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Originally published In Press as doi:10.1074/jbc.M413475200 on June 9, 2005

J. Biol. Chem., Vol. 280, Issue 31, 28663-28674, August 5, 2005
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The CXCR1 and CXCR2 Receptors Form Constitutive Homo- and Heterodimers Selectively and with Equal Apparent Affinities*

Shirley Wilson{ddagger}§, Graeme Wilkinson¶, and Graeme Milligan{ddagger}||

From the {ddagger}Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and Biological Chemistry, AstraZeneca, Mereside, Alderely Park, Cheshire SK10 4TG, United Kingdom

Received for publication, November 30, 2004 , and in revised form, May 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Both homo- and heterodimeric interactions between the CXCR1 and CXCR2 chemokine receptors were observed following co-expression of forms of these receptors in HEK293 cells using assays, including co-immunoprecipitation, single cell imaging of fluorescence resonance energy transfer, cell surface time-resolved fluorescence resonance energy transfer, and bioluminescence resonance energy transfer. These interactions were constitutive and unaffected by the presence of the agonist interleukin 8 and selective as no significant interactions were noted between either the CXCR1 or CXCR2 receptor and the {alpha}1A-adrenoreceptor. Saturation bioluminescence resonance energy transfer indicated that heteromeric interactions between CXCR1 and CXCR2 were of similar affinity as the corresponding homomeric interactions. A novel endoplasmic reticulum trapping strategy demonstrated that these interactions were initiated during protein synthesis and maturation and prior to cell surface delivery. These studies indicated that CXCR1-CXCR2 heterodimers are as likely to form in cells co-expressing these two chemokine receptors as the corresponding homodimers and stand in contrast to previous studies indicating an inability of the CXCR1 receptor to homodimerize or to interact with the CXCR2 receptor (Trettel, F., Di Bartolomeo, S., Lauro, C., Catalano, M., Ciotti, M. T., and Limatola, C. (2003) J. Biol. Chem. 278, 40980-40988).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent times the concept that G protein-coupled receptors (GPCRs)1 can exist as dimers and/or higher order oligomers has become increasingly accepted (1-5). Techniques ranging from the co-immunoprecipitation of co-expressed but differentially epitope-tagged forms of a single receptor species (6, 7) to the use of resonance energy transfer-based methods (8-12) have provided support for the presence of such dimers/oligomers for many GPCRs in transfected cell systems, and the application of atomic force microscopy has shown the presence of dimers and arrays of dimers of rhodopsin in murine rod outer segment discs (13, 14). In many cases, GPCR quaternary structure seems to be defined early in the processes of receptor synthesis and maturation with the GPCR being transported to the cell surface as a preformed dimer/oligomer (15-17), the structure of which is unaffected by the presence of agonist ligands. The potential quaternary structure of a range of chemokine receptors has also been explored by using similar approaches (18-23). Although a substantial number of chemokine receptors have been shown to possess such quaternary structure, a number of features of certain chemokine receptors are either controversial or seem not to follow the general model outlined above. For example, dimerization/oligomerization of a number of chemokine receptors appears to be promoted by the binding of chemokine ligands (18, 19). Equally, it appears that mutation of certain chemokine receptors to prevent dimerization does not restrict membrane delivery (24). Among the chemokine receptors (25), the closely related CXCR1 and CXCR2 receptors share a common agonist ligand in interleukin 8 (IL8, also called CXCL8). They are widely co-expressed on immune cells, including neutrophils, CD8(+) T cells, and mast cells; and noncompetitive allosteric inhibitors of these receptors have been suggested to offer a general means to inhibit polymorphonuclear cell recruitment in vivo (26). Recently, Trettel et al. (23) have reported that the CXCR2 receptor forms a constitutive dimer when expressed in HEK293 cells and also in cerebellar neurons in which it is expressed endogenously. By contrast, these workers reported (23) that the CXCR1 receptor was unable to dimerize. As well as homodimeric/oligomeric interactions, many related GPCRs have been shown to have the capacity to form heterodimers. This can result in alterations in receptor pharmacology and signal transduction characteristics (27-29). A number of chemokine receptors have been reported to have the capacity to form heterodimers (30), but again, Trettel and co-workers (23) reported the lack of interactions between the CXCR1 and CXCR2 receptors following their co-expression. Given the general parsimony of structure and function of closely related proteins, we decided to re-examine the capacity of the CXCR1 and CXCR2 receptors to form homo- and heterodimers. By using a wide range of approaches, we show that both CXCR1 and CXCR2 form homodimer/oligomers at an early stage in synthesis and maturation and that when co-expressed CXCR1 and CXCR2 form heterodimers as effectively as homodimers. The extent of neither the CXCR1-CXCR2 heterodimer nor the corresponding homodimers is affected by the presence of IL8.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
125I-IL8 (2000 Ci/mmol) was from Amersham Biosciences. All reagents for BRET2 studies were from Packard Biosciences. Oligonucleotides were purchased from Interactiva, Ulm, Germany. The anti-human CXCR1 antibody was from R & D Systems, (Abingdon, UK).

Molecular Constructs
The human CXCR1 receptor was used as a PCR template for all CXCR1 constructs. For the N-terminally modified forms of the receptor, primers encoded the appropriate epitope tag sequence and introduced a stop codon after the last amino acid of the receptor sequence.

FLAG-CXCR1—The primers are as follows: sense, 5'-AAAAGAATTCGCCACCATGGACTACAAGGACGACGATGATGATAAGTCAAATATTACAGATCCAC-3'; antisense, 5'-AAAAGAATTCTCAGAGGTTGGAAGAGACATTGAC-3'. The EcoRI sites are underlined, and the amplified fragment was digested and ligated into pcDNA3.

c-myc-CXCR1—The primers are as follows: sense, 5'-AAAAGAATTCGCCACCATGGAACAAAAACTTATTTCTGAAGAAGATCTGTCAAATATTACAGATCCAC-3'; antisense, 5'-AAAAGAATTCTCAGAGGTTGGAAGAGACATTGAC-3'. The EcoRI sites are underlined, and the amplified fragment was digested and ligated into pcDNA3.

HA-CXCR1—The primers are as follows: sense, 5'-AAAAGGTACCGCCACCATGTATCCCTACGACGTCCCCGATTATGCGTCAAATATTACAGATCCAC-3'; antisense, 5'-AAAAGAATTCTCAGAGGTTGGAAGAGACATTGAC-3'. A KpnI site present in the sense primer and an EcoRI site present in the antisense primer are underlined, and the amplified fragment was digested and ligated into pcDNA3.

To generate the endoplasmic reticulum (ER) trapped form of HA-CXCR1 (HA-CXCR1-ER), primers encoding the C-terminal 14-amino acid segment of the {alpha}2c-adrenoreceptor were annealed to HA-CXCR1. The primers are as follows: sense, 5'-AATTCAAGCATATCCTCTTTCGAAGGAGGAGAAGGGGCTTCAGGCAATGAT-3'; antisense, 5'-CTAGATGATTGCCTGAAGCCCCTTCTCCTCCTTCGAAAGAGGATATGCTTG-3'. An EcoRI site present in the sense primer and the XbaI site present in the antisense primer are underlined, and the fragment was digested and ligated downstream of the HA-CXCR1 fragment in-frame in pcDNA3.

C-terminally Tagged Constructs
In each case the primers were designed to amplify the CXCR1 receptor and remove the stop codon.

CXCR1-GFP2—The primers are as follows: sense, 5'-AAAAGAATTCGCCACCATGTCAAATATTACAGATCCAC-3'; antisense, 5'-AAAAGGTACCGAGGTTGGAAGAGACATTGAC-3'. The EcoRI and KpnI sites present in the sense and antisense primers, respectively, are underlined. The amplified fragment was digested and ligated into pGFP2-N2 (Packard Instrument Co.) in-frame with GFP2.

CXCR1-Renilla Luciferase—The primers are as follows: sense, 5'-AAAAAAGCTTGCCACCATGTCAAATATTACAGATCCAC-3'; antisense, 5'-AAAACTCGAGGTTGGAAGAGACATTGAC-3'. The HindIII and XhoI sites present in the sense and antisense primers, respectively, are underlined. The amplified fragment was digested and ligated into pcDNA3 upstream and in-frame with Renilla luciferase ligated between XhoI and XbaI.

CXCR1-YFP—The primers are as follows: sense, 5'-AAAAAAGCTTGCCACCATGTCAAATATTACAGATCCAC-3'; antisense, 5'-AAAAGCGGCCGCGAGGTTGGAAGAGACATTGAC-3'. The HindIII and NotI sites encoded in the sense and antisense primers are underlined. The amplified fragment was digested and ligated into pcDNA3.1 (+) upstream and in-frame with YFP ligated between NotI and XhoI.

