FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.07-8180com.
 (The FASEB Journal. 2008;22:850-860.)
© 2008 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.07-8180comv1
22/3/850    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cordeaux, Y.
Right arrow Articles by Hill, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cordeaux, Y.
Right arrow Articles by Hill, S. J.
(The FASEB Journal. 2008;22:850-860.)
© 2008 FASEB

Agonist-occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells

Y. Cordeaux*,1,2, S. J. Briddon*,1, S. P. H. Alexander*, B. Kellam{dagger} and S. J. Hill*,3

* Institute of Cell Signalling, School of Biomedical Sciences, Medical School, and

{dagger} Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, UK

3Correspondence: Institute of Cell Signalling, C Floor, Medical School, Queen’s Medical Centre, Nottingham, NG7 2UH, UK. E-mail: stephen.hill{at}nottingham.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors are known to be organized within different membrane compartments or microdomains of individual cells. Here, we have used a fluorescent A3 adenosine receptor (A3-AR) agonist, ABEA-X-BY630, and the technique of fluorescence correlation spectroscopy (FCS) to investigate the diffusional characteristics of functional agonist-occupied A3-AR complexes in single living cells. In Chinese hamster ovary cells expressing the human A3-AR, the fluorescent A3-AR agonist was able to inhibit forskolin-stimulated [3H]cAMP production (pEC50=8.57), and this was antagonized by the A3-selective antagonist MRS1220 (pKB=9.32). The fluorescent ligand also stimulated phosphoinositide hydrolysis (pEC50=7.34). Ligand binding to the A3-AR on the membranes of single cells and subsequent increases in single cell [Ca2+]i were monitored simultaneously in real time using confocal microscopy. FCS measurements in small-membrane microdomains (~0.2 µm2) revealed two agonist-occupied A3-AR components with differing diffusion characteristics (diffusion coefficients=2.65x10–8 and 1.19x10–9 cm2/s, respectively). The binding of ligand to these two components was reduced from 5.1 and 14.9 to 2.6 and 3.3 receptors/µm2, respectively, by MRS1220 (100 nM). These data provide direct evidence for at least two populations of agonist-occupied A3-receptor complexes, showing different motilities within the membrane of single living cells. Cordeaux, Y., Briddon, S. J., Alexander, S. P. H., Kellam, B., Hill, S. J. Agonist-occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells.


Key Words: fluorescence correlation spectroscopy • GPCRs • confocal imaging


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ENDOGENOUS NUCLEOSIDE adenosine mediates the majority of its physiological effects via the activation of four G protein-coupled receptors (GPCRs): A1, A2A, A2B, and A3 (1) . The A3 adenosine receptor (A3-AR) is the most recently identified subtype of these receptors (2 3 4) and is widely distributed throughout the body (5 6) . This distribution, coupled with functional characterization, has led to suggested therapeutic use for both agonist and antagonist A3-AR ligands. In particular, areas such as neuroprotection and cardioprotection (5 , 7 , 8) , inflammation, immunity (5 , 9 , 10) , and cancer (11 , 12) have been targeted. Like the A1 adenosine receptor, the A3-AR couples predominantly to pertussis toxin-sensitive G proteins of the Gi/o family (3 , 13 , 14) . This leads to inhibition of adenylyl cyclase, and, via the release of G-protein β{gamma} subunits, this receptor can also stimulate phospholipase C activity (3 , 13 , 14) .

Our current understanding of A3-AR pharmacology has arisen predominantly from the use of conventional techniques for investigating ligand-receptor interactions, such as radioligand binding and measurement of intracellular second messenger generation in large populations of cells (4 , 14 15 16) . As a consequence, the ligand binding and functional properties of GPCRs in a particular cell population represent an average of the receptor occupancy and signaling outcomes of the individual cells within that population. It is now evident, however, that GPCRs are not uniformly distributed at the cell surface, but instead are organized within membrane compartments and microdomains (17 18 19) , providing a mechanism by which intracellular signaling can be orchestrated in different areas of an individual cell. These observations suggest that there may be marked heterogeneity between individual cells in terms of both ligand binding characteristics and the intracellular responses that result. For example, in the study of A1 adenosine receptor-stimulated calcium signaling in primary rat astrocytes, the oscillatory calcium responses induced by agonist stimulation varied markedly between individual cells (20) .

The introduction of fluorescence-based techniques such as confocal microscopy and fluorescence correlation spectroscopy (FCS) has advanced the study of GPCR pharmacology to the single cell level (21 22 23 24) . To apply these techniques to the study of ligand binding to GPCRs in single cells, there is a clear need for fluorescent ligands with appropriate photochemical and pharmacological properties (21 , 25) . With this in mind, we have recently developed a fluorescent derivative of the adenosine receptor antagonist xanthine amine congener (22) . This molecule enabled ligand binding to be monitored in real time in single Chinese hamster ovary (CHO) cells expressing the human A1 adenosine receptor. Furthermore, the use of this ligand in association with the technique of FCS has provided novel information on the molecular properties and diffusional characteristics of the A1-receptor in microdomains of single living cells (22) . Here, we have used a fluorescent adenosine-receptor ligand (ABEA-X-BY630) (26) to investigate the diffusional characteristics of single agonist-occupied A3 adenosine receptor complexes in membranes of living cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
[2-3H]myo-inositol, [2,8-3H]adenine, and [125I]I-AB-MECA were purchased from Amersham Biosciences (Little Chalfont, Bucks, UK). Forskolin, rolipram, 5'-N-ethylcarbamoyladenosine (NECA), and (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine) (I-AB-MECA) were from Sigma-Aldrich (Gillingham, Dorset, UK). MRS1220 (N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide), and IB-MECA (N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine) were purchased from Tocris Cookson (Bristol, UK). ABEA-X-BY630 ((2S,3S,4R,5R,E)-N-ethyl-3,4-dihydroxy-5-(6-(4-(6-(2-(4-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)hexanamido)butylamino)-9H-purin-9-yl)tetrahydrofuran-2-carboxamide; Fig. 1 ) was synthesized as described previously (26) . Pertussis toxin was obtained from Calbiochem (Nottingham, UK). Dulbecco’s modified Eagle’s medium/Nutrient Mix F-12 (1:1) and fetal calf serum were from Sigma-Aldrich. All other chemicals were of analytical grade.


Figure 1
View larger version (5K):
[in this window]
[in a new window]

  		Figure 1. The structure of ABEA-X-BY630.

