Luteinizing Hormone Receptors Are Self-Associated in Slowly Diffusing Complexes during Receptor Desensitization
Regina D. Horvat,
B. George Barisas and
Deborah A. Roess
Cell and Molecular Biology Program (R.D.H.) Department of
Chemistry (B.G.B.) and Department of Physiology (D.A.R.)
Colorado State University Fort Collins, Colorado 80523
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ABSTRACT
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We have previously shown that rat LH receptors
(LHRs) occupied by human CG (hCG) exhibit slow receptor lateral
diffusion and are self-associated. Here we have examined whether LHRs
become self-associated and enter slowly diffusing structures in
response to hormone binding and whether these receptors retain this
organization while in the desensitized state. Before hormone exposure,
wild-type rat LHRs coupled at the C terminus to enhanced green
fluorescent protein (GFP-LHR-wt) exhibited fast lateral diffusion, as
assessed by fluorescent photobleaching recovery (FPR) methods, and most
receptors were laterally mobile. After 30 min exposure to hCG and
subsequent removal of hormone by low pH wash, hormone challenge at any
time within the next 4 h produced no increase in cellular cAMP
levels. During this time, LHRs were either laterally immobile or
exhibited slower lateral diffusion. When LHRs were again responsive to
binding of hormone, the rate of receptor lateral diffusion had become
significantly faster and the fraction of mobile receptors was again
large. Desensitized LHRs were also self-associated and present in
microscopically visible clusters on the plasma membrane. Fluorescence
energy transfer (FET) methods were used to measure the extent of
interaction between receptors coupled to either GFP or to yellow
fluorescent protein (YFP). Before hormone treatment, there was
essentially no energy transfer between LHRs. After desensitization of
the receptors by 30 min exposure to hCG, energy transfer efficiency
increased to 18%. Values for FET efficiency between desensitized
receptors decreased over time, and receptors were responsive to hormone
only after measurable energy transfer had completely disappeared.
Together these results suggest that desensitized LHRs exist in large,
slowly diffusing structures containing self-associated receptors and
that these structures must dissipate before the receptor can again
respond to hormone.
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INTRODUCTION
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Desensitization of LH receptors (LHRs) after brief exposure to
hormone is initially characterized by uncoupling of the receptor from
the signal transduction machinery rather than by a decrease in receptor
number. While desensitized, LHRs found on reproductive tissues
including Leydig, luteal, and granulosa cells are less responsive to
subsequent binding of ligand (1, 2, 3, 4, 5). This process may be important
in vivo where the preovulatory surge of LH results in a
time-dependent decrease in the subsequent response to hormone (6) but
only if hormone doses are sufficient to cause ovulation (1).
Desensitization may also be required for internalization of the
receptor and receptor down- regulation (2, 7, 8).
Despite considerable effort by a number of investigators, little is
known about the molecular mechanisms by which LHR desensitization
occurs. Desensitization for the ß-adrenergic receptor has served as
the model for most G protein-coupled receptors (9). However, unlike the
ß-adrenergic receptor, phosphorylation of the LHR is observed to
accompany, but may not be required for, desensitization (8, 10).
Nonetheless, LHR desensitization may also involve physical interactions
between the receptor and other proteins including ß-arrestin (11)
and/or segregation of the receptor into membrane domains or membrane
rafts containing proteins needed for signaling.
We hypothesize that the desensitized LHR is self-associated within
large, slowly diffusing structures that must dissipate before the
receptor can again respond to hormone. To test this hypothesis, we have
examined the lateral motions of the receptor and fluorescence energy
transfer (FET) between receptors after brief exposure to either
LH or hCG. These studies made use of the fluorescence properties of
enhanced green fluorescent protein (GFP) and its red-shifted variant,
yellow fluorescent protein (YFP). Both these proteins were coupled to
rat LHR at its C terminus and stably expressed either singly or
together in Chinese hamster ovary (CHO) cells. We have previously shown
that hormone binding to
GFP-LHR-wt,1 which is
effectively expressed on the plasma membrane, results in cAMP
accumulation and slower GFP-LHR-wt lateral diffusion (12). Here we
examine both LHR lateral diffusion and receptor self-association during
times (14 h) when the receptor is desensitized but before substantial
internalization has occurred. Results from these studies, together with
fluorescence images of GFP-receptor distribution in the membrane during
desensitization, suggest that desensitized LHRs are self-associated
within large, slowly diffusing complexes. Dissociation of the
self-associated receptors as well as the slowly diffusing complexes
must occur before LHRs are again responsive to binding of hormone.