CXCR1-CFP—The primers are as follows: sense, 5'-AAAAAAGCTTGCCACCATGTCAAATATTACAGATCCAC-3'; antisense, 5'-AAAAGGTACCGAGGTTGGAAGAGACATTGAC-3'. The HindIII and KpnI sites encoded in the sense and antisense primers are underlined. The amplified fragment was digested and ligated into pcDNA3 upstream and in-frame with CFP ligated between HindIII and XhoI.

CXCR2—Human CXCR2 was used as a PCR template for all CXCR2 constructs. These were generated in a similar fashion to the CXCR1 constructs. For the N-terminally modified forms of the receptor, primers encoded the appropriate epitope tag sequence and introduced a stop codon after the last amino acid of the receptor sequence.

FLAG-CXCR2—The primers are as follows: sense, 5'-AAAAGAATTCGCCACCATGGACTACAAGGACGACGATGATAAGGAAGATTTTAACATGGAG-3'; antisense, 5'-AAAAGAATTCGAGAGTGGAAGTGTGCCC-3'. EcoRI sites present in both sense and antisense primers are underlined, and the amplified fragment was digested and ligated into pcDNA3.

c-myc-CXCR2—The primers are as follows: sense, 5'-AAAAGAATTCGCCACCATGGAACAAAAACTTATTTCTGAAGAAGATCTGGAAGATTTTAACATGGAG-3'; antisense, 5'-AAAAGAATTCGAGAGTGGAAGTGTGCCC-3'. EcoRI sites are underlined, and the amplified fragment was digested and ligated into pcDNA3.

VSVG-CXCR2—The primers are as follows: sense, 5'-AAAAGGTACCGCCACCATGTACACCGACATCGAAATGAACCGCCTTGGTAAG-3'; antisense, 5'-AAAAGAATTCGAGAGTGGAAGTGTGCCC-3'. The KpnI and EcoRI sites present in the sense and antisense primers, respectively, are underlined, and the amplified fragment was digested and ligated into pcDNA3.

For the C-terminally modified forms of the receptor, primers were designed to amplify the sequence and remove the stop codon.

CXCR2-GFP2—The primers are as follows: sense, 5'-AAAAGAATTCGCCACCATGGAAGATTTTAACATGGAC-3'; antisense, 5'-AAAAGGTACCGAGAGTAGTGGAAGTGTGCCC-3'. The EcoRI and KpnI sites present in the sense and antisense primers, respectively, are underlined. The amplified fragment was digested and ligated into pGFP2-N2 in-frame with GFP2.

CXCR2-Renilla Luciferase—The primers are as follows: sense, 5'-AAAAAAGCTTGCCACCATGGAAGATTTTAACATGGAG-3'; antisense, 5'-AAAACTCGAGGAGCGTCGTGGAAGTGTG-3'. The HindIII and XhoI sites present in the sense and antisense primers, respectively, are underlined. The amplified fragment was digested and ligated into pcDNA3 upstream and in-frame with Renilla luciferase ligated between XhoI and XbaI.

CXCR2-YFP—The primers are as follows: sense, 5'-AAAAAAGCTTGCCACCATGGAAGATTTTAACATGGAG-3'; antisense, 5'-AAAAGCGGCGCGAGAGTAGTGGAAGTGTGCCC-3'. The HindIII and NotI sites encoded in the sense and antisense primers are underlined. The amplified fragment was digested and ligated into pcDNA3.1 (+) upstream and in-frame with YFP ligated between NotI and XhoI.

CXCR2-CFP—The primers are as follows: sense, 5'-AAAAAAGCTTGCCACCATGGAAGATTTTAACATGGAG-3'; antisense, 5'-AAAAGGTACCGAGAGTAGTGGAAGTGTGCCC-3'. The HindIII and KpnI sites encoded in the sense and antisense primers are underlined. The amplified fragment was digested and ligated into pcDNA3 upstream and in-frame with CFP ligated between HindIII and XhoI.

Cell Membrane Preparation
Pellets of cells were resuspended in 10 mM Tris, 0.1 mM EDTA, pH 7.4 (TE buffer), and the cells were homogenized using 40 strokes of a glass on Teflon homogenizer. Samples were centrifuged at 1000 x g for 10 min at 4 °C to remove unbroken cells and nuclei. The supernatant fraction was removed and passed through a 25-gauge needle 10 times before being transferred to ultracentrifuge tubes and subjected to centrifugation at 50,000 x g for 30 min. The supernatant was discarded, and the pellet was resuspended in TE buffer. Protein concentration was assessed, and membranes were diluted to 1 mg/ml and stored at -80 °C until required.

Radioligand Binding
Reaction mixtures were established in a volume of 100 µl containing 5 µg of membrane protein, 100 pM 125I-IL8, and a range of concentrations of nonradiolabeled IL8. Samples were incubated for 90 min at room temperature prior to filtration through Whatman GF/C filters. Data were analyzed using Graphpad Prism, and IC50 values were determined via nonlinear regression using one-site competition analysis. The equilibrium dissociation constant for the binding of IL8 was calculated using the Cheng-Prusoff equation (31).

Co-immunoprecipitation Studies
Cells were harvested 24 h following transfection and resuspended in RIPA buffer (50 mM HEPES, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM NaF, 5 mM EDTA, 0.1 mM NaPO4, 5% ethylene glycol). The cell pellet was disrupted as above and placed on a rotating wheel for 1 h at 4 °C. Samples were then centrifuged for 10 min at 14,000 x g at 4 °C, and the supernatant was transferred to a fresh tube containing 200 µl of 1x RIPA and 50 µl of protein G beads (Sigma) to pre-clear the samples. Following incubation on a rotating wheel for 1 h at 4 °C, the samples were re-centrifuged at 14,000 x g at 4 °C for 1 min, and the protein concentration of the supernatant was determined. Samples containing equal protein amounts were incubated overnight with 40 µl of protein G beads, 5 µg of M2 anti-FLAG antibody (Sigma) at 4 °C on a rotating wheel, and fractions were reserved to monitor protein expression in the cell lysates. Samples were centrifuged at 14,000 x g for 1 min at 4 °C, and the protein G beads were washed three times with RIPA buffer. Following addition of Laemmli buffer and heating to 85 °C for 4 min, both immunoprecipitated samples and cell lysate controls were revolved by SDS-PAGE using pre-cast 4-12% acrylamide Novex Bistris gels (Invitrogen). Proteins were transferred onto nitrocellulose. These membranes were incubated in 5% (w/v) low fat milk, 0.1% Tween 20/PBS (v/v) solution at room temperature on a rotating shaker for 2 h and then with primary antibody overnight in 5% (w/v) low fat milk, 0.1% Tween 20/PBS (v/v) solution at 4 °C. The membrane was washed three times in 0.1% Tween 20/PBS before addition of secondary antibody. Following further washes, the membrane was subsequently developed using ECL solution (Pierce).

Confocal Laser-scanning Microscopy
Cells were imaged using a laser-scanning confocal microscope (Zeiss LSM 5 pascal) equipped with a 63x oil-immersion Plan Fluor Apochromat objective lens with a numerical aperture of 1.4. A pinhole of 20 and an electronic zoom of 1 or 2.5 were used (Carl Zeiss Inc., Thornwood, NY). The excitation laser line for GFP and YFP was the 488-nm argon laser with detection via a 505-530 band pass filter. Alexa 594 label was detected using a 543-nm helium/neon laser and detected via a 560 long pass filter. The images were manipulated using MetaMorph imaging software (version 6.1.3; Universal Imaging Corp., Downing, PA).

In some experiments, fixed cells were used. Cells grown on coverslips were transiently transfected and washed three times with ice-cold PBS. Cells were fixed for 10 min at room temperature using 4% paraformaldehyde in PBS, 5% sucrose. The cells were washed a further three times in ice-cold PBS prior to being fixed onto microscope slides with 40% glycerol in PBS.