Measurement of [125I]I-AB-MECA binding to CHO-A3 cells
CHO cells expressing the human A3 adenosine receptor (CHO-A3 cells) (26) were grown to confluency in 96-well cluster dishes (Corning, Fisher Scientific, Loughborough, Leics., UK). Competition binding assays were performed in Hank’s buffered salt solution (Sigma) containing 20 mM HEPES (pH 7.4) (HHB). Wells were incubated, in triplicate, with [125I]I-AB-MECA (~0.15 nM) in the presence of increasing concentrations of (unlabeled) I-AB-MECA, in a total volume of 100 µl for 1 h at 37°C. Nonspecific binding was determined in the presence of 1 µM MRS1220. Incubations were terminated by rapid aspiration of buffer and washing cells with ice-cold HHB (3x200 µl). Cells were removed by incubating overnight with 0.5 M NaOH (50 µl), and samples were counted using a Packard gamma counter. Protein was determined by the method of Lowry using bovine serum albumin as a standard (27) .

Measurement of [3H]cAMP and [3H]inositol phosphate accumulation
Samples for [3H]cAMP accumulation were generated from confluent CHO-A3 cells in 24-well plates following labeling with [3H]adenine, as described previously (28) . [3H]cAMP was isolated by single-column alumina chromatography according to the method of Alvarez and Daniels (29) . For [3H]inositol phosphate accumulation assays, confluent cell monolayers were loaded for 24 h with [2-3H]myo-inositol (37 kBq/well) in 24-well cluster dishes in inositol-free DMEM containing 1% fetal calf serum. Cells were stimulated and [3H]inositol phosphates were isolated as described previously (28) . Where stated, cells were incubated overnight with pertussis toxin (100 ng/ml).

Simultaneous imaging of ligand binding and calcium signaling in CHO-A3 cells
For live cell confocal imaging of ligand binding and changes in intracellular calcium, CHO-A3 cells were grown to 70% confluency in 35-mm glass-bottomed microwell dishes (MatTek, Ashland, MA, USA). Where stated, cells were incubated overnight with pertussis toxin (100 ng/ml). Fluo-4AM aliquots (50 µg) were initially prepared as a 1 mM solution in DMSO containing 10% pluronic acid (Molecular Probes, Leiden, The Netherlands). Cells were loaded in 2.3 µM Fluo-4AM in DMEM supplemented with 10% FCS and 2.5 mM probenecid (Sigma) for 1 h at 37°C in a humidified atmosphere of 5% CO2. Cells were then washed twice with Hank’s buffered saline (HBS) solution (147 mM NaCl, 24 mM KCl, 1.3 mM CaCl2, 1 mM MgSO4, 10 mM HEPES, pH 7.4), supplemented with 2.5 mM probenecid, and the incubation continued for 30 min at 37°C before stimulation with ABEA-X-BY630. Where stated, cells were incubated at this stage with the antagonist MRS1220 (1 µM). For experiments carried out in calcium-free conditions, cells were incubated for 30 min in HBS lacking CaCl2 and supplemented with 0.1 mM EGTA.

Images were obtained using a Zeiss LSM510 confocal microscope with an x40 Plan-NeoFluar 1.3NA oil-immersion objective lens (Carl Zeiss, Jena, Germany). Cells were excited simultaneously at 488 and 633 nm, with emission light split through a NFT570 dichroic and collected through LP650 (ligand) and BP505–550 (fluo-4) filters. Images were collected prior to the addition of agonist to confirm that cells showed stable basal calcium levels with minimal oscillations. Experiments were initiated by the addition of agonist (200 µl at 10x final concentration) immediately after capturing the first image, then images were collected every 4 s for 30 min. To avoid cell disruption, agonist was added slowly (over 4 s) and applied to cells distal from the field of view. Images comparing control cells and those pretreated with antagonist or pertussis toxin were collected on the same day using identical laser power, offset, and gain. Images were analyzed using AIM software (Carl Zeiss). Regions of interest (ROIs) were chosen from a cytoplasmic area (for Fluo-4 intensity) and from the cell membrane (for ligand intensity), and changes in fluorescence intensity over time were analyzed. Data were collected from three or more independent experiments, using at least 30 cells in the field of view. Data are expressed as the percentage of cells responding (i.e., showing at least one oscillation in [Ca2+]i in response to agonist addition) or mean number of oscillations per cell.

FCS measurement of ligand binding
CHO-A3 cells were seeded onto Nunc Labtek 8-well plates 48 h prior to experimentation in phenol red-free DMEM-F12 containing 2 mM glutamine and 10% FCS. Where indicated, cells were incubated overnight with pertussis toxin (100 ng/ml). On the day of experiment, cells were washed twice in HEPES-buffered saline solution (HBSS; ref. 22 ), before incubation in the absence or presence of the indicated concentrations of either the A3-AR antagonist MRS1220 (30 min, 37°C) or the agonist NECA (10 min, 22°C). A shorter room-temperature incubation was chosen than that used with the MRS1220 in order to minimize any effect of NECA on receptor desensitization and internalization. Cells were allowed to equilibrate to 22°C, and were then exposed to the indicated concentrations of ABEA-X-BY630 for 10 min. Subsequent FCS measurements were taken on the upper membrane of individual cells using a Zeiss Confocor2 microscope, as described previously (26) . For consistency, the detection volume was always positioned over the nucleus to allow easy identification of the upper membrane peak and to ensure the membrane was consistently oriented for each measurement. For recording of fluctuations, the measurement volume was positioned at the point at which the membrane intensity decreased to 50% of its peak value. These two procedures ensured, as far as was practicable, that the same area of cell membrane was included in each individual measurement. Fluorescence fluctuations were recorded using 633 nm excitation (0.5–1 kW/cm2), and collected through an LP650 filter for 2 x 30 s following a 10-s prebleach step. Data were analyzed using standard autocorrelation analysis within the Zeiss AIM software. Autocorrelation curves were fitted to a model assuming three diffusion components in addition to a triplet state of the fluorophore as described previously (22 , 26) . Binding was quantified using the particle number (N) obtained from the fitted autocorrelation curve and the appropriate contribution of the identified component ({tau}D2 or {tau}D3). Total binding represents the sum of the {tau}D2 and {tau}D3 components. For each experiment, the diffusion time of 10 nM Cy5 was measured. The radius of the detection volume at the beam waist ({omega}0) was then calculated (according to {omega}0=(4.D.{tau}D)1/2; with D=3.16x10–6 cm2/s), and this area was used to determine the value of particle per µm2 (N/µm2). Generally, {omega}0 was determined to be 0.25 µm, giving a measurement area of 0.20 µm2. The n values quoted are for the number of cells measured, over at least 5 independent experiments.

