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Cell and Molecular Biology Program (R.D.H.) Animal Reproduction and Biotechnology Laboratory Department of Physiology (D.A.R., S.E.N., C.M.C.) and Department of Chemistry (B.G.B.) Colorado State University Fort Collins, Colorado 80523
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although the availability of cDNAs encoding the GnRHR has allowed much progress in elucidating structure-function relationships that exist in this protein, significant gaps in our understanding of this molecule remain. In particular, the lack of antibodies capable of recognizing the GnRHR in situ has precluded analyses of the GnRHR in the unbound state and thus any changes that may be induced by hormone binding. For this reason, it has been difficult to monitor GnRHR biosynthesis, its trafficking through intracellular compartments, and its behavior in the plasma membrane. As an alternative to immunological detection, we have constructed a functional GnRHR in which green fluorescent protein (GFP) is fused to the carboxyl terminus of the murine GnRHR (9). This fusion receptor is appropriately trafficked to the plasma membrane, binds hormone with an affinity similar to that of wild-type receptor, and is capable of signal transduction (9). Given the ability of the GnRHR fused to GFP (GnRHR-GFP) to recapitulate these central characteristics of native GnRHRs, this approach represents a powerful tool to address a number of basic questions regarding the biology of the GnRHR. Toward this end, we have used fluorescence photobleach recovery (FPR) to examine lateral dynamics of both the unbound and bound forms of the GnRHR-GFP in the plasma membrane (9). We found that binding of agonist to the GnRHR slowed the rate of lateral movement of the receptor in the plasma membrane and led to an "anchoring" event such that a significant percentage of the receptors became laterally immobile. The binding of the GnRH antagonist Antide also led to a reduction in the rate of lateral diffusion but, in contrast to GnRH, did not affect the fraction of mobile receptors. This striking difference suggests that these two distinct postreceptor binding events reflect a fundamental difference in the behavior of agonist- vs. antagonist-occupied receptor. Since the reduction in the rate of lateral diffusion of the GnRHR-GFP fusion protein was similar with GnRH and Antide, it would appear that this event may reflect ligand binding irrespective of agonist or antagonist. In contrast, the reduction in the mobile fraction observed only with GnRH may reflect additional interactions unique to the fully activated receptor.
There are several potential explanations for the differential effects of agonist and antagonist on the lateral diffusion of the GnRHR. For example, the reduction in the mobile fraction of GnRHRs may simply reflect association of the agonist-occupied receptor with membrane signaling components. Alternatively, the reduced mobile fraction associated with agonist binding may reflect self-association of receptors into microaggregates that allow for G protein coupling. Indeed, receptor dimerization and/or oligomerization has been suggested as a critical event preceding signal transduction by other G protein-coupled receptors including the ß2-adrenergic (10, 11) and M3 muscarinic receptors (12). Similarly, others have suggested that aggregation of GnRHRs occurs as an early and essential step in GnRH signaling (13, 14, 15, 16, 17); however, the inability to examine the aggregated state of the unbound GnRHR has precluded a direct test of this hypothesis. Herein, we examine the relationship between signal transduction and lateral mobility of the GnRHR-GFP fusion protein bound to increasing doses of either the natural ligand GnRH, the super-agonist des-Gly10-D-Ala6-GnRH N-ethyl amide (D-Ala6-GnRH), and Antide, a GnRH antagonist. Additionally, to directly test the hypothesis that binding of agonist but not antagonist leads to self-association of GnRHRs in the plasma membrane, we examine the efficiency of fluorescence energy transfer between the GnRHR-GFP fusion protein and the red-shifted variant of GFP, termed yellow fluorescent protein (YFP).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T3–1 cell line (9). Furthermore, Chinese hamster ovary (CHO) cells
stably expressing the GnRHR-GFP fusion protein exhibited a 4.5-fold
increase in intracellular concentrations of cAMP after treatment with
10 nM GnRH (9). To more directly assess the relationship
between signaling and lateral mobility of the GnRHR in the plasma
membrane, we tested whether the effects of increasing concentrations of
GnRH, D-Ala6-GnRH, and Antide on
generation of cAMP were correlated with changes in either the rate of
lateral diffusion of the receptor or the fraction of laterally mobile
receptors. Specifically, we sought to determine whether lateral
mobility of the GnRHR-GFP fusion protein is, like signaling, dependent
on the concentration of available ligand. A dose-dependent increase in
intracellular cAMP was evident for CHO cells stably expressing
GnRHR-GFP and treated for 1 h with increasing concentrations of
either natural ligand (GnRH) or the superagonist
D-Ala6-GnRH (Fig. 1A
). Consistent with higher affinity
binding to the GnRHR, the dose-response curve for
D-Ala6-GnRH was shifted to the left
as compared with GnRH. As expected, treatment of CHO cells with the
GnRH antagonist Antide did not affect levels of cAMP, which remained
low. Background cAMP levels from nontransfected cells and from CHO
cells expressing GnRHR-GFP were 35 ± 10 and 36 ± 5
pM/106 cells, respectively.