Fluorescent Microscopy and FRET Imaging in Living Cells
HEK293T cells were grown on poly-D-lysine-treated coverslips and transiently transfected with appropriate CFP/YFP fusion proteins. Coverslips were placed into a microscope chamber containing physiological saline solution (130 nM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, 10 mM D-glucose, pH 7.4). Cells were visualized using a Nikon Eclipse TE2000-E fluorescence inverted microscope, and images were obtained individually for enhanced YFP, CFP, and FRET filter channels using an Optoscan monochromator (Cairn Research, Faversham, Kent, UK) and a dichroic mirror 86002v2bs (Chroma Inc., Rockingham, VT). The filter sets used are as follows: YFP (excitation 500/5 nm; emission 535/30 nm), CFP (excitation 430/12 nm; emission 470/30 nm), and FRET (excitation 430/12 nm; emission 535/30 nm). The illumination time was 250 ms and binning modes 2 x 2. MetaMorph imaging software was used to quantify the FRET images using the sensitized FRET method. Corrected FRET was calculated using a pixel-by-pixel methodology using the equation FRETc = FRET - (coefficient B x CFP) - (coefficient A x YFP), where CFP, YFP, and FRET values correspond to background corrected images obtained through the CFP, YFP, and FRET channels. B and A correspond to the values obtained for the CFP (donor) and YFP (acceptor) bleed through coefficients, respectively, calculated using cells singly transfected with either the CFP or YFP protein alone. To correct the FRET levels for the varying amounts of donor (CFP) and acceptor (YFP), normalized FRET was calculated using the equation FRETn = FRETc/CFP x YFP, where FRETc, CFP, and YFP are equal to the fluorescence values obtained from single cells.

Tr-FRET
10-cm2 dishes of HEK293T cells were transfected to express N-terminally c-Myc or FLAG-tagged forms of CXCR1 or CXCR2 individually or in combination. 48 h after transfection, the cells were harvested. Cell pellets were resuspended in 200 µl of ice-cold PBS. Anti-c-Myc Eu3+ and anti-FLAG allophycocyanin (APC) antibodies were diluted in 50% newborn calf serum; 50% PBS to final concentrations of 5 and 15 nM, respectively. Samples were mixed and incubated on a rotating wheel at room temperature for 2 h. Samples were covered in aluminum foil to minimize exposure of the fluorophores to light. Samples were centrifuged at 1000 x g for 1 min, and the antibody mixture was removed from the cell pellet. The pellet was then washed two times in ice-cold PBS and resuspended in 250 µl of PBS. To investigate the agonist effect on energy transfer, 90 µl of cells were transferred to a fresh tube and incubated with the chosen concentration of agonist at 37 °C. To measure the energy transfer, 40 µl of each sample was dispensed in triplicate into a black 384-well plate. Blank wells containing PBS were also included. Tr-FRET was determined using a Victor2 plate reader (Packard Bioscience). Excitation was at 340 nm, and emission filters generated data representing donor (615 nm) and acceptor (665 nm) fluorescence. Normalized FRET was calculated using the following equation: normalized FRET = ((A665 - BLK)/D615) - C, where A665 is the fluorescent emission from the acceptor, D615 is the fluorescent emission from the donor, and BLK represents the background reading at 665 nm from wells containing PBS. C represents the cross-talk between the donor and acceptor windows for the samples incubated with only anti-c-Myc Eu3+ and is equal to A665 - BLK/D615.

BRET2
Single Point BRET2—Cells were washed twice in PBS supplemented with 1 g/liter glucose and resuspended in a final volume of 1 ml. 160 µl of cells were dispensed into a white-walled 96-well plate (Optiplate, PerkinElmer Life Sciences) and either 20 µl of agonist or PBS/glucose added. If agonist was tested then the plate was incubated for 30 min at 37 °C. DeepBlueC (PerkinElmer Life Sciences) substrate was diluted 1:20 in PBS/glucose, and the mixture was kept protected from light until required. 20 µl of substrate was added to each well resulting in a final concentration of 10 µM, and BRET2 was measured using a Mithras LB940 (Berthold Technology, Bad Wildbad, Germany). Readings were taken using 410 nm (band pass 80 nm) corresponding to light emission resulting from Renilla luciferase catalyzing the substrate to coelenteramide. Transferred energy emitted by GFP2 was detected using a 515-nm (band pass 30 nm) filter, and a ratiometric reading was obtained corresponding to the ratio of light intensity (515 nm) to light intensity (410 nm).

Saturation BRET2—In saturation BRET2 experiments cells were transfected with a constant amount of the energy donor (Renilla luciferase) construct and a varying amount of energy acceptor (GFP2) construct. Cells were harvested, and membranes were prepared and diluted to 0.5 mg/ml. BRET2 was assessed as above for intact cells. Luminescence and fluorescence measurements were also obtained. 50 µl of cell membranes were dispensed into white-walled 96-well plates (PerkinElmer Life Sciences) for luminescence measurements and black-walled 386-well plates (Costar, Cambridge, MA) for fluorescence measurement. For luminescence measurement h-coelenterazine (5 µM) was added, and the plate was incubated at room temperature for 30 min prior to measurement at 410 nm using a Mithras LB 940. GFP2 fluorescence was assessed using Victor2 1420 Multilabel counter (PerkinElmer Life Sciences). BRET2 readings were corrected for energy transfer resulting from bleed through of signal from the Renilla luciferase construct expressed alone but detected in the GFP2 channel. Fluorescence readings were corrected for endogenous fluorescence of HEK293T cell membranes alone. Graphpad Prism 4 was used to analyze data using a one-site binding hyperbola equation yielding BRETmax and BRET50 values.

Immunostaining Protocol
Cells were grown onto coverslips and transiently transfected. 24 h later medium was removed, and the cells were incubated with 20 mM HEPES/Dulbecco's modified Eagle's medium containing the appropriate dilution of primary antibody for 40 min at 37 °C in 5% CO2. IL8 (50 nM) was added, and the coverslip was incubated for 30 min. Following three washes with PBS, the cells were fixed by incubating with 4% paraformaldehyde in PBS, 5% sucrose for 10 min at room temperature. Following three further washes the cells were permeabilized with 0.15% Triton-X-100, 3% nonfat milk, PBS for 10 min. The coverslips were incubated with a secondary antibody (5 mg/ml) conjugated to an Alexa 594 fluorophore. Following incubation for 1 h, cells were washed twice in 0.15% Triton X-100, 3% nonfat milk, PBS and three times in PBS. Coverslips were then mounted onto microscope slides with 40% glycerol in PBS.

Endoplasmic Reticulum Trapping and Quantitation Studies
HEK293T cells were transfected to express either HA-CXCR1 or an ER-retained version of this construct that has the C-terminal 14 amino acids of the {alpha}2c-adrenoreceptor attached to the C-terminal tail (HA-CXCR1-ER). Such cells were co-transfected with N-terminally FLAG-tagged forms of various receptors. 48 h following transfection the cells were harvested and counted using a hemocytometer. 5 x 105 cells were dispensed into individual Eppendorf tubes, and the cells were incubated with 15 nM APC-labeled anti-FLAG antibody and 1 µM Hoechst nuclear stain on a rotating wheel for 1 h. The cells were centrifuged at 1000 x g for 1 min, and the cell pellet was washed three times with PBS. The cells were resuspended in 200 µl of PBS, and 40-µl replicates were dispensed into black-walled 384-well plates. Fluorescence corresponding to APC was quantified using a Victor2 1420 Multilabel counter (PerkinElmer Life Sciences). Controls measured fluorescence in similarly treated but nontransfected HEK293T cells, and this value was subtracted from the other readings. To ensure equal cell numbers between wells, fluorescence representing Hoechst staining was measured in parallel. Such data ensured the well to well cell number variation was less than 20%.