For confocal imaging of ligand binding under those conditions used for FCS (Fig. 8) , cells were prepared as described for FCS above. Single equatorial confocal slices were taken from cells exposed to ABEA-X-BY630 (2.5 nM, 10 min, 22°C) on a Zeiss LSM510META NLO confocal microscope. Images were collected through an x63 c-Apochromat 1.2 NA water-immersion objective lens using 633-nm excitation, and emission was collected through a BP650–710 filter. Images within each set of experiments were collected in identical settings of laser power, detector gain, and offset.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		Figure 2. [3H]cAMP and [3H]inositol phosphate accumulation in CHO-A3 cells in response to agonists. A) [3H]cAMP accumulation was measured in cells exposed to ABEA-X-BY630 in the absence (open circles) or presence (solid circles) of 10 nM MRS 1220. Data are expressed as percentage of the [3H]cAMP level obtained with 3 µM forskolin. Each point represents mean ± SE obtained from five independent experiments performed in triplicate. B) Stimulation of [3H]inositol phosphate accumulation by IB-MECA (solid circles), ABEA-X-BY630 (open circles) or NECA (open squares). Data are expressed as a percentage of the response obtained with 10–5M agonist. Results shown are means± SE of triplicates within a single experiment, representative of four independent experiments performed.


Figure 3
View larger version (93K):
[in this window]
[in a new window]

  		Figure 3. Simultaneous live cell confocal imaging of ABEA-X-BY630 binding and changes in [Ca2+]i in CHO-A3 cells. A) CHO-A3 cells were loaded with Fluo-4 (green channel) as described in Materials and Methods and stimulated with 100 nM ABEA-X-BY630 (red channel). The panel shows representative images from a single experiment at the times after agonist addition indicated in each panel. B) Fluorescence intensity changes were recorded from membrane regions of interest (ROIs) in the red channel to indicate ligand binding (red traces) or cytoplasmic ROIs in the green channel to indicate changes in [Ca2+]i (green traces, illustrated in the inset for cell 2). Six examples traces are shown, corresponding to the six cells labeled in A. The example shown is from one experiment representative of three performed.


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Figure 4. Comparison of membrane-bound ABEA-X-BY630 and changes in [Ca2+]i in CHO-A3 cells in the presence of MRS1220 and pertussis toxin. CHO-A3 cells were loaded with Fluo-4 and pretreated with vehicle, MRS1220 (1 µM, 30 min) or 100 ng/ml pertussis toxin (PTx, 18 h), prior to stimulation with 100 nM ABEA-X-BY630. Traces from three representative cells showing membrane binding of ligand (dark trace) and [Ca2+]i (light trace) for each condition are shown. Each of the PTx-treated cells showed a subsequent increase in [Ca2+]i in response to 100 µM UTP. The cells shown are representative of at least 30 analyzed in each of 3 or 4 independent experiments. In each case, a population response is also shown, determined from an ROI containing all cells within the field of view.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Figure 5. FCS measurements of ABEA-X-BY630 binding to the upper membrane of CHO-A3 cells. CHO-A3 cells were incubated with 2.5 nM ABEA-X-BY630 (10 min, 22°C). The confocal volume was positioned using a live image and FCS measurements recorded as described in Materials and Methods. A) The detection volume was positioned 4 µm above the membrane to measure extracellular ligand. Examples of fluorescence fluctuations (top), autocorrelation analysis (middle) and residuals from curve-fitting (bottom) are shown. B) As for A, except the measurement volume has been positioned on the membrane/extracellular boundary, at the point at which the peak membrane intensity has decayed by 50%. C) As for B, performed in cells preincubated with 100 nM MRS1220 (60 min, 37°C). In each case, the particle number (N), the dwell times ({tau}D), and their relative proportions obtained from iterative curve-fitting of the autocorrelation data are given.


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		Figure 6. Quantification of agonist-A3-receptor complexes on the upper membrane of CHO-A3 cells. A) CHO-A3 cells were incubated with 2.5 nM ABEA-X-BY630 for 10 min in the absence (solid bars, n=33) or presence (open bars, n=27) of 100 nM MRS1220 (60 min, 37°C). FCS measurements on the upper cell membrane were used to quantify the total ABEA-X-BY630 bound ({tau}D2+{tau}D3), and the respective amounts of {tau}D2 and {tau}D3 individually. **P < 0.01, ***P < 0.001 vs. control measurement. B) CHO-A3 cells were incubated with 1 nM (n=8, solid bars), 2.5 nM (n=33, open bars), or 5 nM (n=17, hatched bars) ABEA-X-BY630 for 10 min, and FCS measurement taken on the upper cell membrane. The total number of agonist-receptor complexes, along with those representing either {tau}D2 or {tau}D3, were calculated from the autocorrelation analysis. Statistical analysis was via one-way ANOVA with post hoc Newman-Keuls test. *P < 0.001 vs. 1 nM, **P < 0.001 vs. 1 and 2.5 nM, #P < 0.001 vs. 1 nM and P > 0.05 vs. 2.5 nM.


Figure 7
View larger version (25K):
[in this window]
[in a new window]

  		Figure 7. Effect of NECA (A) and MRS1220 (B) on the formation of agonist-A3-receptor complexes in CHO-A3 cell membranes. A) CHO-A3 cells were incubated with the indicated concentrations of NECA (10 min, 22°C) followed by ABEA-X-BY630 (2.5 nM, 10 min, 22°C). Subsequent FCS measurements were performed on the upper cell membrane as described, and the total ABEA-X-BY630 bound ({tau}D2+{tau}D3) was quantified, along with the respective amounts of {tau}D2 and {tau}D3 individually. Results are shown as the mean±SE of single measurements from 12 to 24 individual cells taken over at least 5 independent experiments B) As for A except cells were incubated with the indicated concentrations of MRS1220 (60 min, 37°C) prior to addition of ABEA-X-BY630. Results are shown as the mean±SE of single measurment from 12 to 32 individual cells taken over at least 5 independent experiments. In both cases, results were analyzed by one-way ANOVA and post hoc Newman Keuls test. *P < 0.05, **P < 0.001 vs. control value.


Figure 8
View larger version (55K):
[in this window]
[in a new window]

  		Figure 8. Effect of pertussis toxin on the formation of agonist-A3 receptor complexes on CHO-A3 cell membranes. A) CHO-A3 cells were incubated overnight in the presence (gray and hatched bars) or absence (black and clear bars) of pertussis toxin (100 ng/ml). Cells were subsequently incubated for a further 60 min at 37°C with either buffer (black and gray bars) or 100 nM MRS1220 (clear and hatched bars). FCS measurements were then performed on the upper cell membrane and the total ABEA-X-BY630 bound ({tau}D2+{tau}D3), as well as the respective amounts of {tau}D2 and {tau}D3 quantified. Results are shown as mean ± SE of individual measurements taken on 28–30 cells over the course of at least 6 separate experiments. Results were analyzed by one-way ANOVA and post hoc Newman-Keuls test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. corresponding values in the absence of MRS1220. B) Single equatorial slice confocal images taken of CHO-A3 cells following incubation with 2.5nM ABEA-X-BY630 under identical conditions to those described in A, with the corresponding phase image shown below. Images shown are from the same single experiment, representative of 4 performed.