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RESULTS
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Binding of Ovine (o) LH or Human (h) CG to Receptors on GFP-LHR-wt
or LHR-wt Cells Desensitizes the Receptor
To observe desensitization of LHRs on CHO and 293 cells after
brief hCG exposure, cAMP levels were measured in response to subsequent
hormone challenge. Cells expressing GFP-LHR-wt or LHR-wt were incubated
for 30 min at 37 C with 10 nM hCG and then washed with a
low pH buffer to remove the bound hormone as described in
Materials and Methods. Initiation of hormone treatment is
designated as t = 0. At various times beginning 1 h after
this, cells were challenged with 10 nM hCG for an
additional hour at 37 C. As shown in Fig. 1
, initial treatment of cells expressing
either GFP-LHR-wt (panel A) and LHR-wt (panel B) resulted in a 5- to
7-fold increase in cAMP over basal levels. After receptors were
desensitized by brief exposure to hCG for 4 h, hormone challenge
produced no increase in intracellular cAMP over basal levels. After
5 h, cells responded to hormone challenge with an increase in
intracellular cAMP to levels comparable to those seen after initial
exposure to hCG. Recovery from desensitization of LHR-wt treated with
10 nM LH was essentially identical to that of
hCG-treated cells both in terms of the time required and the magnitude
of the cAMP response (data not shown). The effects of LH exposure on
cAMP levels in LHR-wt cells at 1 h and 5 h following
desensitizing hormone treatment are shown in Table 1
and do not differ significantly from
the effects of hCG treatment at either time.

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Figure 1. The Time Course of Recovery of LHR Signaling for
CHO Cells Expressing rLHR-GFP ( ) and HEK 293 Cells Expressing
Wild-Type LHR ( ) after Receptor Desensitization
To determine initial levels of cAMP in response to hCG treatment
(t = 0), GFP-LHR-wt or LHR-wt were treated with hCG for 1 h
before measurement of intracellular cAMP. To measure cAMP levels after
desensitization of the receptor by hormone as described in
Materials and Methods, 10 nM hCG was added
to the cell suspension at the indicated times (t= 1, 2, 3, 4, or 5
h) for a 1-h incubation before measurement of intracellular cAMP
levels. In response to initial treatment with hCG, there was a 4- to
6-fold increase in intracellular cAMP. After desensitization of the
receptor, subsequent hormone treatment for 1 h had no effect on
intracellular cAMP until 5 h following removal of desensitizing
hormone. Results are the average and SD for four
experiments performed in triplicate.
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Lateral Diffusion of Desensitized GFP-LHR-wt Receptors on CHO Cells
Is Slow
Before binding of ligand, GFP-LHR-wt exhibited fast lateral
diffusion (16 ± 0.3 x
10-10cm2sec-1)
with a high fraction of mobile receptors (% r =
58 ± 4). After desensitization of the receptor by LH, the
diffusion coefficients for the unoccupied receptor were reduced more
than 3-fold to 4.4 ± 1.0 x
10-10cm2sec-1
and fluorescence recovery decreased to 26 ± 3% (Fig. 2
). Between 2 and 4 h after initial
hormone treatment, lateral diffusion of the unoccupied receptor became
progressively faster and the extent of fluorescence recovery increased.
However, only when receptors were again hormone responsive at 5 h
were the rate of receptor lateral diffusion and the fraction of
laterally mobile receptors comparable to values measured on untreated
cells. When receptors were desensitized by brief exposure to hCG, the
fraction of mobile receptors dropped to 16% at 1 h indicating
that most receptors were laterally immobile (13). At 2 and 4 h
following initiation of hormone treatment, the extent of fluorescence
recovery after photobleaching, and thus the relative fraction of mobile
receptors, increased to 42% by 4 h. Diffusion coefficients
for hCG-desensitized receptors were significantly slower than those of
LH- desensitized receptors at each time point (Table 2
). As was the case for receptors
desensitized by LH, diffusion coefficients for hCG-desensitized
receptors were again comparable to those of untreated receptors after 5
h.