cAMP Measurements
The inhibition of forskolin-stimulated cAMP generation was determined using the HitHunterTM cAMP XS assay kit (32) (DiscoverX, Birmingham, UK). Transfected cells were harvested and resuspended in 1x PBS containing 0.5 mM isobutylmethylxanthine. Cells were dispensed into white-walled 96-well plates (Optiplate, PerkinElmer Life Sciences) at ~30,000 cells/well and incubated with 10 µM forskolin and varying concentrations of GRO-{alpha} for 30 min at 37 °C. The assay kit was then used in accordance with the manufacturers' instructions, and luminescence was detected by using a Mithras LB940 (Berthold Technology, Bad Wildbad, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The CXCR1 and CXCR2 receptors share interleukin 8 (IL8) as a high affinity agonist ligand. Because a significant number of both N- and C-terminally modified variants of the human forms of these two receptors were to be utilized in these studies, we initially demonstrated that such modifications did not prevent IL8-mediated binding and internalization of the receptors. Following introduction of the c-Myc epitope tag sequence into the extreme N terminus of both the CXCR1 and CXCR2 receptors, these constructs were expressed transiently in HEK293T cells. Both constructs were shown to be located predominantly at the cell surface (Fig. 1, a and b). Following treatment of these cells with 50 nM IL8 for 30 min, a substantial fraction of both modified receptor constructs was re-located into punctate intracellular vesicles (Fig. 1, a and b). Equivalent experiments using N-terminally FLAG-tagged forms of the receptors produced similar results (data not shown). Both the CXCR1 and CXCR2 receptors were also modified by the addition of a variety of forms of the Aequoria victoria green fluorescent protein (GFP) to the C-terminal tail. As with the N-terminally modified variants, transient expression in HEK293T cells of the CXCR1 and CXCR2 receptors tagged with, for example, the BRET acceptor-competent fluorescent protein GFP2 resulted in a predominantly plasma membrane localization (Fig. 1, c and d), and treatment with 50 nM IL8 for 30 min also caused marked internalization into punctate vesicles (Fig. 1, c and d). Forms of the CXCR1 and CXCR2 receptors with the BRET energy donor Renilla luciferase linked in-frame to the C terminus were also effectively delivered to the cell surface following transient expression in HEK293T cells (Fig. 1e and data not shown). Modification of the receptors did not alter the binding affinity for IL8. Direct comparisons of the specific binding of 125I-IL8 to the unmodified CXCR1 receptor and its self-competition by nonradiolabeled IL8, with forms of this receptor N-terminally modified to include the c-Myc, FLAG, or HA epitope tags or with forms of the receptor C-terminally labeled with GFP2, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), or Renilla luciferase, indicated each binds the ligand with a pKi close to -9.5 M (Table I). Similarly, N-terminal modification of the CXCR2 receptor with each of the FLAG, c-Myc, or vesicular stomatitis virus epitope tags or addition of CFP, YFP, GFP2, or Renilla luciferase to the C-terminal tail did not alter the affinity of this receptor to bind IL8 (Table I). Based on the specific binding of 100 pM 125I-IL8 and the calculated Ki values for IL8, expression levels of the various forms of CXCR1 and CXCR2 were estimated. These ranged from 50 to 500 fmol/mg membrane protein, and as noted previously (33), for equal amounts of transfected cDNAs, levels of the CXCR1 constructs were higher than for the equivalent CXCR2 variant.


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  		TABLE I
N- and C-terminal modifications of the CXCR1 and CXCR2 receptors have little effect on affinity for IL8

The affinity of IL8 to bind to wild type and each of the modified forms of the CXCR1 and CXCR2 receptors was assessed from analysis of the capacity of IL8 to compete with a single concentration (100 pM) of 125I-IL8 for binding. All experiments were performed on a minimum of three occasions using separate membrane preparations. No significant differences were noted for each of the modified constructs compared with the wild type (one-way analysis of variance with Dunnetts' post-test).

 



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  		FIG. 1.
IL8 induces internalization of N-terminally and C-terminally modified forms of the CXCR1 and CXCR2 receptors. N-terminally c-Myc-(a and b) or C-terminally GFP2 (c and d)-tagged forms of the CXCR1 (a and c) or CXCR2 (b and d) receptors were expressed in HEK293T cells. Cells were challenged with vehicle (left-hand panels) or IL8 (50 nM, 30 min, 37 °C) (right-hand panels) fixed and stained with anti-c-Myc (a and b) or directly visualized (c and d). e, CXCR1 C-terminally tagged with Renilla luciferase was expressed transiently in HEK293 cells. Detection employed an anti-human CXCR1 antibody (R & D Systems, Abingdon, UK) directed to an extracellular epitope.

 
Protein/protein interactions indicative of the quaternary structure of GPCRs have been monitored in a range of ways. The most widely used has been the co-immunoprecipitation of differentially tagged but co-expressed polypeptides. Co-expression of both N-terminally c-Myc- and FLAG-tagged forms of the CXCR1 receptor allowed their co-immunoprecipitation (Fig. 2A). Following immunoprecipitation with anti-FLAG antibody, samples were resolved by SDS-PAGE and immunoblotted with anti-c-Myc antibody. This resulted in the detection of principally a 38-kDa polypeptide with small amounts of a band of some 80 kDa that suggests a fraction of the immunoprecipitated dimer/oligomer was not effectively separated by the SDS-PAGE conditions employed (Fig. 2A). No anti-c-Myc-reactive bands were present in the immunoprecipitates when either the FLAG- or c-Myc-tagged forms of CXCR1 were expressed individually, although direct immunoblots of lysates produced from such cells confirmed the expression of the individual forms of the CXCR1 receptor in each of the samples anticipated (Fig. 2A). Equally, mixing of cell lysates expressing either FLAG-CXCR1 or c-myc-CXCR1 prior to immunoprecipitation with the anti-FLAG antibody did not result in co-immunoprecipitation, indicating that co-expression was required to allow interaction. Equivalent experiments using FLAG and c-Myc-tagged forms of CXCR2 produced equivalent data (Fig. 2b), with the monomer migrating as a polypeptide of some 35 kDa. Again, a small fraction of the co-immunoprecipitated anti-c-Myc immunoreactivity remained as a dimer following migration through SDS-PAGE. Co-expression of FLAG-CXCR1 with c-myc-CXCR2 also allowed their co-immunoprecipitation (Fig. 2C), consistent with the capacity of these two closely related GPCRs to form a heterodimer-oligomer complex in cells in which they are co-expressed.

Co-immunoprecipitation experiments require cellular fragmentation and detergent-mediated dissolution of membranes. Therefore, in recent times a series of approaches based on resonance energy transfer techniques have been employed to examine potential protein-protein interactions in living cells. Initially, we employed imaging of cells expressing forms of the CXCR1 and CXCR2 receptors tagged at the C terminus with either CFP or YFP, as these are well established fluorescence resonance energy transfer (FRET) partners. Expression of either CXCR1-CFP (Fig. 3) or CXCR1-YFP (Fig. 3) in HEK293 cells allowed the selective imaging of each construct in individual cells. Co-expression of CXCR1-CFP and CXCR1-YFP resulted in FRET (Fig. 3). Co-transfection of the isolated forms of CFP and YFP did not result in significant levels of FRET, although imaging studies confirmed their co-expression in individual cells (data not shown but see Ref. 34). These results define that the positive FRET signals obtained with co-expression of CXCR1-CFP and CXCR1-YFP did not reflect significant mutual affinity between the two fluorescent proteins and therefore rather support dimeric/oligomeric protein/protein interactions involving the CXCR1 receptor. This was a selective interaction because in cells co-transfected with CXCR1-CFP and {alpha}1A-adrenoreceptor-YFP, only low levels of FRET were recorded (Fig. 3), although imaging of individual cells demonstrated their co-expression, and we ensured levels of expression of the {alpha}1A-adrenoreceptor-YFP were similar to the levels of expression of CXCR1-YFP in the earlier experiments by direct measures of the levels of YFP fluorescence (Fig. 3). Equivalent studies with CFP- and YFP-tagged forms of the CXCR2 receptor also resulted in positive FRET signals when the two forms of this receptor were co-expressed (Fig. 4). As with the CXCR1 receptor, these effects were selective as only weak interactions could be recorded between the CXCR2 receptor and the {alpha}1A-adrenoreceptor-YFP (Fig. 4). Finally, co-expression of CXCR1-CFP and CXCR2-YFP, or co-expression of the alternate CXCR2-CFP and CXCR1-YFP pairing, also generated strong positive FRET signals indicative of the capacity of these two related receptors to form a heterodimer-oligomer complex (Fig. 5). As all of these experiments were conducted in the absence of IL8, these studies are also consistent with both homo- and heterodimer/oligomers of these receptors forming constitutively without need for agonist.