Data analysis
pEC50 (and similarly pIC50 for inhibitory responses) values were obtained by computer-assisted curve fitting (GraphPad Prism, San Diego, CA, USA) of data to the equation:

Formula
The same program was also used to perform nonlinear regression analysis of homologous radioligand binding competition experiments. The pKi for [125I]I-AB-MECA was calculated from the IC50 of I-AB-MECA using the equation:

Formula
where [L*] is the concentration of [125I]I-AB-MECA. BMAX was calculated from the specific binding obtained with the fixed concentration of [125I]I-AB-MECA employed using the equation:

Formula
Statistical significance was determined by either Student’s unpaired t test or ANOVA with post hoc Neuman-Keuls analysis. All data are presented as means ± SE. The n in the text refers to the number of separate experiments performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of [3H]cAMP accumulation in CHO-A3 cells
A single CHO-A3 cell line (26) was used throughout the present study ([125I]I-AB-MECA binding: BMAX=765±22 fmol/mg protein, pKi=8.36±0.01, n=3). In these cells, the A3 receptor-selective agonist, IB-MECA, the nonselective agonist NECA, and its fluorescent derivative ABEA-X-BY630 (Fig. 2 A) produced concentration-dependent inhibitions of forskolin-stimulated cAMP accumulation (pEC50: 9.31±0.20, n=4, 8.34±0.12, n=4, and 8.57±0.22, n=5, respectively; pEC50 value for ABEA-X-BY630 as given in ref. 26 ). The maximal extent of the inhibition of forksolin-stimulated cAMP production obtained with each agonist was 89 ± 4, 96 ± 2, and 96 ± 2% for IB-MECA, NECA, and ABEA-X-BY630, respectively. The responses were mediated through activation of the A3-receptor, since preincubation of the cells with the A3 receptor-selective antagonist MRS1220 caused a rightward shift in the concentration-response curve, yielding a similar affinity in each case (pKB: 9.45±0.21, n=4, 9.47±0.07, n=4, and 9.32±0.09, n=5 respectively; Fig. 2A ).

Stimulation of [3H]inositol phosphates accumulation
The agonists IB-MECA, NECA, and ABEA-X-BY630 also stimulated the accumulation of [3H]inositol phosphates in CHO-A3 cells (Fig. 2B ). In each case, this was achieved with a lower potency than for adenylyl cyclase inhibition (pEC50: 8.10±0.02, 7.03±0.06, and 7.34±0.07, n=4, respectively) but with the same rank order of potency. Interestingly, ABEA-X-BY630 was significantly more potent than NECA in this assay (Fig. 2B , P<0.05). For all of the three agonists, the response could be inhibited by preincubating the cells with MRS1220 (pKB: 9.02±0.13, n=4, 8.99±0.14, n=4 and 8.91±0.05, n=3, respectively). In each case, inositol phosphate accumulation was increased by ~3-fold (compared to basal levels). However, in experiments in which the maximal response achieved with 10 µM ABEA-X-BY630 was compared directly with that of 10 µM NECA or 10 µM IB-MECA, ABEA-X-BY630 was seen to be as efficacious as NECA (108±11% NECA response, n=3) but more efficacious than IB-MECA (128±5% IB-MECA response, P<0.05, Student’s t test, n=3).

Simultaneous imaging of ABEA-X-BY630 binding and changes in intracellular calcium
In CHO-A3 cells loaded with the calcium indicator, Fluo-4, no residual oscillations in intracellular calcium ([Ca2+]i) were seen following resting of the cells for 30 min. The addition of the agonist ABEA-X-BY630 (100 nM) resulted in a clear labeling of the cell membrane (Fig. 3 ). Although labeling of the cell membrane was clearly heterogeneous, all cells in the fields of view bound ABEA-X-BY630. This binding preceded a series of oscillations in [Ca2+]i in 72 ± 3% of cells measured (30 cells analyzed per experiment; n=3 separate experiments). In those cells that responded, the number of oscillations seen was variable, and these were generally asynchronous (8±1 in 30-min period following agonist addition, mean±SE, n=3; Fig. 3 ). As illustrated in Fig. 4 , this led to the interesting observation that when the cells were analyzed as a population, no change in [Ca2+]i was detected. A video illustrating these data is available in the Supplemental Data. Preincubation of the cells with the selective A3 receptor antagonist MRS1220 (1 µM, 30 min) substantially reduced both the binding of ABEA-X-BY630 and the number of cells producing at least one oscillation in [Ca2+]i (to 7±1%, n=3; Fig. 4 ). The response to ABEA-X-BY630 was also substantially decreased in cells pretreated with pertussis toxin (100 ng/ml, overnight) with only 6 ± 3% (n=3) of cells showing fluctuations in [Ca2+]i (Fig. 4) . Interestingly, the binding of ABEA-X-BY630 was unaffected following treatment with pertussis toxin. Similar experiments performed in the presence of pertussis toxin and MRS1220 showed that this binding was displaceable and specific (data not shown, but see also Fig. 8 ). Cells that did not respond to ABEA-X-BY630 did, however, produce a synchronized increase in [Ca2+]i in response to UTP (100 µM) (data not shown). When cells were stimulated with ABEA-X-BY630 in the absence of extracellular calcium, a large majority of cells responded (83±5%, n=5), but the pattern of response differed from that seen with extracellular calcium present, with the number of oscillations observed being reduced to 2.0 ± 0.3 (mean±SE), and with 48 ± 11% of cells producing a single synchronous increase in [Ca2+]i (n=5).