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Figure 2. Spot FPR Measurements of rLHR-GFP Lateral Diffusion
before Hormone Binding and after oLH- ( ) or hCG-Induced ( )
Receptor Desensitization
Panel A shows the diffusion coefficient (D) for the GFP-LHR-wt over the
time course of recovery from receptor desensitization. Panel B shows
fluorescence recovery (%R) of GFP-LHR-wt at the corresponding times.
Receptors desensitized by either ligand exhibited significantly slower
D 1 h following desensitization. D increased over the next 4
h until D was indistinguishable from that of untreated LHR. %R
decreased upon binding of hormone and then increased over time until
5 h when %R did not differ significantly from untreated receptor.
The magnitude of the decrease in D and %R in response to receptor
desensitization was dependent on whether LH or hCG was used to
desensitize the receptor. Values for diffusion coefficients and % R
reflect three separate repetitions of an experiment in which 20
measurements on individual cells were made on a given day.
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The Fraction of Mobile, Desensitized Receptors Depends upon the
Desensitizing Hormone
We then examined the complexes formed after desensitization of
LHR-wt receptors on 293 cells using fringe fluorescence photobleaching
recovery (FPR) methods. We had previously shown that the fractions of
mobile receptors after binding of tetramethylrhodamine isothiocyanate
(TrITC)-LH or TrITC-hCG differed significantly and that values for
receptor lateral diffusion were not linked to the extent of
fluorescence recovery after photobleaching (14). Although the diffusion
coefficients for hCG- and LH-occupied receptors on LHR-wt cells were
35 x
10-10cm2sec-1,
the fraction of mobile LHRs, as indicated by percent mobility (%M),
was significantly higher after binding of LH (69 ± 13%) when
compared with hCG-occupied receptors (43 ± 3%). As shown in
Table 1
, at 1 h following initiation of hormone treatment, values
for %M always reflected the hormone initially used to desensitize
receptors regardless of whether LH or hCG was later used as the
fluorescent probe. After 5 h, values for %M were characteristic
of the fluorescent ligand bound to the receptor and, thus, were
independent of the hormone used initially to desensitize the receptor.
In control experiments in which cells were treated with buffer rather
than hormone, acid washed, and then labeled with TrITC-LH or TrITC-hCG,
the diffusion coefficients and mobile fractions for these hormones did
not differ significantly from those of hormone-occupied LHRs on LHR-wt
cells that had not received low pH treatment. These data suggest that
the rebinding of ligand to a desensitized receptor does not induce
formation of a new receptor-containing structure but rather that the
complexes are stable and characteristic of the hormone used to
desensitize the receptor.
FET Occurs between GFP-LHR-wt and YFP-LHR-wt during Receptor
Desensitization
We have previously shown that LHR-wt receptors on 293 cells are
self-associated after binding of LH or hCG (14). To investigate whether
this occurs only in response to hormone binding and whether receptors
remain self-associated while desensitized, we measured the FET between
hCG-treated LHRs coupled to either GFP and YFP. Before binding of hCG
there was no significant energy transfer between GFP-LHR-wt and
YFP-LHR-wt fusion proteins (Fig. 3
).
After desensitization of the receptor by 30 min exposure to 10
nM hCG and hormone removal, energy transfer efficiency
between unoccupied LHRs increased to 18 ± 1% at 1 h. Values
for energy transfer efficiency between unoccupied, desensitized LHRs
decreased over the next 3 h but did not reach 0 until after 5
h when receptors were again responsive to hCG challenge.

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Figure 3. Percent Energy Transfer Efficiency (%E) between
Unoccupied LHR Fusion Proteins before (0 h) and after Initiation of
Receptor Desensitization (15 h) by 10 nM hCG
At 0 and 5 h when receptors are hormone responsive, there is no
significant energy transfer between LHRs. When receptors were not
hormone responsive, there was measurable energy transfer between LHRs.
The results are the average and SD of a total of 40
measurements made on individual cells with 20 measurements per day made
on two separate days.
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Positive values for energy transfer efficiency were accompanied by
microscopically visible clustering or patches of the GFP-LHRs. As shown
in Fig. 4
, fluorescence from GFP-LHRs was
distributed uniformly in the plasma membrane before hormone binding.