Both the co-immunoprecipitation and FRET imaging studies offer initial insights into the capability of the CXCR1 and CXCR2 to form homodimeric-oligomeric and, when co-expressed, heterodimeric-oligomeric complexes. However, such studies can offer little insight into the relative propensity of pairs of GPCRs to interact. To assess this, we employed saturation BRET2 experiments. Proteins tagged with Renilla luciferase and with GFP2 can allow BRET upon addition of an appropriate substrate for the luciferase if interactions between the partner proteins bring the luciferase and the GFP2 into proximity (35). By varying the ratio of energy acceptor (the GFP2-tagged protein) and energy donor (the luciferase-tagged protein), BRET2 saturation curves can be generated in which half-maximal signal provides a measure of the relative affinity of protein/protein interactions (12, 36). Following expression in HEK293 cells of varying ratios of CXCR1-Renilla luciferase and CXCR1-GFP2 addition of the luciferase substrate DeepBlueC to intact cells resulted in BRET2 signals that approached an asymptote with increasing acceptor/donor ratios and with an estimated BRET2 50% of 3.9 ± 0.6 (Fig. 6A). By contrast, when CXCR1-Renilla luciferase was co-expressed with the isolated GFP2 in varying ratios, no measurable BRET2 signal was obtained (Fig. 6A), confirming that the BRET2 signals obtained with co-expression of CXCR1-Renilla luciferase and CXCR1-GFP2 reflect protein/protein interactions involving the CXCR1 receptor rather than direct interactions between Renilla luciferase and GFP2. Saturation BRET2 experiments in which CXCR2-Renilla luciferase and CXCR2-GFP2 were coexpressed also confirmed the capacity of this receptor to form homodimer/oligomers, and the measured BRET2 50% of 2.2 ± 0.1 (Fig. 6A) indicated that the affinity of interactions between these forms of the CXCR2 receptor is at least as high as for the CXCR1 receptor. Co-expression of various ratios of CXCR1-Renilla luciferase and CXCR2-GFP2 confirmed the capacity of these two receptors to heterodimerize/oligomerize, and because the BRET2 50% ratio in these experiments, 3.6 ± 0.1, was highly similar to those obtained for the homodimer pairings, it suggests the propensity of CXCR1 and CXCR2 to heterodimerize is similar to that of the individual receptors to form homodimers. Again, it was important to obtain clear negative controls. To do so we co-expressed CXCR2-Renilla luciferase along with a GFP2-tagged form of the {alpha}1A-adrenoreceptor that we have used in previous studies (12). BRET2 signals produced from this pairing were low and, most importantly, were fit adequately by a straight line in which signal increase was a direct reflection of the energy acceptor to energy donor ratios (Fig. 6A), suggesting that this pair of GPCRs has no substantial mutual affinity. As a ratiometric measure, BRET2 signals that reflect specific protein/protein interactions should be independent of absolute expression levels. Increasing signals with receptor expression levels may reflect physical crowding that is not related to true interactions. Such effects have been termed bystander effects (36). Although the expression levels measured by the specific binding of 125I-IL8 (see earlier) were far lower than those reported previously to be required to observe bystander effects (36), we tested this directly. HEK293 cells were transfected with different amounts of CXCR1-Renilla luciferase (energy donor) and CXCR1-GFP2 (energy acceptor) cDNAs but in each case in a 1:1 ratio. Differing expression levels of CXCR1-Renilla luciferase were monitored by direct measurement of luciferase activity using h-coelenterazine as substrate to generate the luminescent signal because the emission spectrum from oxidation of this substrate is not suited for energy transfer to GFP2 (12). Luminescence increased across the full range of cDNA amounts employed (Fig. 6B). Similarly, relative levels of CXCR1-GFP2 were monitored by direct fluorescence and increased linearly with the cDNA amount transfected (Fig. 6B). Despite this, the BRET2 ratios recorded in such cells upon addition of DeepBlueC as luciferase substrate were not different for the varying levels of the constructs expressed (Fig. 6B).



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  		FIG. 2.
Constitutive homo- and heterodimeric/oligomeric interactions between co-expressed forms of CXCR1 and CXCR2 receptors revealed by co-immunoprecipitation. A, HEK293T cells were mock-transfected (lane 1) or transfected to transiently express FLAG-CXCR1 (lanes 2 and 4) and/or c-myc-CXCR1 (lanes 3 and 4). A mix control (lane 5) was included representing cells individually expressing the constructs and mixed prior to immunoprecipitation. Upper panel, cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, and samples were resolved by SDS-PAGE and then immunoblotted with anti-c-Myc antibody. Lower panels, Western blot analysis of cell lysates using anti-FLAG and anti-c-Myc antibodies was also performed to confirm the anticipated pattern of protein expression. B, experiments similar to A were performed except that CXCR1 was replaced with CXCR2. C, HEK293T cells were mock-transfected (lane 1) or transfected to transiently express FLAG-CXCR1 (lanes 2 and 4) and/or c-myc-CXCR2 (lanes 3 and 4). A mix control (lane 5) was included representing cells individually expressing the constructs and mixed prior to immunoprecipitation. Upper panel, cell lysates were immunoprecipitated with anti-FLAG antibody, and samples were resolved by SDS-PAGE and then immunoblotted with anti-c-Myc antibody. Lower panels, Western blot analysis of cell lysates using anti-FLAG and anti-c-Myc antibodies was also performed to confirm the anticipated pattern of protein expression.

 



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  		FIG. 3.
FRET imaging of constitutive CXCR1 homomeric interactions in single cells. CXCR1-CFP and CXCR1-YFP were co-expressed (a) or expressed individually (b, CXCR1-CFP; c, CXCR1-YFP). {alpha}1a-Adrenoreceptor-YFP was also expressed individually (d) or co-expressed with CXCR1-CFP (e) in HEK293T cells. Individual cells and cell groups were imaged. Left-hand panels, CFP; center panels, YFP; right-hand panels, corrected FRET. The FRET signals from such images were then quantitated as under "Experimental Procedures" (f). Data are means ± S.E. from three experiments.

 



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  		FIG. 4.
FRET imaging of selective and constitutive CXCR2 homomeric interactions in single cells. CXCR2-CFP and CXCR2-YFP were co-expressed (a) or expressed individually (b, CXCR2-CFP; c, CXCR2-YFP). {alpha}1a-Adrenoreceptor-YFP was also expressed individually (d) or co-expressed with CXCR2-CFP (e) in HEK293T cells. Individual cells and cell groups were imaged. Left-hand panels, CFP; center panels, YFP; right-hand panels, corrected FRET. The FRET signals from such images were then quantitated as under "Experimental Procedures" (f). Data are means ± S.E. from three experiments.

 



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  		FIG. 5.
FRET imaging of constitutive CXCR1-CXCR2 heteromeric interactions in single cells. CXCR1-CFP (a and b) was expressed individually (b) or co-expressed with CXCR2-YFP (a). CXCR2-CFP was also co-expressed with CXCR1-YFP (c) or expressed individually (d) in HEK293T cells. Individual cells and cell groups were imaged. Left-hand panels, CFP; center panels, YFP; right-hand panels, corrected FRET. The FRET signals from such images were then quantitated as under "Experimental Procedures" (e). Data are means ± S.E. from three experiments.

 



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  		FIG. 6.
BRET2 analysis of the selectivity and specificity of CXCR1 and CXCR2 interactions. A, CXCR1 (filled circles) and CXCR2 (open circles) homodimeric interactions were assessed following transient expression of various ratios of receptor-GFP2 (acceptor) and receptor-Renilla luciferase (donor) in HEK293T cells and addition of DeepBlueC as a luciferase substrate. CXCR1-CXCR2 heteromeric interactions (filled squares) were assessed following expression of various ratios of CXCR1-Renilla luciferase and CXCR2-GFP2. The ratio on the x axis reflects direct measures of fluorescence (GFP2) and luminescence (luciferase) (see Ref. 12 for details). The specificity of CXCR2 receptor interactions (filled diamonds) was assessed by co-expression of CXCR2-Renilla luciferase and {alpha}1A-adrenoreceptor-GFP2. Lack of inherent interactions between Renilla luciferase and GFP2 (open squares) was obtained by co-expression of CXCR1-Renilla luciferase and GFP2. Data from a single experiment are displayed, and the error bars represent mean ± S.E. of measurements of BRET2 in three separate wells. Two further experiments produced similar results. B, intact HEK293T cells transiently transfected with increasing amounts of CXCR1-Renilla luciferase (donor) and CXCR1-GFP2 (acceptor) cDNAs in a 1:1 ratio. The amount of each cDNA used is noted. i, donor and acceptor receptor conjugate relative expression levels were monitored by measuring luminescence (triangles) and fluorescence (squares) (see "Results"). Data from triplicate assays in a single experiment are displayed. Two further experiments produced similar results. ii, BRET2 experiments were performed on these samples.

 
As monitored using BRET2, neither homodimeric CXCR1 and CXCR2 nor heterodimeric CXCR1/CXCR2 interactions were modified by the addition of IL8 (Fig. 7), indicating, as in the FRET imaging experiments, that each of these interactions is generated constitutively and not in response to agonist binding.



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  		FIG. 7.
IL8 does not modulate CXCR1 or CXCR2 homo- or heteromeric interactions. BRET2 experiments were performed in the absence (open bars) or presence (filled bars) of 50 nM IL8 following co-expression of CXCR1-Renilla luciferase and CXCR1-GFP2, CXCR2-Renilla luciferase, and CXCR2-GFP2 or CXCR2-Renilla luciferase and CXCR1-GFP2. Data from triplicate assays in a single experiment are displayed. Two further experiments produced similar results.