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). A significant reduction in the diffusion coefficient was
evident at 0.1 nM GnRH and continued to decrease to a
minima of 5.5 x
10-10cm2sec-1
at 10 nM GnRH. A similar dose-dependent decrease in the
rate of receptor lateral diffusion was evident for
D-Ala6-GnRH. However, as with
signaling, the dose-response curve was shifted to the left relative to
GnRH. Treatment with Antide also reduced the rate of lateral diffusion
in a concentration-dependent manner and, like the dose-response curve
for D-Ala6-GnRH, was left-shifted
relative to that for GnRH. This observation is consistent with higher
affinity binding of both D-Ala6-GnRH
and Antide for the GnRHR as compared with GnRH (18, 19).
As previously reported, the fraction of mobile receptors for
the unoccupied GnRHR was high (%r = 73 ± 1%)
(Fig. 1C). Thus, the majority of GnRHRs are laterally mobile and
display rapid lateral diffusion in the membrane. Increasing doses of
GnRH led to a dose-dependent reduction in the fraction of mobile
receptors (Fig. 1C). Similarly,
D-Ala6-GnRH also decreased
the fraction of laterally mobile receptors in a dose-dependent manner;
however, as with signaling and rate of lateral diffusion a lower dose
of the superagonist caused a significant reduction in the mobile
fraction (0.01 nM for
D-Ala6-GnRH vs.
1.0 nM for GnRH). The fraction of laterally
mobile receptors was unaffected at any dose of Antide tested. Thus, a
reduced rate of lateral diffusion of the GnRHR-GFP fusion protein
appears to be independent of the "nature" of the ligand
(i.e. agonist, superagonist, or antagonist). In contrast,
only agonist or superagonist (GnRH or
D-Ala6-GnRH) is capable of
effectively reducing the percentage of laterally mobile receptors in
the plasma membrane and activating signal transduction. Furthermore, it
is interesting to note the relationship between signaling and the
reduction in the fraction of mobile receptors. In Fig. 2, data for cAMP production or signaling
(S) and percentage of laterally mobile receptors (%R) were normalized
to the maximal values for each parameter. For GnRH and
D-Ala6-GnRH, the point of
convergence of the two lines (R/S50) is
coincident with a 50% reduction in %R and a 50% increase in cAMP
production. Consistent with the increased affinity of the superagonist,
the R/S50 for
D-Ala6-GnRH is
approximately 0.1 nM, as compared with 1.0
nM for GnRH.
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T3–1 cells (9). Thus, both GnRHR-GFP and GnRHR-YFP
bind D-Ala6-GnRH with affinities
similar to the wild-type GnRHR. No specific binding was detected for
nontransfected CHO cells.
To confirm that the fusion receptors were capable of signal
transduction, the intracellular concentration of cAMP in the CHO cells
expressing GnRHR-YFP or GnRHR-GFP/GnRHR-YFP was measured after 1 h
incubation with increasing concentrations of GnRH,
D-Ala6-GnRH, or Antide (Fig. 3). In both cell lines, the addition of
GnRH and D-Ala6-GnRH to the cells
resulted in a dose-dependent increase in intracellular cAMP
concentration with an ED50 similar to the
Kd of the wild- type GnRHR and the GnRHR-GFP
fusion receptor (9). There was no effect of Antide on intracellular
concentrations of cAMP at any dose tested. There was no effect of GnRH
on cAMP levels in nontransfected CHO cells (data not shown).