After treatment with 10 nM hCG to desensitize the receptor,
unoccupied GFP-LHR-wt was organized in clusters that appeared largest
within 1 h after initiation of hormone treatment. Over the next
3 h, the size of clusters containing GFP-LHR-wt became
progressively smaller and, by 5 h, the receptors were again
uniformly distributed over the cell surface.

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Figure 4. Fluorescence Images of GFP-LHR-wt before
(Untreated) and after hCG-Induced Desensitization of the Receptor
When the receptors were capable of signal transduction, fluorescence
from the unoccupied receptors was diffusely distributed over the
membrane (untreated, 5 h). When receptors were desensitized,
fluorescence from GFP was distributed in discrete patches. At 1 and
2 h the patches appeared larger than those seen 3 and 4 h
after hormone treatment. The images presented here were obtained
sequentially on one day.
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DISCUSSION
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These studies demonstrate that desensitized LHRs are situated in
large, slowly diffusing complexes containing self-associated receptors.
Before binding of hormone, GFP-LHR-wt appear to be uniformly
distributed throughout the plasma membrane and are not self-associated
until after brief hormone treatment. In addition, rebinding of hormone,
either LH or hCG, does not alter the diffusive properties of the
desensitized receptor.
In FPR measurements of receptors occupied by TrITC-derivatized
hormones, the mobile fractions for receptors desensitized by LH or hCG
were characteristic of the desensitizing hormone complex regardless of
which hormone was subsequently used as a receptor probe at 1 h
(Table 1
). Thus, any additional interactions between LHRs and, as
examples, membrane lectins (15) or the cytoskeleton (16), must occur as
the receptor-containing complex is forming. Once receptor-containing
complexes have formed, the structure of these complexes seems to be
unaffected by hormone rebinding until the complexes have dissociated at
approximately 5 h. At early times after desensitization by LH or
hCG, the diffusion coefficients measured for GFP-LHR-wt were similar to
those of LH and hCG-occupied receptors on ovine (17) and rat (18)
luteal cells. In spot FPR measurements of LHRs on ovine luteal cells,
most hCG-occupied receptors were immobile on the cell surface,
exhibiting less than 20% fluorescence recovery after photobleaching
(17). The few mobile LHRs occupied by hCG had a 7-fold slower diffusion
coefficient than did LH-occupied receptors (19). Although more ovine LH
(oLH)-occupied receptors were mobile, fluorescence recovery after
photobleaching was only about 30% (17), which is considerably less
than the 60% fluorescence recovery typical of most membrane proteins
(20).
Desensitization of the LHR occurred in less than 1 h, independent
of the type of hormone used to desensitize the receptor. At 1 h,
after desensitization by either LH or hCG, there was no response to
hormone challenge, a result consistent with in vitro studies
by Hunzicker-Dunn and co-workers (6), who have demonstrated in a
cell-free system that LHRs on pig Graafian follicles are fully
uncoupled from adenylate cyclase within 30 min. Similarly, Segaloff and
co-workers (21) have shown in intact cells that rat wild-type LHR
expressed in 293 cells is desensitized within 1 h.
Resensitization of the GFP-LHR-wt and LHR-wt is slow, requiring about
5 h. This rate is comparable to that reported by Ulaner et
al. (22) for rat LHRs transfected in Y-1 cells and treated with
either LH or hCG. Interestingly, resensitization of LHRs is
significantly slower than that of the ß-adrenergic receptors, which
occurs within 1520 min following removal of hormone agonist with low
pH buffer (23). Although LHR desensitization has been studied in
vivo, it cannot be examined independently of down-regulation of
receptor number (24) and degradation of mRNA transcripts (25, 26).
Thus, the reappearance in vivo of functional LHRs on the
plasma membrane is slow with times varying from 72 h (27) to 7
days (24), depending on cell type. This in vivo phenomenon
must thus involve receptor replenishment through a very different
mechanism than is involved in LHR resensitization in this study.
FET between receptors is indicative of dimerization or oligomerization
of the receptor, a process that occurs in response to hCG binding and
persists while the LHR is nonresponsive. Hebert and co-workers (28)
have suggested that dimerization of the ß-adrenergic receptor is
essential for receptor signaling. Nonfunctional receptors can be
rescued by antibody-induced receptor dimerization (29), which is
mediated by a dimerization sequence in the sixth transmembrane domain
(28). Conn and co-workers (30) have shown that antibody-mediated
dimerization of GnRH receptors is sufficient to stimulate LH secretion
by pituitary cells. As is the case for ß-adrenergic receptor,
self-association of LHRs may be required for receptor signaling. Rat
LHRs containing a specific single point mutation are able to bind LH or
hCG but do not signal or have measurable levels of FET between
receptors after hormone binding (14).