 
Because the Renilla luciferase and GFP2 tags that act as BRET2 partners are both inside intact cells, it is impossible to determine the cellular location of the dimeric/oligomeric GPCRs from such studies. Although the distribution of GFP2-tagged forms of both CXCR1 and CXCR2 monitored by confocal microscopy indicated significant plasma membrane delivery (Fig. 1), this can only be fully established by three-dimensional cellular reconstruction from z-plane confocal slices through a single cell. We therefore wished to confirm the quaternary structure of CXCR1 and CXCR2 homo- and heterodimers at the surface of transfected cells and to assess whether interactions between at least this population of these receptors might be modulated by IL8. To do so we employed Tr-FRET following expression of N-terminally FLAG and c-Myc epitope-tagged forms of the CXCR1 and CXCR2 receptors. Addition of a combination of Eu3+-labeled anti-c-Myc antibody, as a long lived energy donor, and APC-labeled anti-FLAG antibody as an energy acceptor to intact HEK293 cells co-expressing N-terminally c-Myc- and FLAG-tagged forms of the CXCR1 receptor resulted in a substantial signal corresponding to Tr-FRET (Fig. 8). This was not observed with addition of only the Eu3+-labeled anti-c-Myc antibody or the APC-labeled anti-FLAG antibody (data not shown). Signals were also negligible when both labeled antibodies were added to mixed cell populations each expressing either c-myc-CXCR1 or FLAG-CXCR1 alone (Fig. 8). Equivalent results were obtained with expression of c-Myc and FLAG-tagged forms of CXCR2 (Fig. 8). Equally strong Tr-FRET signals were also obtained from cells co-expressing pairs of N-terminally tagged CXCR1 and CXCR2 receptors (Fig. 8), with results being equivalent whether CXCR1 or CXCR2 acted to bind the energy donating Eu3+-labeled anti-c-Myc antibody (Fig. 8). As in the BRET2 experiments, addition of IL8 (100 nM, 15 min) did not modify the Tr-FRET signal (Fig. 8).



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  		FIG. 8.
Tr-FRET detects constitutive CXCR1 and CXCR2 homomeric complexes at the surface of HEK293 cells. Combinations of N-terminally FLAG and c-Myc forms of CXCR1 and CXCR2 were transfected individually and the cells mixed (Mix) or co-expressed (Co) in HEK293 cells. Cells were treated with vehicle (open bars) or 100 nM IL8 (filled bars). Tr-FRET was then measured in these cells, as under "Experimental Procedures," to monitor homo- and heteromeric interactions at the cell surface. Data represent means ± S.E. from three independent experiments.

 
Recent studies have provided evidence that protein/protein interactions involving GPCRs can occur at an early stage in biogenesis and in advance of delivery to the cell surface (16, 17). To examine this for CXCR1/CXCR2 receptors, we employed an endoplasmic reticulum trapping strategy. As shown earlier (Fig. 1), following transient expression in HEK293 cells, a substantial fraction of both N- and C-terminally modified forms of the CXCR1 receptor is delivered to the cell surface. Following transient expression of N-terminally HA-tagged CXCR1 in HEK293 cells, antibody staining in nonpermeabilized and permeabilized cells confirmed substantial membrane delivery of this construct (Fig. 9A). By contrast, although a fraction of a N-terminally HA-tagged form of {alpha}2C-adrenoreceptor was able to reach the cell surface and hence be detected without cellular permeabilization (Fig. 9A), the vast majority of this construct was retained intracellularly, with a distribution pattern consistent with labeling of the endoplasmic reticulum (Fig. 9A). Indeed, previous work has shown this to reflect the presence of an endoplasmic reticulum retention motif within the C-terminal 14 amino acids of the {alpha}2C-adrenoreceptor (37, 38). We generated a form of the CXCR1 receptor with these 14 amino acids from the {alpha}2C-adrenoreceptor added to the C-terminal tail. In contrast to the wild type CXCR1 receptor, little of the modified CXCR1 receptor was trafficked to the cell surface (Fig. 9A). This was not a reflection of the poor expression of this construct. Following permeabilization of the cells, the modified CXCR1 receptor (HA-CXCR1-ER) was shown to be largely intracellular (Fig. 9A). The cell surface expression of FLAG-CXCR1 was monitored when this construct was co-expressed with either HA-tagged wild type CXCR1 or HA-CXCR1-ER. Only some 50% as much of the FLAG-tagged wild type CXCR1 reached the cell surface when its expression partner was HA-CXCR1-ER (Fig. 9B). The same was true when the ability of FLAG-CXCR2 to reach the cell surface was monitored. Coexpression with HA-CXCR1-ER resulted in a substantial reduction of cell surface FLAG-CXCR2 compared to when this receptor was co-expressed with HA-CXCR1 (Fig. 9B). As in the BRET2 experiments, we wished to establish the selectivity of the endoplasmic reticulum trapping strategy. When FLAG-{alpha}1A-adrenoreceptor was employed, cell surface delivery of this construct was the same whether co-expressed with HA-CXCR1 or HA-CXCR1-ER (Fig. 9B), confirming a lack of significant interactions between the CXCR1 receptor and the {alpha}1A-adrenoreceptor.



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  		FIG. 9.
An endoplasmic reticulum trapping strategy to identify CXCR1 receptor interacting GPCRs. A, CXCR1 (a), HA-{alpha}2C-adrenoreceptor (b), and HA-CXCR1-ER retained (c) constructs were expressed transiently in HEK293T cells grown on coverslips. Immunostaining with anti-CXCR1 antibody was performed in nonpermeabilized (left-hand panels) and permeabilized (right-hand panels) cells. B, HEK293T cells were transiently co-transfected with either HA-CXCR1 (open bars) or the ER-retained version of this construct (HA-CXCR1-ER) (filled bars) along with N-terminally FLAG-tagged forms of CXCR1, CXCR2, or the {alpha}1A-adrenoreceptor. Equal numbers of cells were incubated with anti-FLAG antibody conjugated to APC for 2 h prior to washing. Fluorescence corresponding to the APC-labeled antibody was then quantified. Background fluorescence of HEK293T cells was measured and removed from the values obtained. ***, p < 0.001. Data are means ± S.E. from three experiments.

 
To assess if interactions between the CXCR1 and CXCR2 receptors altered potency or functionality of ligands, we examined the ability of the CXCR2-selective agonist GRO-{alpha} to inhibit cAMP generation stimulated by forskolin (10 µM) in cells individually expressing CXCR1 or CXCR2, cells individually expressing the two receptors but mixed prior to the assay or cells co-expressing CXCR1 and CXCR2. As anticipated, GRO-{alpha}-mediated inhibition of cAMP production was 30-fold more potent in cells expressing CXCR2 than CXCR1 (Fig. 10). In cells co-expressing CXCR1 and CXCR2, the potency of GRO-{alpha} was intermediate between the values for the individually expressed receptors and was not different from that noted for cells individually expressing CXCR1 or CXCR2 that were mixed prior to assay (Fig. 10).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It has become widely accepted that many members of the rhodopsin-like family A class of GPCRs have the capacity to homodimerize. As both direct experimental data (39) and models based on the dimensions of GPCRs and G proteins (14) are consistent with a GPCR dimer binding a single G protein heterotrimer, dimerization may be integral to GPCR function (6). There is increasing evidence that dimers of many GPCRs form during synthesis and maturation and that this can be important for cell surface delivery (16, 17). Equally, there are many examples in which the extent of GPCR dimerization is unaffected by the binding of agonist ligands and, indeed, where the GPCR is internalized into the cell as a dimer/oligomer in response to agonist challenge (40, 41).

The chemokine receptors are members of the class of family A GPCRs (25). However, although the literature is complex (for review see Ref. 5), a number of reports on specific chemokine receptors are inconsistent with the simple pattern outlined above. For example, early studies indicated the chemokine SDF-1{alpha} to produce dimerization of the CXCR4 receptor that was almost undetectable in the absence of the ligand (19). In complete contrast, combinations of BRET and sedimentation studies indicated the CXCR4 receptor to be a constitutive dimer that was unaffected by the presence of SDF-1{alpha} (22). Ligand-induced dimerization has also been reported for the CCR2 receptor on addition of monocyte chemoattractant protein-1 (18), and the capacity of human immunodeficiency virus, type 1, to utilize the CCR5 receptor as a "co-receptor" for cell entry is blocked by dimerization of the receptor produced by chemokine agonists (42). However, the CCR5 receptor has also been reported to be both a ligand-independent constitutive dimer/oligomer (21) and a monomer (22). At this time, a clear pattern is therefore difficult to discern. In many other aspects of structure, function, and regulation, family A GPCRs show considerable parsimony, as might be expected for a family of homologous proteins (43), and thus we wished to use as wide a range of approaches as possible to re-examine aspects of the interactions between CXCR1 and CXCR2 receptors. In contrast to work published previously (23), here we demonstrate the ability of the CXCR1 receptor, as well as the CXCR2 receptor, to form a homodimer/oligomer and that when co-expressed the CXCR1 and CXCR2 receptors are able to heterodimerize/oligomerize. Such conclusions are based on data from five distinct techniques, including co-immunoprecipitation, resonance energy transfer approaches, and intracellular trapping by an ER-retained version of the CXCR1 receptor. Three of these approaches were particularly enlightening. First, the ER trapping strategy showed that an ER-retained form of the CXCR1 receptor limited cell surface delivery of N-terminally FLAG-tagged forms of both CXCR1 and CXCR2. Specificity of this assay was established by unaltered cell surface delivery of an N-terminally FLAG-tagged form of the {alpha}1A-adrenoreceptor, a receptor shown by other means to interact with the CXCR1 and CXCR2 receptors with minimal affinity. This assay established that CXCR1 homodimerization and CXCR1-CXCR2 heterodimerization occurs, as established previously for the {beta}2-adrenoreceptor (17), during receptor synthesis and maturation. Second, the application of cell surface Tr-FRET showed that constitutively established forms of each of CXCR1 and CXCR2 homodimers/oligomers and CXCR1-CXCR2 heterodimers were present at the surface of cells in the absence of IL8 and that such interactions were not modified substantially by the presence of the agonist. Third, use of saturation BRET2 techniques (36) showed that the propensity of CXCR1 and CXCR2 to generate hetero-interactions was not different from their ability to homodimerize. Thus, unless specific cellular mechanisms exist in particular cell types to ensure that mRNAs encoding these GPCRs are trafficking to different sections of the ER machinery, then it must be expected that CXCR1-CXCR2 heterodimers as well as the corresponding homodimers will exist and in ratios determined by expression levels of the individual receptors. Although experimental evidence indicates the hypothesis that the propensity of pairs of distinct GPCRs to form heterodimers will simply reflect the sequence homology between individual GPCRs (44) is too simplistic (for review see Ref. 5), the high similarity between the CXCR1 and CXCR2 would certainly be consistent with the equivalent apparent interaction affinities for homo- and heterodimerization that we observed in the BRET saturation studies. Similarly, for the three opioid receptor subtypes, very recent BRET saturation studies have indicated similar interaction affinities between all possible heterodimeric pairs (45), although earlier and technically more limited studies had indicated that certain pairs were unable to heterodimerize (46). In each of these then the CXCR1 and CXCR2 receptors behave as "typical" family A GPCRs. As noted above, the issue of ligand dependence of GPCR dimerization has been particularly contentious for the chemokine receptors. A substantial number of resonance energy transfer-based experiments have shown small effects of agonist ligands above substantial signals corresponding to constitutive dimers/oligomers (5). Despite this, an emerging consensus favors family A GPCRs with small endogenous ligands that bind within the seven transmembrane domains predominantly as constitutive dimers. The observed effects of ligands are then most likely because of conformational alterations within the complex resulting in small changes in the orientation or distance between the resonance energy transfer reporters (47). However, for family A GPCRs with larger peptide and small protein ligands that have contact points for binding out with the topology of the seven transmembrane helix bundle, the situation is more complex. Each of the neuropeptide Y Y4 receptor (48), thyrotropin receptor (49), and type A cholecystokinin receptor (50) dimers have been reported to be constitutively formed but dissociated by agonists, whereas, by contrast, the gonadotrophin-releasing hormone (51), lutropin (52), and a number of other receptors have been reported to increase in aggregation state in response to agonists. As such, heterodimerization between GPCR pairs might alter ligand pharmacology and function, particularly for receptors such as the chemokine receptors where ligand binding is defined at least in part by elements in the extracellular N-terminal region of the receptor. Indeed, recent studies (53) have provided evidence for negative binding cooperativity within the CCR5-CCR2b heterodimers. As such, in the current studies, we examined if the effectiveness or potency of the CXCR2-selective agonist ligand GRO-{alpha} to inhibit cAMP production was affected by co-expression of CXCR1 and CXCR2. It was not, and thus at least for this ligand, which has relatively modest selectivity between the two GPCRs, there was no indication of the heterodimer displaying a distinct pharmacology or function. Further work, perhaps with less closely related GPCRs, will be required to explore such possibilities more effectively. For example, the ability of the CCR5 chemokine receptors to heterodimerize with other co-expressed GPCRs, including opioid receptors, has been reported recently (54). At the moment there is no information on the relative affinity of such interactions and therefore their likely importance for physiology and function. However, as other examples of GPCR heterodimerization, including those involving interactions between the angiotensin AT1 receptor and each of the AT2 receptors (55), the bradykinin B2 receptor (56), and the mas proto-oncogene (57), have been reported to have physiological and pathophysiological consequences, application of the type of techniques used in these studies would provide a useful starting point to understand the importance of interactions between chemokine receptors with other members of the GPCR family.



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  		FIG. 10.
The potency of GRO-{alpha} to inhibit cAMP production is unaffected by co-expression of CXCR1 and CXCR2. The potency of GRO-{alpha} to inhibit cAMP generation stimulated by forskolin (10 µM) was measured in cells individually expressing CXCR1 and CXCR2 (closed squares and closed circles, respectively), cells individually expressing the two receptors and mixed prior to the assay (open circles), and cells co-expressing CXCR1 and CXCR2 (open diamonds). pIC50 values represent the potency of GRO-{alpha} to inhibit cAMP production. Results represent means ± S.E. of five individual experiments. No significant difference in potency was found between mixed CXCR1- and CXCR2-expressing cells and those co-expressing CXCR1 and CXCR2 (Student's t test).

 

   FOOTNOTES
 
* This work was supported in part by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a CASE studentship from the Biotechnology and Biosciences Research Council. Back

|| To whom correspondence should be addressed: Davidson Bldg., University of Glasgow, Glasgow G12 8QQ, Scotland, UK. Tel.: 44-141-330-5557; Fax: 44-141-330-4620; E-mail: g.milligan{at}bio.gla.ac.uk.

1 The abbreviations used are: GPCRs, G protein-coupled receptors; APC, allophycocyanin; BRET, bioluminescence resonance energy transfer; CFP, cyan fluorescent protein; ER, endoplasmic reticulum; GFP, green fluorescent protein; Tr, time-resolved; FRET, fluorescence resonance energy transfer; IL8, interleukin 8/CXCL8; TR-FRET, time-resolved fluorescence resonance energy transfer; YFP, yellow fluorescent protein; HA, hemagglutinin; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Angers, S., Salahpour, A., and Bouvier, M. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 409-435[CrossRef][Medline] [Order article via Infotrieve]
  2. Milligan, G., Ramsay, D., Pascal, G., and Carrillo, J. J. (2003) Life Sci. 74, 181-188[CrossRef][Medline] [Order article via Infotrieve]
  3. Breitwieser, G. E. (2004) Circ. Res. 94, 17-27[Abstract/Free Full Text]
  4. Javitch, J. A. (2004) Mol. Pharmacol. 66, 1077-1082[Abstract/Free Full Text]
  5. Milligan, G. (2004) Mol. Pharmacol. 66, 1-7[Abstract/Free Full Text]
  6. Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. (1996) J. Biol. Chem. 271, 16384-16392[Abstract/Free Full Text]
  7. Cvejic, S., and Devi, L. A. (1997) J. Biol. Chem. 272, 26959-26964[Abstract/Free Full Text]
  8. Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3684-3689[Abstract/Free Full Text]
  9. Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344[CrossRef][Medline] [Order article via Infotrieve]
  10. Rocheville, M., Lange, D. C., Kumar, U., Patel, S. C., Patel, R. C., and Patel, Y. C. (2000) Science 288, 154-157[Abstract/Free Full Text]
  11. McVey, M., Ramsay, D., Kellett, E., Rees, S., Wilson, S., Pope, A. J., and Milligan, G. (2001) J. Biol. Chem. 276, 14092-14099[Abstract/Free Full Text]
  12. Ramsay, D., Carr, I. C., Pediani, J., Lopez-Gimenez, J. F., Thurlow, R., Fidock, M., and Milligan, G. (2004) Mol. Pharmacol. 66, 228-239[Abstract/Free Full Text]
  13. Liang, Y., Fotiadis, D., Filipek, S., Saperstein, D. A., Palczewski, K., and Engel, A. (2003) J. Biol. Chem. 278, 21655-21662[Abstract/Free Full Text]
  14. Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2004) FEBS Lett. 564, 281-288[CrossRef][Medline] [Order article via Infotrieve]
  15. White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M., and Marshall, F. H. (1998) Nature 396, 679-682[CrossRef][Medline] [Order article via Infotrieve]
  16. Terrillon, S., Durroux, T., Mouillac, B., Breit, A., Ayoub, M. A., Taulan, M., Jockers, R., Barberis, C., and Bouvier, M. (2003) Mol. Endocrinol. 17, 677-691[Abstract/Free Full Text]
  17. Salahpour, A., Angers, S., Mercier, J. F., Lagace, M., Marullo, S., and Bouvier, M. (2004) J. Biol. Chem. 279, 33390-33397[Abstract/Free Full Text]
  18. Rodriguez-Frade, J. M., Vila-Coro, A. J., de Ana, A. M., Albar, J. P., and Martinez-A, C., and Mellado, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3628-3633[Abstract/Free Full Text]
  19. Vila-Coro, A. J., Rodriguez-Frade, J. M., Martin De Ana, A., Moreno-Ortiz, M. C., Martinez-A, C., and Mellado, M. (1999) FASEB J. 13, 1699-1710[Abstract/Free Full Text]
  20. Mellado, M., Rodriguez-Frade, J. M., Vila-Coro, A. J., Fernandez, S., Martin de Ana, A., Jones, D. R., Toran, J. L., and Martinez-A, C. (2001) EMBO J. 20, 2497-2507[CrossRef][Medline] [Order article via Infotrieve]
  21. Issafras, H., Angers, S., Bulenger, S., Blanpain, C., Parmentier, M., Labbe-Jullie, C., Bouvier, M., and Marullo, S. (2002) J. Biol. Chem. 277, 34666-34673[Abstract/Free Full Text]
  22. Babcock, G. J., Farzan, M., and Sodroski, J. (2003) J. Biol. Chem. 278, 3378-3385[Abstract/Free Full Text]
  23. Trettel, F., Di Bartolomeo, S., Lauro, C., Catalano, M., Ciotti, M. T., and Limatola, C. (2003) J. Biol. Chem. 278, 40980-40988[Abstract/Free Full Text]
  24. Hernanz-Falcon, P., Rodriguez-Frade, J. M., Serrano, A., Juan, D., del Sol, A., Soriano, S. F., Roncal, F., Gomez, L., Valencia, A., Martinez-A, C., and Mellado, M. (2004) Nat. Immun. 5, 216-223
  25. Horuk, R. (2001) Cytokine Growth Factor Rev. 12, 313-335[CrossRef][Medline] [Order article via Infotrieve]
  26. Bertini, R., Allegretti, M., Bizzarri, C., Moriconi, A., Locati, M., Zampella, G., Cervellera, M. N., Di Cioccio, V., Cesta, M. C., Galliera, E., Martinez, F. O., Di Bitondo, R., Troiani, G., Sabbatini, V., D'Anniballe, G., Anacardio, R., Cutrin, J. C., Cavalieri, B., Mainiero, F., Strippoli, R., Villa, P., Di Girolamo, M., Martin, F., Gentile, M., Santoni, A., Corda, D., Poli, G., Mantovani, A., Ghezzi, P., and Colotta, F. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 11791-11796[Abstract/Free Full Text]
  27. Levac, B. A., O'Dowd, B. F., and George, S. R. (2002) Curr. Opin. Pharmacol. 2, 76-81[CrossRef][Medline] [Order article via Infotrieve]
  28. Breit, A., Lagace, M., and Bouvier, M. (2004) J. Biol. Chem. 279, 28756-28765[Abstract/Free Full Text]
  29. Lee, S. P., So, C. H., Rashid, A. J., Varghese, G., Cheng, R., Lanca, A. J., O'Dowd, B. F., and George, S. R. (2004) J. Biol. Chem. 279, 35671-35678[Abstract/Free Full Text]
  30. Rodriguez-Frade, J. M., Mellado, M., and Martinez-A, C. (2001) Trends Immunol. 22, 612-617[CrossRef][Medline] [Order article via Infotrieve]
  31. Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108[CrossRef][Medline] [Order article via Infotrieve]
  32. Eglen, R. M. (2002) Assay Drug Dev. Technol. 1, 97-104[CrossRef][Medline] [Order article via Infotrieve]
  33. Hall, D. A., Beresford, I. J. M., Browning, C., and Giles, H. (1999) Br. J. Pharmacol. 126, 810-818[CrossRef][Medline] [Order article via Infotrieve]
  34. Carrillo, J. J., López-Gimenez, J. F., and Milligan, G. (2004) Mol. Pharmacol. 66, 1123-1137[Abstract/Free Full Text]
  35. Milligan, G. (2004) Eur. J. Pharm. Sci. 21, 397-405[CrossRef][Medline] [Order article via Infotrieve]
  36. Mercier, J. F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. (2002) J. Biol. Chem. 277, 44925-44931[Abstract/Free Full Text]
  37. Zerangue, N., Schwappach, B., Jan, Y. N., and Jan, L. Y. (1999) Neuron 22, 537-548[CrossRef][Medline] [Order article via Infotrieve]
  38. Margeta-Mitrovic, M. (2002) Methods 27, 311-317[CrossRef][Medline] [Order article via Infotrieve]
  39. Baneres, J. L., and Parello, J. (2003) J. Mol. Biol. 329, 815-829[CrossRef][Medline] [Order article via Infotrieve]
  40. Yesilaltay, A., and Jenness, D. D. (2000) Mol. Biol. Cell 11, 2873-2884[Abstract/Free Full Text]
  41. Cao, T., Brelot, A., and von Zastrow, M. (2004) Mol. Pharmacol. 67, 288-297[CrossRef][Medline] [Order article via Infotrieve]
  42. Vila-Coro, A. J., Mellado, M., Martin de Ana, A., Lucas, P., del Real, G., Martinez-A, C., and Rodriguez-Frade, J. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3388-3393[Abstract/Free Full Text]
  43. Ballesteros, J. A., Shi, L., and Javitch, J. A. (2001) Mol. Pharmacol. 60, 1-19[Abstract/Free Full Text]
  44. Ramsay, D., Kellett, E., McVey, M., Rees, S., and Milligan, G. (2002) Biochem. J. 365, 429-440[CrossRef][Medline] [Order article via Infotrieve]
  45. Wang, D., Sun, X., Bohn, L. M., and Sadee, W. (2005) Mol. Pharmacol. 67, 2173-2184[Abstract/Free Full Text]
  46. Jordan, B. A., and Devi, L. A. (1999) Nature 399, 697-700[CrossRef][Medline] [Order article via Infotrieve]
  47. Ayoub, M. A., Couturier, C., Lucas-Meunier, E., Angers, S., Fossier, P., Bouvier, M., and Jockers, R. (2002) J. Biol. Chem. 277, 21522-21528[Abstract/Free Full Text]
  48. Berglund, M. M., Schober, D. A., Esterman, M. A., and Gehlert, D. R. (2003) J. Pharmacol. Exp. Ther. 307, 1120-1126[Abstract/Free Full Text]
  49. Latif, R., Graves, P., and Davies, T. F. (2001) J. Biol. Chem. 277, 45059-45067
  50. Cheng, Z. J., and Miller, L. J. (2001) J. Biol. Chem. 276, 48040-48047[Abstract/Free Full Text]
  51. Cornea, A., Janovick, J. A., Maya-Nunez, G., and Conn, P. M. (2001) J. Biol. Chem. 276, 2153-2158[Abstract/Free Full Text]
  52. Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904-5914[Abstract/Free Full Text]
  53. El-Asmar, L., Springael, J. Y., Ballet, S., Andrieu, E. U., Vassart, G., and Parmentier, M. (2005) Mol. Pharmacol. 67, 460-469[Abstract/Free Full Text]
  54. Suzuki, S., Chuang, L. F., Yau, P., Doi, R. H., and Chuang, R. Y. (2002) Exp. Cell Res. 280, 192-200[CrossRef][Medline] [Order article via Infotrieve]
  55. AbdAlla, S., Lother, H., Abdel-tawab, A. M., and Quitterer, U. (2001) J. Biol. Chem. 276, 39721-39726[Abstract/Free Full Text]
  56. AbdAlla, S., Lother, H., el Massiery, A., and Quitterer, U. (2001) Nat. Med. 7, 1003-1009[CrossRef][Medline] [Order article via Infotrieve]
  57. Kostenis, E., Milligan, G., Christopoulos, A., Sanchez-Ferrer, C. F., Heringer-Walther, S., Sexton, P., Gembardt, F., Kellett, E., Martini, L., Vanderheyden, P., Schultheiss, H. P., and Walther, T. (2005) Circulation 111, 1806-1813[Abstract/Free Full Text]

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