilaments, cells were treated with cytochalasin D for
1 h before labeling of cells for fluorescence photobleaching
recovery measurements. Cytochalasin D treatment significantly affected
the measured rate of receptor lateral diffusion and the fraction of
mobile receptors on LHR-wt but not LHR-K583R cells (Table 1
). After
treatment with cytochalasin D, the fractions of mobile hCG-occupied
receptors on LHR-wt cells increased from 43 ± 3% to 66 ±
13%, whereas the fraction of mobile LH-occupied receptors
decreased.
Energy transfer occurs between receptors on LHR-wt cells
We then used single-cell fluorescence energy transfer methods to
evaluate whether different extents of receptor self-association
accompanied different fractions of mobile receptors. Representative
data traces showing fluorescence energy transfer between LH- and
hCG-occupied receptors are presented in Fig. 2. As summarized in Table 2, energy transfer efficiency was
significantly higher for hCG-occupied receptors, compared with LH
occupied receptors (17% and 13%, respectively) on LHR-wt cells. On
LHR-K583R cells, energy transfer efficiencies between LH- and
hCG-occupied receptors were 1% and 5%, respectively. These values are
thus considerably lower than those cells expressing wild-type receptor
and, in fact, do not differ significantly from zero.
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). Significant effects of cytochalasin D
treatment on fluorescence energy transfer between LH receptors were
observed only for LH-occupied receptors on LHR-wt cells, where energy
transfer efficiency between LH-occupied receptors increased from
13 ± 2% to 19 ± 2%. Thus, diffusion characteristics and
energy transfer between LH-occupied receptors on LHR-wt cells, after
cytochalasin D treatment, did not differ significantly from those of
untreated, hCG-occupied receptors. This suggests that there may be
limits to both the extent of energy transfer efficiency and the
relative number of mobile receptors on LHR-wt cells and that
microfilaments interact in a differential fashion with the complexes
formed after binding of LH and hCG. In the case of LH-occupied
receptors, the cytoskeleton restricts receptor-receptor interactions.
For hCG-occupied receptors, the cytoskeleton restricts the lateral
diffusion of a significant fraction of the hCG-occupied receptor
population. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lateral dynamics of wild-type LH receptors on LHR-wt cells were typical of those measured for other membrane proteins, including, for example, the major histocompatibility complex class I antigens (22) and lymphocyte membrane glycoproteins (23). The diffusion coefficient measured for both LH- and hCG-occupied receptors was also similar to that measured for LH-occupied native LH receptors on ovine luteal cells (15) and rat luteal cells (24). However, in contrast to these other cell types, where hCG-occupied receptors seemed laterally immobile, hCG-occupied receptors on LHR-wt cells exhibited measurable rates for lateral diffusion. These results for hCG-occupied receptors differed from those previously reported in which the hCG-occupied LH receptor was laterally immobile on luteal cell membranes at 37 C (15, 24), with values for fluorescence recovery, after photobleaching, of less than 20%. Nonetheless, binding of either LH or hCG to receptors on LHR-wt cells affected both the diffusion parameters and fluorescence energy transfer efficiency between hormone-occupied receptors. LH-occupied wild-type receptors had larger mobile fractions and less energy transfer between receptors than did the hCG-occupied receptors. Together with studies showing slower rotational diffusion after binding of hCG (and thus, presumably large complexes) than on LHR-wt cells (4), these results suggest that the receptor-containing structures formed after binding of LH or hCG differ structurally. Because receptor number remained constant throughout these studies, differences in receptor number do not contribute to the differential effects of hormone binding on receptor self-association or the magnitude of the laterally immobile fraction. It is more likely that there are additional interactions between hCG and nearby membrane proteins, perhaps as a result of the additional glycosylation of the hCG molecule (25). As observed previously, binding of chemically deglycosylated hCG to LH receptors does not slow receptor rotational diffusion (4) or produce a large fraction of laterally immobile receptors (26).
The components of the slowly diffusing complexes are not known, but it is likely that these structures contain other nonreceptor proteins. LH receptors exhibit very slow rotational motion in time-resolved phosphorescence anisotropy studies on ovine and bovine luteal cell membranes (3), and these slower motions are observed only when the hormone-receptor pair is functional, i.e. capable of activating adenylate cyclase (4). LH receptors on bovine luteal cell plasma membranes are located within perhaps 100A of various nonreceptor proteins (27), and this may be true in other species as well.
The differences between the lateral diffusion of functional hormone receptor complexes on LHR-wt cells and nonfunctional hormone-receptor complexes on LHR-K583R cells raise questions as to whether receptor-receptor interactions may be necessary for signal transduction. These measurements do not, however, resolve whether receptor self-association precedes signaling. The biophysical methods applied here required a finite time to label the receptor with fluorescent probes and to initiate measurements. Within that time, receptor self-association has occurred, and the receptor exhibits slow lateral diffusion. Nonetheless, nonfunctional LH receptors on LHR-K583R cells were highly mobile, exhibited little or no interreceptor energy transfer, and, in rotational diffusion studies (4), had fast rotational correlation times that were consistent with small complex sizes. Thus, in the absence of receptor function, either as a result of receptor mutation or binding of hormone antagonist, there is no interaction between receptors and no signaling. Functional receptors on LHR-wt cells have slow rotational correlation times (4) and exhibit substantial energy transfer between receptors. This suggests that microaggregation of LH receptors on LHR-wt cells may accompany, or be required for, productive signal transduction. In addition, receptor self-association may persist into the times when LH receptors are nonresponsive to hormone challenge and thus desensitized (28).
Receptor self-association has been proposed as a early event in the function of another structurally-related hormone receptor involved in reproductive function. Janovick and Conn (29), using lactoperoxidase-conjugated hormones to iodinate proximal GnRH receptors , have demonstrated that agonist, but not antagonist, binding to the GnRH receptor results in formation of GnRH microaggregates and have identified nonreceptor proteins in the vicinity of the receptor. We have directly demonstrated such microaggregates in studies of fluorescence energy transfer between GnRH receptors (30). GnRH agonists increase energy transfer efficiency between receptors in a dose-dependent fashion, but binding of a GnRH antagonist results in no energy transfer between receptors. Lateral diffusion of the GnRH receptor also depends on whether the receptor has bound GnRH agonist or antagonist. There is a significant decrease in the fraction of mobile receptors in response to agonist (but not antagonist) binding. The picture that emerges from Janovick and Conn’s work (29), as well as from our biophysical studies of GnRH receptors (30) and LH receptors, is that hormone-responsive receptors seem to cluster in structures that contain both self-associated receptors and nonreceptor proteins and that are sufficiently large to exhibit reduced lateral mobility.
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Acknowledgments |
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Footnotes |
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Received April 18, 2000.
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References |
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and ferritin-LH. Endocrinology 115:2217–2225
opiod receptor: implication for a role in receptor internalization.
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