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are representative fluorescence data
and the fitted decay curves for CHO cells expressing fluorescence donor
GnRHR-GFP only or the fluorescence donor and acceptor, GnRHR-GFP and
GnRHR-YFP. In the absence of ligand, there was essentially no energy
transfer between donor and acceptor fluorophores. Treatment of CHO
cells with 10 nM GnRH resulted in energy transfer
efficiency of approximately 16 ± 1%, a value consistent with
aggregation of the donor and acceptor molecules in the plasma membrane
(Fig. 4). In contrast, 10 nM Antide had no
significant effect on %E. Therefore, the binding of agonist, but not
antagonist, induced self-association of GnRHR-GFP and GnRHR-YFP.
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, we observed essentially no energy transfer between GnRHR-GFP and
GnRHR-YFP before the introduction of ligand (Fig. 5). The addition of increasing
concentrations of GnRH led to a dose-dependent increase in %E to a
maximum value of approximately 16 ± 1% at 10 nM
GnRH. Increasing concentrations of
D-Ala6-GnRH produced a similar
increase in %E. However, as with signaling, rate of lateral
diffusion, and fraction of laterally mobile receptors, the %E
dose-response curve for D-Ala6-GnRH
was shifted to the left relative to GnRH. The GnRH antagonist Antide
had no significant effect on energy transfer efficiency at any
concentration tested (1.0, 10, and 100 nM).
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, GnRHR exhibited maximum
energy transfer efficiency when cells were treated with 9
nM
D-Ala-6-GnRH and 1
nM Antide suggesting that the presence of Antide
at low concentrations did not block energy transfer between receptors.
However, energy transfer efficiency decreased by nearly 50% when
the amount of D-Ala6-GnRH
was reduced to 7 nM and Antide was increased to 3
nM. As the concentration of Antide was increased
and D-Ala6-GnRH was
decreased, energy transfer efficiency decreased further. No significant
energy transfer was detected when concentrations of Antide were 9
nM even in the presence of 1
nM
D-Ala6-GnRH which, as shown
in Fig. 5, was sufficient to cause maximum energy transfer between
GnRHR in the absence of Antide. Together, these results suggest
that within the population of GnRHRs only agonist-occupied receptors
will self-aggregate and that the presence of Antide, even at low
concentrations, significantly reduces receptor-receptor
interactions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We find that the majority of unoccupied GnRHRs display lateral movement in the plasma membrane. Additionally, the rate of lateral diffusion of the unoccupied GnRHR is very rapid. Increasing concentrations of either agonist or antagonist slows the rate of lateral diffusion in a dose-dependent fashion. In contrast, only agonist and superagonist lead to a dose-dependent decrease in the fraction of laterally mobile receptors. Thus, a reduced rate of lateral diffusion of the GnRHR is apparent after the binding of either agonist, superagonist, or antagonist whereas a reduction in the percentage of laterally mobile receptors is observed only after the binding of a functional ligand that is capable of initiating intracellular signaling.
The slowing of diffusion could be attributed, at least in part, to incorporation of the GnRHR into protein complexes within the membrane. Even though antagonist-occupied receptors slow their rate of lateral diffusion, perhaps by incorporation into protein complexes, the mobile fraction does not change significantly. These results would be consistent with formation of small, nonfunctional complexes containing GnRHR.
The reduction in the fraction of mobile receptors may, alternatively, result from "trapping" of the receptor within microdomains in the plasma membrane. Recently, there has been increased speculation that the plasma membrane contains small domains with specialized functions (30). A number of plasma membrane receptors, including the IgA receptor (31), epidermal growth factor receptor (32), and the tissue factor receptor (31), are preferentially distributed into discrete domains. In some cases these microdomains are detergent-insoluble membrane fragments that exhibit high buoyancy after isopycnic centrifugation and can contain high concentrations of sphingomyelin and cholesterol as well as membrane proteins involved in cell signaling such as G proteins (31). It is possible that binding of hormone to the GnRHRs not only drives receptors into microdomains within which receptor motions are constrained. If a large number of receptors were constrained within these domains, the fraction of immobile receptors would appear to be large in measurements of fluorescence photobleaching recovery.