Analysis of the diffusion of A3-receptor-agonist complexes in CHO-A3 cells
To quantify the binding of ABEA-X-BY630 to the A3 receptor in single cells, and subsequently the diffusion characteristics of A3 receptor-agonist complexes, we used the technique of FCS. As described previously (22 , 23 , 30) , this technique analyzes fluctuations in fluorescence obtained from a small detection volume (0.5 fl) placed on or above the cell membrane. Autocorrelation analysis of these data gives information about the average dwell time in the confocal volume of both ligand-occupied receptors and also fast-moving free ligand itself. Initial FCS measurements of ABEA-X-BY630 (50 nM in HBSS) indicated that free ligand in solution had a diffusion time ({tau}D1) of 64 ± 1 µs (diffusion coefficient, D=2.44±0.40x10–6 cm2/s, n=4) (Fig. 5 A). Subsequent FCS measurements were performed on the upper membrane of single CHO-A3 cells following incubation with ABEA-X-BY630 (2.5 nM, 10 min, 22°C). Here, in addition to free ligand diffusion, two slower-diffusing species were detected ({tau}D2=5.9±0.7 ms and {tau}D3=131±15 ms, n=49; D=2.65±0.28x10–8 cm2/s, and 1.19±0.12x10–9 cm2/s, respectively) (Fig. 5B ). Quantification of this binding indicated that the very slow moving component, {tau}D3, represented the majority (75%) of the total binding (total binding=20.0±3.1, {tau}D2=5.1±0.6, and {tau}D3=14.9±2.5 receptors/µm2 (N/µm2), n=33) (Fig. 6 A). To confirm that these slower-diffusing species ({tau}D2 and {tau}D3) represented agonist-occupied A3 receptors, similar experiments were performed in cells preincubated with MRS1220 (100 nM, 37°C; Fig. 5C ). In these experiments, total binding was reduced to 5.8 ± 0.8 receptors/µm2 (n=27; P<0.001 vs. control) (Fig. 6A ). This consisted of reductions in both {tau}D2 (to 2.6±0.4 receptors/µm2, P<0.01) and {tau}D3 (to 3.3±0.4 receptors/µm2, P<0.001). MRS1220 had no significant effect on the diffusion times obtained for {tau}D2 and {tau}D3 (6.4±0.8 and 117±26 ms, D=2.44±0.27x10–8 cm2/s and 1.34±0.26x10–9 cm2/s, respectively). Thus, under these conditions, FCS was able to detect 71, 50, and 78% specific binding for total binding, {tau}D2 and {tau}D3, respectively. FCS measurements were also taken on cells that had been exposed to a range of concentrations of ABEA-X-BY630 close to its EC50 value for adenylyl cyclase inhibition (1–5 nM). As illustrated in Fig. 6B , the increase in total binding seen over this concentration range was due to a significant increase in {tau}D3, with {tau}D2 relatively unaffected.

Pharmacological characterization of slow-diffusing agonist-receptor complexes ({tau}D3)
As shown in Fig. 6A , the majority of the agonist-A3 receptor complexes in the membrane existed in a slow-diffusing form that we have designated {tau}D3. These results (Fig. 6B ) also demonstrated that the amount of {tau}D3 increased significantly over a range of agonist concentrations, which would suggest that agonist binds to this component with high affinity. Because of the limit on the highest concentrations which can be used for FCS experiments, it was difficult to assess this binding at concentrations of ABEA-X-BY630 above those shown. Instead, we performed competition experiments by preincubating CHO cells with the nonfluorescent agonist NECA (10 min, 22°C) prior to addition of ABEA-X-BY630 (2.5 nM). Preincubation with 0.3–300 nM NECA led to a concentration-dependent decrease in the amount of fluorescent agonist bound to the A3-receptor, causing a significant decrease in both the total binding and specifically, {tau}D3 (Fig. 7 A). The amount of {tau}D3 was significantly reduced at concentrations as low as 3 nM, confirming that these complexes have a high affinity for agonist ligands. Faster-diffusing complexes ({tau}D2) were less affected by NECA, especially at lower concentrations. Similarly, MRS1220 also caused substantial inhibition of total binding at concentrations of 0.3 nM and above. This inhibition was observed for both {tau}D3 and the faster-diffusing {tau}D2 component. These results confirm the specific nature of the binding represented by {tau}D2 and {tau}D3 but show that {tau}D3 has high affinity for both agonist and antagonist ligands. To investigate further the nature of this agonist binding to {tau}D3, experiments were performed in CHO-A3 cells that had been exposed to pertussis toxin overnight. Pertussis toxin ADP ribosylates a cysteine residue in the {alpha}-subunit of Gi/o G proteins and prevents their coupling to the receptor. As shown in Fig. 8 , exposure to pertussis toxin (100 ng/ml) did not reduce the specific binding of ABEA-X-BY630 to CHO-A3 cell membranes, and more specifically, the formation of {tau}D2 or {tau}D3 complexes. These results, obtained at low agonist concentrations, were confirmed using confocal microscopy (Fig. 8B ), and are consistent with those experiments described earlier using 100 nM ABEA-X-BY630 (see Fig. 4 ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our current understanding of GPCR pharmacology is largely based on data obtained from conventional biochemical studies of ligand-binding and second messenger generation in large populations of cells. These have provided information on ligand occupancy and receptor signaling characteristics that represent an average of the parameters for individual cells within that cell population. However, there is an increasing awareness that cells may vary dramatically in their individual ability to respond to stimuli (20 , 31) . Coupled with the fact that GPCRs are not distributed uniformly at the cell surface within an individual cell (18 , 19) , this has raised the need to undertake experiments that can provide detailed information on ligand-binding characteristics at the single cell and subcellular level. In the present study, we have used a fluorescent A3 receptor agonist and the FCS technique to undertake such an investigation.

The fluorescent NECA derivative ABEA-X-BY630 (26) proved to be a potent and effective agonist at the human A3-AR expressed in CHO cells. Indeed, it was more potent than NECA in both inhibiting forskolin-stimulated cAMP accumulation (pIC50: 8.57 vs. 8.34 for NECA) and in stimulating [3H]inositol phosphate accumulation (pEC50: 7.34 vs. 7.03). This latter response is less well coupled than inhibition of adenylyl cyclase and probably reflects the less efficient signal amplification mediated by Gβ{gamma} subunits (32 33 34) . A similar observation has been made for both NECA and ABEA-X-BY630 at the A1-adenosine receptor (26 , 28) . At this latter receptor, ABEA-X-BY630 is less efficacious than NECA and behaves as a partial agonist for stimulation of inositol phospholipid hydrolysis (26) . However, at the A3-receptor it has the advantage that it has the same efficacy as NECA for both responses. The availability of a high-efficacy fluorescent A3-receptor agonist, capable of stimulating inositol phospholipid hydrolysis, provided the means by which ligand binding and functional responses could be monitored simultaneously at the single living cell level using A3 receptor-mediated changes in [Ca2+]i as the functional readout.

The spectral characteristics of the red emitting ABEA-X-BY630 (26) allowed simultaneous measurement of ligand binding and [Ca2+]i (with the green emitting calcium dye fluo-4; ref. 35 ) within single CHO cells expressing the human A3-receptor. This ligand should, therefore, find great utility for high content analysis of A3-receptor pharmacology using confocal imaging plate readers. As would probably be predicted from previous studies of [Ca2+]i following GPCR stimulation at the single cell level (20 , 31) , A3 adenosine receptors were not uniformly distributed between individual cells. Instead, there was a marked heterogeneity in the intensity of specific binding with ABEA-X-BY630 in different cells. This was also reflected in the heterogeneity of calcium responses observed in the same cells, although it was notable that some 28% of cells bound ABEA-X-BY630 but did not exhibit a calcium response. The calcium change produced in response to either ABEA-X-BY630 or NECA was oscillatory in nature and not coordinated between neighboring cells. This was a very similar profile to that previously observed for A1-receptor stimulation in rat astrocytes and CHO cells (ref. 20 and unpublished observations). The calcium response (but not ABEA-X-BY630 binding) was attenuated by pertussis toxin treatment (confirming a role for Gi/o-proteins), while the selective A3-receptor antagonist, MRS1220, reduced membrane binding and prevented calcium signaling (confirming the A3-receptor-mediated nature of the effects). The uncoordinated nature of the oscillatory calcium response to A3-receptor stimulation in CHO-A3 cells emphasizes the importance of measurements at the single cell level since the mean population response appears to be negligible (Fig. 4) . Furthermore, the fact that a number of cells express A3-receptors but do not couple to calcium signaling suggests that the local membrane environment of the receptor, in particular cells, may be a major determinant of subsequent signaling activity. It is now well established that GPCRs can associate with different compartments and accessory proteins (17 , 18 , 36 , 37) and that different ligands can produce signaling pathway-dependent effects (38 39 40 41 42) . This implies that GPCRs can exist in different signaling complexes even within the same cell. There is, therefore, an urgent need to study the molecular nature of these receptor signaling complexes in membrane microdomains of a single living cell.