There are, however, critical differences between the extent of LHR and
ß-adrenergic receptor self-association. The ß-adrenergic receptor
apparently forms discrete homodimers (28) that appear in unresolved
small, punctate structures in fluorescence micrographs (31). In
contrast, the LHR, which lacks this dimerization sequence in its TM6
domain, is present in larger clusters after binding of hormone.
Luborsky et al. (32) have observed clusters of about 1020
receptors on rat granulosa cells using electron microscopy after
binding of high concentrations of LH. In addition, LHRs desensitized by
hCG appear in larger scale macroscopic patches on the membranes of rat
granulosa cells (33). The components of the large molecular weight
complexes formed during receptor desensitization are not known, but it
likely that these structures contain other nonreceptor proteins. LHRs
exhibit very slow rotational motion in time-resolved phosphorescence
anisotropy studies on ovine and bovine luteal cell membranes (34), and
these slower motions are observed only when the hormone-receptor pair
is functional, i.e. capable of activating adenylate cyclase
(35). LHRs on bovine luteal cell plasma membranes are associated with a
family of nonreceptor proteins (36), and this is presumably true in
other species. On membranes from porcine granulosa cells, a number of
signaling molecules, including, notably, ß-arrestin, must also be
available for desensitization of the receptor (11).
We speculate that ligand binding to LHRs may also be associated with a
redistribution of receptors in the membrane into small membrane domains
in which signaling and/or receptor desensitization can occur. However,
it does not appear that desensitized receptors are sequestered in
membrane vesicles. First, there was no decrease in the number of LHRs
on the plasma membrane either 1 or 5 h following brief hormone
treatment, suggesting that there was no significant internalization of
receptors. Second, although sequestration of LHRs is suggested by
aggregation of the receptor into fluorescent clusters (Fig. 4
), many
LH-treated receptors remain laterally mobile after hormone treatment.
If receptors were clustered into small vesicles, there would be
essentially no receptor diffusion measured on the time scale of our
experiments and no measurable recovery of fluorescence after
photobleaching. Finally, the process initiated by exposure to either LH
or hCG was reversible. The observed decreases in the rate of receptor
lateral diffusion and the fraction of mobile receptors were transient
and, upon recovery from receptor desensitization, receptor lateral
diffusion was fast and the fraction of mobile receptors was high.
Together these results suggest that the LHR forms clusters on the
membrane that dissipate over time but that receptors present in these
clusters are not internalized within membrane vesicles.
LHR dimerization or oligomerization may arguably be the initial step in
signal transduction, although the sequence of events after binding of
hormone to receptor is not clear. This could be followed by the
movement of LHRs into membrane regions containing proteins required for
signaling including, for example, G proteins, effector proteins, and
proteins for desensitization or, alternatively, signal transduction
could occur outside of membrane domains and be followed by movement of
receptors into membrane domains containing proteins necessary for
desensitization of the receptor. In addition to interactions with ß-
arrestins, clustering of receptors into very large complexes may,
as Amsterdam et al. (33) have suggested, interfere with
receptor response to hormone. In either case, one would predict that
the fraction of immobile receptors would increase upon desensitization
of the receptor as we, in fact, observe. Dissociation of the receptor
from complexes would result in an increase in the fraction of mobile
receptors and the average diffusion coefficient for the receptor
population. However, there is no productive signaling until a
sufficiently large population of freely diffusing receptors and/or
molecules necessary for signal transduction were again available
outside specialized membrane domains.
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MATERIALS AND METHODS
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Materials
DMEM and DMEM containing high glucose were purchased from Irvine
Scientific (Santa Ana, CA). Geneticin was purchased from Life Technologies, Inc., (Gaithersburg, MD). HEPES and nonessential
amino acids were purchased from Sigma (St. Louis, MO).
Horse serum was purchased from Summit Biotechnology (Fort
Collins, CO) and FBS was purchased from HyClone Laboratories, Inc. (Logan, UT). oLH (NIH 28) was obtained from the National
Hormone and Pituitary Program, NIADDK (Baltimore, MD) and hCG was
purchased from Research Diagnostics Inc. (Flanders, NJ). TrITC and
erythrosin isothiocyanate (ErITC) were purchased from Molecular Probes, Inc. (Eugene, OR).