Another critical difference between functional and
nonfunctional complexes was the appearance of large extents of receptor
self-association under conditions in which receptors were capable of
activating adenylate cyclase. Janovick and Conn (17) have hypothesized
that the GnRHR is found within microaggregates after binding of agonist
and that it is the presence of the receptor within these protein
complexes that allows for the initiation of signal transduction. This
appears to be the case in our studies. However, by stably coexpressing
GnRHRs fused to GFP or YFP in CHO cells, we were able to examine
whether GnRHRs were self-associated both before and after exposure to
ligand. As predicted, the unoccupied GnRHR was not self-associated, and
treatment of cells with agonist led to an dose-dependent increase in
energy transfer efficiency. There was no significant increase in energy
transfer between receptors when treated with Antide, even at the
highest concentration. Interestingly, when GnRHRs are simultaneously
treated with both agonist and antagonist, the presence of excess
antagonist blocks energy transfer between agonist-occupied receptors
(Fig. 6). Collectively these data are direct evidence that the GnRHR
self-associates due to binding of agonist but not antagonist.
From these data, we can suggest a general mechanism for signal transduction by GnRHRs. We hypothesize that GnRHR exists as isolated membrane proteins that diffuse freely in the plasma membrane. Upon binding of hormone agonists, GnRHRs undergo a conformational change that exposes contact sites on one or more of the receptor’s transmembrane domains. A dimerization motif has been identified for the ß-adrenergic receptor on the sixth transmembrane domain (11). Interactions between GnRHRs via similar sites may result in the formation of receptor aggregates. During receptor self-association, or perhaps as a result of receptor self-association, other proteins involved in signaling interact with the receptor. Thus, a complex is formed containing both receptors and nonreceptor proteins that is sufficiently large to exhibit significantly slower rates of lateral diffusion than does the isolated, monomeric receptor. When the receptor is functional, a large fraction of these complexes become laterally immobile.
The hypothetical situation for nonsignaling receptors would be
somewhat different. GnRHRs bound with antagonist have diffusion
coefficients similar to those of their functional counterparts and
significantly slower diffusion than unoccupied receptors. We suggest
that GnRHRs, when bound by antagonist, are present in slowly diffusing
complexes that do not contain the full complement of proteins required
for signaling or, alternatively, do not reach membrane microdomains
containing necessary proteins for initiation of productive signaling.
In either case, the ability of GnRHR to transduce signal appears to
require receptor self-association. Further, our biophysical assessment
of GnRHR self-association is in agreement with other biochemical
studies in which G protein-coupled receptors, such as the
ß-adrenergic receptor and the 
-opioid receptor (11, 33), form
dimers after binding of ligand. That this process is important in
receptor function can be demonstrated by functional rescue of
M3 muscarinic receptors containing two reciprocal
nonfunctional mutations (12).
These studies of receptor self-association and membrane domains, together with the development of new biophysical methods to examine membrane proteins in living cells, should considerably advance our understanding of GnRHR signaling mechanisms. In particular, methods for monitoring the organization and distribution of single receptors within the membrane will establish whether the plasma membrane environment of the GnRHRs and the organization of receptors within that environment differs significantly after the binding of GnRH agonists vs. antagonists.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of pGnRHR-YFP
Plasmid pEYFP (CLONTECH Laboratories, Inc. Palo
Alto CA) was digested with NotI and NcoI to
liberate the YFP coding sequence. The YFP cDNA was then ligated into
pEGFP-N2 (CLONTECH Laboratories, Inc.), which had been
digested to completion with NotI followed by partial
digestion with NcoI to remove the GFP coding sequence. This
ligation yielded pEYFP-N2 in which YFP is placed in the correct reading
frame for fusion to the carboxyl terminus of the murine GnRHR cDNA.
The mGnRHR cDNA was liberated from pGnRHR-GFP (9) by digestion with
EcoRI and BamHI and then ligated into pEYFP-N2
digested with the same enzymes yielding pGnRHR-YFP.
Construction of Stable Cell Lines
pGnRHR-GFP (0.4 µ g) and pGnRHR-YFP (1.2 µg) were
transfected into CHO cells using Lipofectamine Plus Reagent (Life Technologies, Inc., Gaithersburg, MD) in a 35-mm plate (ISC
Bioexpress, Kaysville, UT) (9). After overnight culture, the cells were
transferred into a 150-mm plate and cultured with 600 µg/ml G418
(Gemini Bioproducts, Woodland CA) for 7 days, washed with PBS, and
cloned by limiting dilution in 96-well plates (ISC Bioexpress). Wells
were examined with epifluorescence for fluorescent cells. Clonal
cell lines expressing fluorescence were expanded and used for further
studies. CHO cells expressing GnRHR-YFP were produced by the same
method omitting pGnRHR-GFP from the transfection.