In this study, we have used the efficacious fluorescent A3-agonist described above (ABEA-X-BY630) and single molecule imaging approaches (FCS) to undertake the first study of the diffusional characteristics of agonist occupied A3-AR complexes in very small (~0.2 µm2) regions of a single cell membrane. This approach has provided clear evidence for two agonist-occupied A3-receptor species with markedly different diffusional characteristics ({tau}D2=5.9 ms and {tau}D3=131 ms), with the slow-diffusing species representing the majority (75%) of the total binding of ABEA-X-BY630. Both {tau}D2 and {tau}D3 components were sensitive to inhibition by the A3-receptor selective antagonist MRS1220, which significantly reduced ABEA-X-BY630 binding. These data confirmed that both diffusional species contained specific binding that was consistent with labeling of the A3-adenosine receptor.

The presence of at least two different receptor populations, distinguished by their membrane diffusion coefficients, is consistent with previous studies using FCS to investigate binding to other transmembrane receptors (43 44 45) . These studies also assigned diffusion times to ligand-receptor complexes similar to those found in our study. The average dwell time in the FCS confocal volume ({tau}D) can be related to either the relative molecular sizes or the local membrane environment of the A3-AR complexes identified. Large molecular species will clearly diffuse much more slowly through the FCS confocal volume than smaller complexes. As a rule of thumb, the dwell time, {tau}D, is doubled by an 8-fold increase in molecular mass (30 , 46) , so the differences between {tau}D2 and {tau}D3 point to a very large difference in the size of the agonist-receptor complexes. Such a large difference would indicate that the slowest A3-receptor species ({tau}D3) could equate to the diffusion of a large complex structure such as a caveola or clathrin pit, or perhaps be a consequence of scaffolding to components of the cytoskeleton. Differences in the local membrane environment will also substantially affect the translational diffusion of a receptor, and the dwell times obtained for {tau}D3 are similar to those found for markers of detergent-resistant membranes and for cytoskeletal-bound transmembrane proteins in COS-7 cells (47) . Thus the differing components may represent receptor partitioned in ({tau}D3) or outside ({tau}D2) these membrane domains.

As indicated above, the concentrations of ABEA-X-BY630 required to inhibit forskolin-stimulated adenylyl cyclase activity (pIC50=8.57; 2.7 nM) and to activate phospholipase C (pEC50=7.34; 46 nM) are very different. FCS measurements were generally conducted at 2.5 nM since the technique is most sensitive at low concentrations of fluorescent ligand. At higher ligand concentrations, the number of molecules in the confocal volume increases substantially, and this consequently limits the extent of the fluorescent fluctuations on which the FCS analysis depends (30) . Interestingly, the concentration used is very similar to the IC50 for inhibition of forskolin-stimulated [3H]cAMP accumulation. To determine how A3-receptor agonist binding (detected by FCS) varied with concentration in the range required for inhibition of adenylyl cyclase, experiments were also conducted at concentrations of ABEA-X-BY630 either side of the IC50 value for this response (1–5 nM). It was notable, however, that the binding of ABEA-X-BY630 to the slow diffusing species (i.e., {tau}D3; Fig. 5B ) increased significantly between 1 and 2.5 nM and then appeared to saturate. In contrast, the binding to the faster diffusing species ({tau}D2) appeared to increase linearly between 1 and 5 nM. This differential effect on {tau}D2 and {tau}D3, and the greater particle number for the {tau}D3 component, strongly suggests that the {tau}D2 species does not simply reflect a faster diffusion of agonist-occupied A3-receptors complexes superimposed on the diffusion ({tau}D3) of a slowly moving compartment within which they are contained (e.g., caveolae or clathrin-coated pits); the two components appear to represent discrete populations of agonist-receptor complexes. It is, therefore, tempting to speculate that the subset of agonist-occupied A3 receptors represented by {tau}D3 may be intimately involved in Gi-mediated inhibition of adenylyl cyclase activity.

Some insight into the nature of the precise receptor conformation represented by {tau}D3 is provided by the data obtained when NECA was used to inhibit the binding of ABEA-X-BY630 to this diffusional species. According to the cubic extended ternary complex model of GPCR activation (Fig. 9 ; ref. 48 ), GPCRs can exist in resting (R) or activated (R*) conformations, which may also be coupled to the G protein (RG and R*G). By definition, FCS uses low concentrations of fluorescent ligands and, therefore, operates at low receptor occupancies. In intact cells, in which the intracellular GTP concentration is high (and will therefore rapidly uncouple R*G as soon as it is formed), low concentrations of fluorescent agonists should detect predominantly AR*. The potent inhibition of the binding of the fluorescent agonist to {tau}D3 by low nanomolar concentrations of NECA is completely consistent with this. In addition, the lack of effect of pertussis toxin on agonist binding to {tau}D3 confirms that it represents a form that is not coupled to G{alpha}i. Note that {tau}D2 is much less sensitive to inhibition by NECA (but is still displaceable by the A3-antagonist MRS 1220). It is tempting to speculate that this is consistent with {tau}D2 representing binding to R rather than R*. This would also be consistent with the fact that the binding of the fluorescent agonist to {tau}D2 increased linearly between 1 and 5 nM. However, given the small size of {tau}D2, (detected in this concentration range of fluorescent agonist), a firm conclusion on this will need to await the development of a fluorescent A3-receptor antagonist (which will have a higher affinity for both R and R*) that will be able to detect R in significant amounts at the low fluorescent ligand concentrations required for FCS.


Figure 9
View larger version (12K):
[in this window]
[in a new window]

  		Figure 9. The cubic ternary complex model. This diagram, based on the cubic ternary complex model (48) , illustrates the receptor species likely to be present in cells expressing a receptor R in the presence of a ligand A. At the low concentrations of agonist ligand used in FCS experiments, the presence of high GTP concentrations in living cells will mean that mainly R* is detected.