Cell Lines
Dr. Tae Ji kindly provided 293 cells stably transfected with the
wild-type rat LHR (LHR-wt cells). These cells, as well as untransfected
293 cells, were maintained in DMEM containing 10% horse serum, 100 U
penicillin, 1,000 µg/ml streptomycin, and 10 mM HEPES, pH
7.4. Medium for LHR-wt cells was supplemented with 400 µg/ml
geneticin. Untransfected CHO cells were maintained in DMEM supplemented
with 4,500 µg/ml glucose and containing 10% FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 1x MEM nonessential amino
acids (Sigma).
Four CHO cell lines were used in this study including 1) untransfected
cells; 2) cells expressing GFP-LHR-wt; 3) cells expressing YFP-LHR-wt;
and 4) both GFP-LHR-wt and YFP-LHR-wt. Cells stably transfected with
the GFP-LHR-wt construct were prepared as described previously (12) and
maintained in CHO cell medium containing 200 µ g/ml G418. CHO cells
expressing YFP-LHR-wt alone or expressing TFP-LHR-wt and GFP-LHR-wt
were prepared using the same strategy described in detail for
GFP-LHR-wt (12). To construct the YFP-LHR vector, the full-length
receptor cDNA for rat LHR (rLHR), a gift from Dr. Deborah Segaloff, was
subcloned into enhanced EYFP-N2 vector (CLONTECH Laboratories, Inc. Palo Alto, CA). A fragment of the LHR beginning at the
intrinsic ScaI site (ACTATAACCACGCCATAGAC) and ending at the
receptor 3'-end was removed, thus removing an intrinsic stop codon, and
replaced with an in-frame BamHI site (underlined)
at the 3'-end (CGGGATCCAACGCTCTCGGTGGTATGG). This fragment
was amplified by PCR. The PCR product was digested with
BamHI, as was the eYFP-N2plasmid, and ligated into
ScaI and BamHI plasmid. The final fusion protein
DNA sequence consisted of rLHR, a spacer sequence of the amino acids
IHRPVAT, and enhanced YFP. The ScaI/BamHI
fragment was confirmed by cDNA sequencing by Macromolecular Resources
(Fort Collins, CO). CHO cells expressing YFP-LHR-wt alone were
transfected with 1 µg of the rLHR-YFP vector using Lipofectamine-Plus
(Life Technologies, Inc.) according to the manufacturers
instructions. CHO cells coexpressing both GFP-LHR-wt and YFP-LHR-wt
were transfected with 0.4 µg of GFP-LHR-wt and 1.2 µg of
YFP-LHR-wt. After overnight culture, transfected cells were transferred
into 150-mm plates and cultured with CHO cell medium containing 600
µg/ml G418 (Gemini Biological Products, Woodland CA) for
7 days. At this time, cells were washed with PBS and cloned by limiting
dilution in 96-well plates (ISC BioExpress, Kaysville, UT) where they
were incubated for 2 weeks before selection of fluorescent colonies and
expansion of those colonies.
Preparation of TrITC and ErITC-Derivatized Hormones
Hormones were derivatized with ErITC or TrITC using a
modification of methods described by Brinkley et al. (37)
and described in detail elsewhere (34). The molar ratios for
dye-hormone were determined spectrophotometrically. Hormone
preparations used in these experiments had 1.01.5 mol of ErITC or
TrITC per mole of oLH or hCG. Before use, all derivatized hormones in
PBS were centrifuged at 130,000 x g for 10 min in a
Beckman Coulter, Inc. Airfuge (Beckman Coulter, Inc., Palo Alto, CA) to remove any protein aggregates formed
during storage at 4 C.
LHR Desensitization
LHRs were desensitized using a protocol that has been described
in detail by others (21). Briefly, CHO cells or 293 cells expressing
LHRs were incubated for 30 min with 10 nM LH or hCG at 37 C
and then treated for 5 min at 4 C with 50 mM glycine, 100
mM NaCl, pH 3.0, PBS to remove hormone bound to the LHR.