Cell Culture
Cells were cultured in Eagle’s modified media with high glucose
(DMEM) containing 10% FBS, 100 U/ml penicillin, 100 µg/ml
streptomycin sulfate, 292 µg/ml glutamine (Gemini Bioproducts), 1x nonessential amino acids, in
high-glucose DMEM (Life Technologies, Inc.), in a 5%
CO2, humidified atmosphere at 37 C.
Scatchard Analysis
Approximately 100,000 cells per well of the CHO GnRHR-YFP and
CHO GnRHR-GFP/YFP cell lines were plated in 24-well plates (BD
Biosciences, Bedford, MA) and cultured overnight. Varying
concentrations of freshly prepared
[125I]-des-Gly10,
D-Ala6-GnRH N-ethyl amide
(specific activity of 1.5 µCi/pmol) in the range of 4.5
nM to 20 pM in 150 µl of
ice-cold complete media were then added to each well in the presence or
absence of 260 nM of unlabeled
D-Ala6-GnRH
N-ethyl amide (9). Cells were incubated on ice for 4 h
and then washed twice with 1 ml of 170 mM NaCl, 3
mM KCl, 10 mM
Na2HPO4, and 4
mM
K2HPO4, pH 7.4 (PBS)
containing 2 mg/ml BSA. The cells were lysed in 100 µl of 1% SDS
(sodium dodecyl lauryl sulfate) and counted on an Apex Automatic Gamma
Counter (Micromedic Systems, Inc., Horsham, PA). Specific counts were
determined as total counts per minute bound less the counts per minute
bound in the presence of 260 nM of unlabeled
D-Ala6-GnRH N-ethyl amide.
Data were analyzed by a nonlinear regression using Prism Software
(GraphPad Software, Inc., San Diego, CA) using a one-site
model. A minimum of two independent experiments were conducted.
cAMP Assays
CHO (1 x 106) cells expressing
GnRHR-GFP, GnRHR-YFP, or GnRHR-GFP/GnRHR-YFP were incubated with 0,
0.01, 0.1, 1, 10, or 100 nM GnRH,
D-ala6-GnRH or Antide in a total
volume of 0.5 ml in HBSS for 1 h at 37 C. Cells were lysed with
ice-cold 10% trichloroacetic acid and the cAMP was extracted with
ether and dried under N2. The cAMP was assayed
for by enzyme-linked immunosorbent assay (ELISA) (PE Applied Biosystems, Framingham, MA) (9), and data were analyzed
by ANOVA (SAS Institute, Inc., Cary, NC). 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
cAMP kits.
Spot Fluorescence Photobleaching Recovery Analysis of GnRHR-GFP
Lateral Diffusion
The equipment and methods used for performing spot fluorescence
photobleaching recovery (FPR) measurements have been described in
detail elsewhere (34). In these studies, all measurements were
performed at room temperature using a Carl Zeiss
Axiomat-based instrument and a 40x microscope objective (Carl Zeiss, Thornwood, NY). For spot FPR measurements on individual
cells, a Coherent Radiation Innova 100 Argon ion laser interrogated an
area of the cell with a 1/e2 radius of 0.41 µm.
The laser provided power of 53 mW in the bleaching beam and 0.2 mW in
the probe beam at 488 nm. Fluorophore bleaching time was 150 msec in
the spot FPR measurements. In an individual FPR experiment, data were
acquired for 20 sec before fluorophore bleaching and for 30 sec
postbleach at a rate of 50 msec/point. FPR data were processed as
described previously (34). To assess the effect of GnRH,
D-Ala6-GnRH, or Antide on the
GnRHR-GFP lateral dynamics, each ligand was added to a final
concentration of 0, 0.01, 1.0, 10, or 100 nM 5 min before
the acquisition of data and remained at this concentration for the
duration of the data acquisition, which was approximately 45 min.
Spot Fluorescent Energy Transfer between GnRHR-GFP and
GnRHR-YFP
Fluorescence energy transfer between GnRHR-GFP and GnRHR-YFP
was evaluated based on the reduced rate of irreversible photobleaching
of GFP fluorophores when YFP fluorophores were present (20). Slower
rates of fluorescence decay for cells expressing the GnRHR-GFP donor
and GnRHR-YFP acceptor (D+A) than for cells expressing
GnRHR-GFP alone (D) are indicative of energy transfer from
fluorescence donor to acceptor and occurs only when the donor and
acceptor are separated by a distance less than approximately 100 Å.