In summary, the present study has shown that the combined use of a fluorescent A3 adenosine receptor agonist, and the techniques of FCS can provide novel information on the molecular characteristics of agonist-occupied human A3 adenosine receptors in small submicron volumes of the plasma membrane of an individual living cell. This approach has shown that the agonist-occupied forms of the human A3 adenosine receptor exist in at least two different complexes of vastly different motilities and/or molecular size. This technique has the potential to be applied to native human cells in health and disease and to provide important information on pharmacological characteristics of GPCRs in specific membrane microdomains.


   ACKNOWLEDGMENTS
 
We thank Mr. Tim Self for his assistance with the confocal microscopy. This work was supported by Wellcome Trust grants 66817 and 57199.


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Current address: University of Cambridge, Department of Obstetrics and Gynaecology, Rosie Hospital, Addenbrookes, Cambridge, CB2 2SW, UK.

Received for publication February 5, 2007. Accepted for publication September 20, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fredholm, B. B., Ijzerman, A. P., Jacobson, K. A., Klotz, K.N., Linden, J. (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol. Rev. 53,527-552[Abstract/Free Full Text]
  2. Meyerhof, W., Müller-Brechlin, R., Richter, D. (1991) Molecular cloning of a novel putative G protein-coupled receptor expressed during rat spermiogenesis. FEBS Lett. 284,155-160[CrossRef][Medline]
  3. Zhou, Q. Y., Li, C., Olah, ME, Johnson, R. A., Stiles, G. L., Civelli, O. (1992) Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor. Proc. Natl. Acad. Sci. U. S. A. 89,7432-7436[Abstract/Free Full Text]
  4. Salvatore, C. A., Jacobson, M. A., Taylor, H. E., Linden, J., Johnson, R. G. (1993) Molecular cloning and characterization of the human A3 adenosine receptor. Proc. Natl. Acad. Sci. U. S. A. 90,10365-10369[Abstract/Free Full Text]
  5. Ralevic, V., Burnstock, G. (1998) Receptors for purines and pyrimidines. Pharmacol. Rev. 5,413-492
  6. Fishman, P., Bar-Yehuda, S. (2003) Pharmacology and therapeutic applications of the A3 receptor subtype. Curr. Top. Med. Chem. 3,463-469[CrossRef][Medline]
  7. Shneyvays, V., Mamedova, L., Zinman, T., Jacobson, K., Shainberg, A. (2001) Activation of A3 adenosine receptor protects against doxorubicin-induced cardiotoxicity. J. Mol. Cell. Cardiol. 33,1249-1261[CrossRef][Medline]
  8. Maddock, H. L., Gardner, NM, Khandoudi, N., Bril, A., Broadley, K. J. (2003) Protection from myocardial stunning by ischemia and hypoxia with the adenosine A3 receptor agonist, IB-MECA. Eur. J. Pharmacol. 477,235-245[CrossRef][Medline]
  9. Livingston, M., Heaney, L. G., Ennis, M. (2004) Adenosine, inflammation and asthma—a review. Inflamm. Res. 53,171-178[CrossRef][Medline]
  10. Gessi, S., Varani, K., Merighi, S., Cattabriga, E., Avitabile, A., Gavioli, R., Fortini, C., Leung, E., MacLennan, S., Borea, PA (2004) Expression of A3 adenosine receptors in human lymphocytes: up-regulation in T cell activation. Mol. Pharmacol. 65,711-719[Abstract/Free Full Text]
  11. Madi, L., Ochaion, A., Rath-Wolfson, L., Bar-Yehuda, S., Erlanger, A., Ohana, G., Harish, A., Merimski, O., Barer, F., Fishman, P. (2004) The A3 adenosine receptor is highly expressed in tumor vs. normal cells: potential target for tumor growth inhibition. Clin. Cancer Res. 10,4472-4479[Abstract/Free Full Text]
  12. Gessi, S., Cattabriga, E., Avitabile, A., Gafa, R., Lanza, G., Cavazzini, L., Bianchi, N., Gambari, R., Feo, C., Liboni, A., Gullini, S., Leung, E., Mac-Lennan, S., Borea, P. A. (2004) Elevated expression of A3 adenosine receptors in human colorectal cancer is reflected in peripheral blood cells. Clin. Cancer Res. 10,5895-5901[Abstract/Free Full Text]
  13. Ramkumar, V., Stiles, G. L., Beaven, M. A., Ali, H. (1993) The A3 adenosine receptor is the unique adenosine receptor which facilitates release of allergic mediators in mast cells. J. Biol. Chem. 268,16887-16890[Abstract/Free Full Text]
  14. Abbracchio, M. P., Brambilla, R., Ceruti, S., Kim, H. O., von Lubitz, D. K., Jacobson, K. A., Cattabeni, F. (1995) G protein-dependent activation of phospholipase C by adenosine A3 receptors in rat brain. Mol. Pharmacol. 48,1038-1045[Abstract]
  15. Palmer, T. M., Gettys, T. W., Stiles, G. L. (1995) Differential interaction with and regulation of multiple G-proteins by the rat A3 adenosine receptor. J. Biol. Chem. 270,16895-16902[Abstract/Free Full Text]
  16. Klotz, K.N., Hessling, J., Hegler, J., Owman, C., Kull, B., Fredholm, B.B., Lohse, M.J. (1998) Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells. Naunyn Schmiedebergs Arch. Pharmacol. 357,1-9[Medline]
  17. Gines, S., Ciruela, F., Burgueno, J., Casado, V., Canela, E. I., Mallol, J., Lluis, C., Franco, R. (2001) Involvement of caveolin in ligand-induced recruitment and internalization of A1 adenosine receptor and adenosine deaminase in an epithelial cell line. Mol. Pharmacol. 59,1314-1323[Abstract/Free Full Text]
  18. Ostrom, R. S., Insel, P. A. (2004) The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br. J. Pharmacol. 143,235-245[CrossRef][Medline]
  19. Insel, P. A., Head, B. P., Ostrom, R. S., Patel, H. H., Swaney, J. S., Tang, C. M., Roth, D. M. (2005) Caveolae and lipid rafts: G protein-coupled receptor signaling microdomains in cardiac myocytes. Ann. N.Y. Acad. Sci. 1047,166-172[CrossRef][Medline]
  20. Peakman, M. C., Hill, S. J. (1995) Adenosine A1-receptor-mediated changes in basal and histamine-stimulated levels of intracellular calcium in primary rat astrocytes. Br. J. Pharmacol. 115,801-810[Medline]
  21. Daly, C. J., McGrath, J. C. (2003) Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol. Ther. 100,101-118[CrossRef][Medline]