The cells were centrifuged at 300 x g and resuspended
in fresh PBS. The extent of LHR desensitization was evaluated by
measuring cellular cAMP production. At 1-h intervals after the time 0
when hormone was initially introduced to cell samples, either LH or hCG
was again added for 1 h to cell suspensions containing 1 x
106 cells. At the end of this incubation,
cellular cAMP was measured using a TiterFluor cAMP EIA kit
(Perkin-Elmer Corp., Norwalk, CT) according to the
manufacturers instructions. To reduce well-to-well variations in
measured levels of cAMP, 96-well plates coated with antirabbit IgG were
obtained from Pierce Chemical Co. (Rockford, IL) and were
substituted for those provided in the TiterFluor cAMP kits.
After hormone treatment to desensitize the LHR, we verified that
receptor number was unchanged and receptors were able to rebind
hormone. This was done by measuring the phosphorescence intensity from
ErITC-derivatized hormones bound to LHRs on LHR-wt cells. Cells were
incubated with 10 nM ErITC-hCG or ErITC-LH for 1 h and
washed two times by centrifugation at 300 x g for 3
min in balanced salt solution (BSS) to remove any unbound ligand. The
sample was then deoxygenated for 15 min by purging with argon gas to
eliminate phosphorescence quenching caused by O2
and placed in a 5-mm Suprasil quartz cuvette (Helma Cells, Inc.,
Jamaica, NY), which was inserted in a thermostatted cuvette holder. The
frequency-doubled 532-nm output of a Spectra-Physics DCR-11 Nd:YAG
laser was used to excite ErITC. The laser was operated at 10 Hz with a
vertically polarized TEM00 output of 0.19 mJ and a beam
1/e2 radius of 2.5 mm at the sample.
Phosphorescence emission from the sample was collected by an EMI 9816A
photomultiplier tube, amplified by a Tektronix 476 oscilloscope
(Tektronix, Inc., Beaverton, OR) and a 35 MHZ buffer amplifier, and
digitized by a Nicolet 12/70 signal averager (Nicolet Instrument Corp.,
Madison, WI). After data acquisition was complete, the data were
downloaded into a Pentium II microcomputer (Santa Clara, CA) (34). In a
typical experiment, 105 cells labeled with
ErITC-oLH or ErITC-hCG exhibited phosphorescence intensity of 5.8
± 0.4 and 6.2 ± 0.1 in arbitrary units, respectively, while
untreated cells had only 0.5 ± 0.3 U phosphorescence. Treating
cells with low pH buffer removed hormone from the receptor as indicated
by a decrease in the phosphorescence signal to baseline values
(0.530.57). Rebinding ErTIC-LH or ErITC-hCG to LHRs at 1 h and
5 h after removal of hormone with low pH buffer increased the
signals to 4.6 ± 1.5 and 6.2 ± 0.1, respectively, which did
not differ significantly from signals measured after initial binding of
either ErITC-LH or ErITC-hCG. To verify that there were no nonspecific
interactions of the ErITC-hormones with human kidney 293 cells, 293
cells that did not contain expression vectors for the LHR were treated
with 10 nM ErITC-rLH or -hCG. To determine
whether binding of the ErITC-derivatized hormones was specific, cells
were preincubated with excess oLH before labeling with ErITC-oLH in
some experiments. In both cases, there was no detectable
phosphorescence signal from the cell sample.
Spot and Fringe FPR Measurements
The optical system for spot and fringe FPR measurements and the
methods used for data analysis have been described in detail (38). The
microscope objective used in these studies was a 40x objective of
NA 0.65 (Carl Zeiss, Thornwood, NY). Standard
Carl Zeiss filter and dichroic mirror sets for fluorescein
isothiocyanate (FITC) and TrITC fluorescence were used. Cells were
examined under coverslip on well slides while temperature was
maintained by a thermal stage with a temperature range of 0 C to 40 C.
For spot measurements of unoccupied GFP-LHR-wt lateral diffusion, an
attenuated Coherent Radiation Innova 100 argon ion laser beam at 488 nm
was focused to a spot on the plasma membrane of 0.41 µm
1/e2 radius. Bleaching and probe beam powers were
1.4 mW and 1.7 µW, respectively. Data were acquired at 50 msec/point
for 20 sec before, and for 30 sec after, a 150 msec bleaching pulse.
For fringe measurements of TrITC-LH or TrITC-hCG lateral diffusion, the
region illuminated at the sample had a 1/e2
radius of at least 18 µm, and the photometer acceptance region was
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 and a function
representing the recovery kinetics in terms of a decay half-time. The
desired diffusion coefficient and the extent of fluorophores mobile on
the timescale of the experiment 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 evaluated (38, 39, 40).
A detailed comparison of the methods used to analyze results from spot
and fringe FPR measurements is presented in Munnelly et al.
(40). A review of biophysical methods for measuring translational
diffusion is presented by Jovin and Vaz (41). Each data point presented
in either spot or fringe FPR measurements represented a total of 60
measurements with 20 measurements made on three different days.
Single Cell FET
Slower rates of fluorescence decay for cells expressing both
GFP-LHR donor and YFP-LHR acceptor (D+A) than for cells expressing
GFP-GnRHR only (D) were indicative of energy transfer from fluorescence
donor to acceptor. For this donor-acceptor pair, Försters
r0 is calculated to be 56 A (42); therefore,
energy transfer occurs to a measurable extent only when the donor and
acceptor are separated by distances less than about 100 A. FET
measurements were made using a fluorescence microscope photometer based
on an inverted-configuration Carl Zeiss Axiomat microscope
and associated components used for spot FPR measurements. Fluorescence
excitation was provided by a Coherent 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 45 mW,
and this was held constant between measurements on LHR-GFP cells or on
LHR-GFP/YFP expressing cells. The 1/e2 Gaussian
spot diameter was 18 µm. Donor fluorescence from GFP was isolated
with a standard fluorescein filter set together including a short pass
fluorescein-selective filter to remove yellow fluorescence contributed
by YFP-LHR-wt. This combination was highly effective in rejecting YFP
fluorescence. 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 illuminate
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. In each
experiment, four sets of identically handled cells were examined
including untransfected CHO cells, CHO cells expressing GFP-LHR-wt
alone, CHO cells expressing YFP-LHR-wt alone, and cells expressing both
GFP-LHR-wt and YFP-LHR-wt. Cells expressing LHR-YFP alone produced
signals that did not differ significantly from those of untransfected
CHO cells using the fluorescein-selective filter set. Signal from CHO
cells expressing LHR-GFP or LHR-GFP/YFP was greater than 8-fold higher
than background levels. Thus, the rate constants for photobleaching of
GFP on cells expressing LHR-GFP alone (kD) or
LHR-GFP/YFP (kDA) were analyzed from data traces
as described in detail previously (43). The energy transfer efficiency
was expressed as a percent (%E) and was calculated from these rate
constants using %E = (1 -
kDA/kD) x 100
(44).
Fluorescence Imaging of GFP-LHR-wt
GFP-LHR-wt fluorescence from individual cells was
measured on a Carl Zeiss Axiovert microscope
equipped with a 1.4 NA oil immersion condenser and a 1.3 NA 63x
Plan-Apochromat objective. A 100 W arc lamp was used to excite the
sample and a standard FITC filter set was used to isolate the green
(GFP) signal. Fluorescent images were obtained using a Dage-MTI CCD300
digital camera (Dage-MTI, Inc., Michigan City, IN) using an
integration time of 30 sec, digitized, pseudo-colored via Metamorph
imaging software (Universal Imaging, West Chester, PA), and exported to
Adobe PhotoShop (Adobe Systems, Inc., San Jose, CA) for further image
processing. On two separate days, a minimum of 10 cells for each sample
were imaged and analyzed.
 |
ACKNOWLEDGMENTS
|
---|
We thank the National Hormone and Pituitary Program, NIDDKD, for
providing the oLH and hCG. We would also like to thank Dr. Scott Nelson
for his help in the preparation of the YFP-LHR-wt cell line.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Deborah A. Roess, Department of Physiology, Colorado State University, Fort Collins, Colorado. E-mail:
Deborah.Roess{at}ColoState.edu
This work was supported by NIH Grants HD-23236 and HD-01067
(D.A.R.).
1 Abbreviations: LHR-wt, 293 human embryonic
kidney cells with an expression vector for the wild-type LH receptor;
GFP-LHR-wt, CHO cells expressing the rat LH receptor-GFP fusion
protein; YFP-LHR-wt, CHO cells expressing the rat LH receptor-YFP
fusion protein. 
Revision received December 27, 2000.
Accepted for publication January 2, 2001.
 |
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