The Forster distance or Ro for the GFP-YFP pair
is 51 Å (21). To perform these experiments, we used a fluorescence
microscope photometer based on the inverted-configuration Axiomat
microscope (Carl Zeiss). Fluorescence excitation was
provided by a Coherent Radiation Innova 100 argon ion laser operating
under light control at 488 nm. The intensity of the laser radiation
focused on the cell was 30 mW, and this was held constant between
measurements on GnRHR-GFP cells or on GnRHR-GFP/YFP cells. The
1/e2 Gaussian spot diameter was 0.41 µm. Donor
fluorescence from GFP was isolated with a standard fluorescein filter
set together with a short pass fluorescein-selective filter to remove
yellow fluorescence from the YFP-tagged GnRHR. This combination was
effective in eliminating 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 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. In each experiment, four sets of identically handled cells were
examined including untransfected CHO cells, CHO cells expressing
GnRHR-GFP alone, CHO cells expressing GnRH-YFP alone, and cells
expressing both GnRHR-GFP and GnRHR-YFP. Cells expressing GnRHR-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 GnRHR-GFP or GnRHR-GFP/YFP was greater
than 9-fold higher than background levels from untransfected CHO cells
or cells expressing GnRHR-YFP. Thus, the rate constants for
photobleaching of GFP on cells expressing GnRHR-GFP alone
(kD) or GnRHR-GFP/YFP (kDA)
were analyzed from data traces as described in detail previously
(35). There was no significant difference (P < 0.05)
between the rate constants for photobleaching of GFP in cells
expressing GnRHR-GFP alone. These rate constants were consistently
5.2–5.7 sec-1 and were not affected by binding
of ligand. The energy transfer efficiency was expressed as a percent
(%E) and was calculated from these rate constants using %E =
(1 - kDA/kD) x
100 (36). To assess the effect of GnRH,
D-Ala6-GnRH, or Antide on
the self-association of GnRHRs, each ligand was added to a final
concentration of 0, 0.01, 1.0, 10, or 100 nM 5
min before the acquisition of data and remained at this concentration
for the duration of the data acquisition, which was approximately 20
min.
Statistical Analysis
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 (20). Since
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>±
2 s 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 multiple treatment
groups were made using single classification ANOVA methods (SigmaStat,
Jandel Scientific, San Rafael, CA).
The average cAMP, lateral diffusion, and energy transfer data were analyzed by one-way ANOVA using SigmaStat (Jandel Scientific). If the F test was significant (P < 0.01), means were separated using the least significant difference (LSD) criterion. Data are presented as the mean ± SD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by NIH Grants R01-HD-32416 (C.M.C.) and R01-HD-23236 (D.A.R.) and the Colorado State University Experiment Station.
Received for publication August 17, 2000. Revision received January 19, 2001. Accepted for publication February 6, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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R. C. Patel, U. Kumar, D. C. Lamb, J. S. Eid, M. Rocheville, M. Grant, A. Rani, T. Hazlett, S. C. Patel, E. Gratton, et al. Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells PNAS, March 5, 2002; 99(5): 3294 - 3299. [Abstract] [Full Text] [PDF]
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),
D-Ala6-GnRH
(
), or Antide
(
). Intracellular
concentrations of cAMP were determined by ELISA after 1-h treatment
(A). To assess the effects of ligand binding on rate of lateral
diffusion (diffusion coefficient) (B) and percentage of laterally
mobile receptors (% Recovery) (C), each ligand was added at the
indicated concentration 5 min before data acquisition and remained at
this concentration for the duration of data acquisition, which was
approximately 45 min. Data were analyzed by one-way ANOVA, and means
were separated using least significant differences (LSD) criteria.
Values indicated with a * were different (P <
0.01) from untreated cells. The mean and SDs for diffusion
coefficient and %R were calculated from at least 60 measurements on
individual cells.
). Fluorescence decay
(cps) was measured and % energy transfer efficiency calculated as
described in Materials and Methods. Hormone treatments
were applied 5 min before data acquisition and remained at the
indicated concentrations for the duration of data acquisition (
20
min). Data (mean ± SD) were analyzed by one-way
ANOVA, and means were separated using LSD criteria. *, Values were
different (P < 0.01) from untreated cells.