  22. Briddon, S. J., Middleton, R. J., Cordeaux, Y., Flavin, F. M., Weinstein, J. A., George, M. W., Kellam, B., Hill, S. J. (2004) Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells. Proc. Natl. Acad. Sci. U. S. A. 101,4673-4678[Abstract/Free Full Text]
  23. Pramanik, A. (2004) Ligand-receptor interactions in live cells by fluorescence correlation spectroscopy. Curr. Pharm. Biotechnol. 5,205-212[CrossRef][Medline]
  24. Miquel, M. R., Segura, V., Ali, Z., D'ocon, M. P., McGrath, J. C., Daly, C. J. (2005) 3-D imaging of fluorescent drug binding. Mol. Imaging 4,40-52[Medline]
  25. Middleton, R. J., Kellam, B. (2005) Fluorophore-tagged GPCR ligands. Curr. Opin. Chem. Biol. 9,517-525[CrossRef][Medline]
  26. Middleton, R. J., Briddon, S. J., Cordeaux, Y., Yates, A. S., Dale, C., George, M. W., Baker, J. G., Hill, S. J., Kellam, B. (2007) New fluorescent adenosine A1-receptor agonists which allow quantification of ligand-receptor interactions in microdomains of single living cells. J. Med. Chem. 50,782-793[CrossRef][Medline]
  27. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275[Free Full Text]
  28. Cordeaux, Y., Briddon, S. J., Megson, A. E., McDonnell, J., Dickenson, J. M., Hill, S. J. (2000) Influence of receptor number on functional responses elicited by agonists acting at the human adenosine A1 receptor: evidence for signaling pathway-dependent changes in agonist potency and relative intrinsic activity. Mol. Pharmacol. 58,1075-1084.[Abstract/Free Full Text]
  29. Alvarez, D. V., Daniels, R. (1990) A single column method for the assay of adenylate cyclase. Anal. Biochem. 187,98-103[CrossRef][Medline]
  30. Schwille, P. (2001) Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem. Biophys. 34,383-408[CrossRef][Medline]
  31. Marsh, K. A., Hill, S. J. (1994) Des-Arg9-bradykinin-induced increases in intracellular calcium ion concentration in single bovine tracheal smooth muscle cells. Br. J. Pharmacol. 112,934-938[Medline]
  32. Selbie, L. A., Hill, S. J. (1998) G-protein-coupled receptor crosstalk: the fine-tuning of multiple receptor-signaling pathways. Trends Pharmacol. Sci. 19,87-93[CrossRef][Medline]
  33. Dickenson, J. M., Hill, S. J. (1998) Involvement of G protein β{gamma}-subunits in coupling the adenosine A1-receptor to phospholipase C in transfected CHO cells. Eur. J. Pharmacol. 355,85-93[CrossRef][Medline]
  34. Dickenson, J. M., Camps, M., Gierschik, P., Hill, S. J. (1995) Activation of phospholipase C by G-protein β{gamma} subunits in DDT1MF-2 cells. Eur. J. Pharmacol. 288,393-398[CrossRef][Medline]
  35. Gee, K. R., Brown, K. A., Chen, W. N., Bishop-Stewart, J., Gray, D., Johnson, I. (2000) Chemical and physiological characterization of fluo-4 Ca2+ indicator dyes. Cell Calcium 27,97-106[CrossRef][Medline]
  36. Fraser, J. D., Cong, M., Kim, J., Rollins, E. N., Daaka, Y., Lefkowitz, R. J., Scott, J. D. (2000) Assembly of an A kinase-anchoring protein-β2-adrenergic receptor complex facilitates receptor phosphorylation and signaling. Curr. Biol. 10,409-412[CrossRef][Medline]
  37. Hall, R. A., Premont, R. T., Chow, C. W., Blitzer, J. T., Pitcher, J. A., Claing, A., Stoffe, L. R. H., Barak, L. S., Shenolikar, S., Weinman, E. J., Grinstein, S., Lefkowitz, R. J. (1998) The β2-adrenergic receptor interacts with the Na+/H+ exchanger regulatory factor to control Na+/H+ exchange. Nature 392,626-630[CrossRef][Medline]
  38. Azzi, M., Charest, P. G., Angers, S., Rousseau, G., Kohout, T., Bouvier, M., Pineyro, G. (2003) β-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc. Natl. Acad. Sci. U. S. A. 100,11406-11411[Abstract/Free Full Text]
  39. Baker, J. G., Hall, I. P., Hill, S. J. (2003) Agonist and inverse agonist actions of "β-blockers" at the human β2-adrenoreceptor provide evidence for agonist-directed signaling. Mol. Pharmacol. 64,1357-1369[Abstract/Free Full Text]
  40. Cordeaux, Y., Ijzerman, A.P., Hill, S.J. (2004) Coupling of the human A1 adenosine receptor to different heterotrimeric G proteins: evidence for agonist-specific G protein activation. Br. J. Pharmacol. 143,705-714[CrossRef][Medline]
  41. Galandrin, S., Bouvier, M. (2006) Distinct signaling profiles of β1 and β2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol. Pharmacol. 70,1575-1584[Abstract/Free Full Text]
  42. Charest, P. G., Oligny-Longpre, G., Bonin, H., Azzi, M., Bouvier, M. (2007) The V2 vasopressin receptor stimulates ERK 1/2 activity independently of heterotrimeric G protein signaling. Cell Signal. 19,32-41[CrossRef][Medline]
  43. Pramanik, A., Olsson, M., Langel, U., Bartfai, T., Rigler, R. (2001) Fluorescence correlation spectroscopy detects galanin receptor diversity on insulinoma cells. Biochemistry 40,10839-10845[CrossRef][Medline]
  44. Rigler, R., Pramanik, A., Jonasson, P., Kratz, G., Jansson, O. T., Nygren, P.-Å., Ståhl, S., Ekberg, K., Johansson, B.-L., Uhlén, S., Uhlén, M., Jörnvall, H., Wahren, J. (1999) Specific binding of proinsulin C-peptide to human cell membranes. Proc. Natl. Acad. Sci. U. S. A. 96,13318-13323[Abstract/Free Full Text]
  45. Zhong, Z.-H., Pramanik, A., Ekberg, K., Jansson, O.T., Jörnvall, H., Wahren, J., Rigler, R. (2001) Insulin binding monitored by fluorescence correlation spectroscopy. Daibetilogia 44,1184-1188[CrossRef]
  46. Meseth, U., Wohland, T., Rigler, R., Vogel, H. (1999) Resolution of fluorescence correlation measurements. Biophys. J. 76,1619-1631[Medline]
  47. Lenne, P.-F., Wawrezinieck, L., Conchonaud, F., Wurtz, O., Boned, A., Guo, X.-J., Rigneault, H., He, H.-T., Margeut, D. (2006) Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork. EMBO J. 25,3245-3256[CrossRef][Medline]
  48. Kenakin, T. (2001) Inverse, protean and ligand-selective agonism: matters of receptor conformation. FASEB J. 15,598-611[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.07-8180comv1
22/3/850    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cordeaux, Y.
Right arrow Articles by Hill, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cordeaux, Y.
Right arrow Articles by Hill, S. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS