Single-Molecule Imaging Demonstrates Ligand Regulation of

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Single-Molecule Imaging Demonstrates Ligand Regulation of Oligomeric Status of CXCR4 in Living Cells Jun Lao, Hua He, Xiaojuan Wang, Zhencai Wang, Yanzhuo Song, Bin Yang, Naseer Ullah Khan, Baosheng Ge, and Fang Huang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10969 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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The Journal of Physical Chemistry

Single-molecule Imaging Demonstrates Ligand Regulation of Oligomeric Status of CXCR4 in Living Cells Jun Lao,† Hua He,† Xiaojuan Wang, Zhencai Wang, Yanzhuo Song, Bin Yang, Naseer Ullahkhan, Baosheng Ge,* Fang Huang*

State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580, P. R. China

†

These authors contributed equally

*

To whom correspondence may be addressed: [email protected]; [email protected] Tel: 0086-532-86981560, FAX: 0086-532-86981560

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ABSTRACT: The role of dimerization and oligomerization of G-protein coupled receptors on their signal transduction is highly controversial. Delineating this issue can greatly facilitate rational drug design. With single-molecule imaging, we show that chemokine receptor CXCR4 exists mainly as monomer in normal mammalian living cells and forms dimers and higher-order oligomers at high expression level, such as in cancer cells. Chemotaxis tests demonstrate that the signal transduction activity of CXCR4 does not only depend on its expression level, indicating a close relation to the oligomeric status of CXCR4. Moreover, binding ligands can effectively up-regulate or down-regulate the oligomeric level of CXCR4, which suggests that binding ligands may realize their pivotal roles by regulating the oligomeric status of CXCR4 rather than simply inducing conformational changes.


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INTRODUCTION

G-protein-coupled receptors (GPCRs) are a large family of transmembrane proteins with seven α-helices,1 which play key roles in signal transduction in various processes and are the target of about 50% of the current therapeutic drugs on the market.2 Recently, dimerization and oligomerization have been demonstrated to be critical for many GPCRs to realize their functions. Therefore, better understanding on the structure, oligomerization and functions of GPCRs is expected to greatly facilitate rational design of drugs with increased efficacy and selectivity.3-4 Biophysical studies on GPCRs have therefore attracted enormous attention. Based on their sequence homology, GPCRs are classically divided into three subfamilies: family A (Rhodopsin-like receptors), family B (secretin receptors) and family C (metabotropic glutamate receptors).5 For family C GPCRs, which have been observed to exist as homodimer, heterodimer or even higher-order oligomeric status, and their oligomeric status are closely related to their transduction functions.6-7 However, the role of dimerization and oligomerization for some other GPCRs, such as those from family A, is still controversial. For example, although a growing body of evidence suggests that many family A GPCRs exist and function as dimers in vivo,6, 8-12 some other evidence supports that these proteins work as monomers.13-16 The controversy may come from the difference in experimental conditions or the nature of different detection technologies.



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CXCR4 is a member of family A GPCRs, which is expressed in a wide variety of cell types. As the natural receptor of stromal cell-derived growth factor (SDF-1α),17-18 CXCR4 plays a key role in leukocyte trafficking, hematopoiesis, organs development and cancer metastases.19-22 More importantly, CXCR4 is also one of the principle co-receptors for human immunodeficiency virus type 1(HIV-1),23 and thus are important therapeutic target.24 The diverse role of CXCR4 in physiology and disease underscore the importance of our better understanding of the biochemical or biophysical characteristics and regulatory mechanism of CXCR4 functions in living cells. Dimerization of CXCR4 has been characterized using fluorescence resonance energy transfer (FRET)25 and bioluminescence resonance energy transfer (BRET).26-27 Previous results suggest that CXCR4 can form constitutive homo- or heterodimers under basal conditions without ligand binding.26-27 However, it is still controversial whether ligands can regulate dimerization/oligomerization of CXCR4. BRET experiments suggest that ligand binding may not affect the oligomeric status of CXCR426 but induce conformational changes.27 It is noticed that previous studies were based on ensemble BRET or FRET experiments, which may not give a clear-cut result since the apparent change in energy transfer efficiency cannot tell whether it is due to the conformational change of CXCR4 or the variation in oligomeric status.28 Single-molecule fluorescence techniques have shown advantages over conventional ensemble methods in study of conformational changes and oligomeric status of 4 ACS Paragon Plus Environment

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proteins.29-30 Recently, single-molecule fluorescence imaging has been successfully applied on membrane protein studies in living cells.30-35 In these work, the target proteins were labelled either with fluorophores or fluorescent proteins, which could be photobleached in a total internal reflection fluorescence microscopy (TIRFM) experiments. By counting the number of photobleaching steps of a single molecule or complex, the oligomeric status can be well characterized.31-35 The number of photobleaching steps cannot be affected by conformational change so that it can be directly linked to the number of proteins in a complex. In the present study, we take the advantage of single-molecule fluorescence imaging to study dimerization of CXCR4 in the course of ligands binding. CXCR4 was expressed as fusion with enhanced green fluorescent protein (EGFP) and CXCR4-EGFP molecules on the living cell membrane were detected directly by TIRFM at single molecular level. We have found that CXCR4 exists as mixture of monomers and dimers in living cell membrane, where the oligomeric status of CXCR4 strongly depends on its expression level and is regulated obviously by ligands binding. Chemotaxis test shows that migration index of T-REx-293 cells with CXCR4 gene stably transfected changes with the oligomeric status of CXCR4, suggesting the correlation between functions and oligomeric status of CXCR4.

MATERIALS AND METHODS Plasmid Construct.

CXCR4-EGFP plasmid construct: CXCR4-EGFP fusion 5 ACS Paragon Plus Environment


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construct was generated by overlap PCR using the following primers: CXCR4 forward primer: 5’-AAAAAGCTTATGTCCATTCCTTTGCCTCTTTTG-3’, linker primer1: 5’-CAAGTTTTCACTCCAGCGGCGGCGCGGCCGCTGGCAGCATGGTGAGCA A-3’,

linker

primer2:

5’-ATGCTGCCAGCGGCCGCGCCGCCGCTGGAGTGAAAACTTG-3’ and EGFP reverse primer: 5’-AAAGAATTCTTACTTGTACAGCTCGTCCATGCCGAGA-3’. The PCR product was double digested with DNA restriction enzymes HindIII and EcoRI and then cloned into the pcDNA4.0/TO vector digested with the same DNA restriction enzymes. CD86-EGFP plasmid were constructed by sub-cloning the full-length CD86 DNA fragment into the XhoI and BamHI sites of pEGFP-N1, yielding the CD86-EGFP expression plasmid. All resulting constructs were confirmed by DNA sequencing. Cell Culture and Transfection. T-REx-293 cell line containing pcDNA6/TR gene that encodes the Tet repressor protein (TetR) was purchased from Invitrogen (Waltha, MA). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX (Invitrogen, USA) and supplemented with 10% fetal bovine serum (Sijiqing, China), HEPES (15 mM), non-essential amino acids (0.1 mM), penicillin (100 µg /ml), streptomycin (100 µg/ml) and grown at 37 ºC under 5% (v/v) CO2. The pcDNA4/TO/CXCR4-EGFP vector was transfected into T-REx-293 cells using Lipofectamine 2000 (Invitrogen, USA) according to manufacturer’s instruction. After transfection, cells were further cultured for 48 h, and then selective medium containing 6 ACS Paragon Plus Environment

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5 µg/ml Blasticidin and 60 µg/ml Zeocin was added. Cells were continuously cultured in the selective medium for another 4 weeks. Colonies stably integrating the recombinant vectors were picked up and screened for better receptor expression. To observe CXCR4-EGFP molecules, stably transfected cells were induced by 1 µg/ml Tetracycline, washed by PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), and then imaged in DMEM without phenol red using TIRFM. For the SDF-1α (Prospec, USA), AMD3100 (Selleck, USA) and TC14012 (Toris, USA) stimulation experiments, before imaging stably transfected cells were firstly incubated with the above agonists respectively in the DMEM without FBS for 10 min at 4 ºC, and then washed twice by PBS. In order to exclude the interference of protein density on photobleaching analysis, T-REx-293 cells was transiently transfected with CD86-EGFP plasmid in the serum-free and phenol red-free DMEM, and cells with expression time as 4 h and 8 h were imaged under TIRFM as control. Single Molecular Fluorescence Imaging. Single molecular fluorescence imaging was performed with objective-type TIRFM using an inverted Nikon Ti series microscope equipped with a total internal reflective fluorescence illuminator, 100×/1.49NA Plan Apochromat TIR objective and an intensified electron multiplying charge coupled device (EMCCD) camera (DU897, Andor, USA). EGFP was excited at 488 nm by a solid-state laser (Cobolt MLDTM488 nm) with the power of 7 mW and detected after the laser passing through the objective. The collected fluorescent signals were passed through two filters, B-2A cubes BA510IF and HQ 525/50 (Nikon, 7 ACS Paragon Plus Environment


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Japan), before directed to the EMCCD camera. The gain of the EMCCD camera was set as 296. Movies of 100-500 frames were acquired for each sample at a frame rate of 10 Hz. Image Analysis. For analysis of single-molecule fluorescence intensity in a movie acquired from living cells, the background fluorescence was first subtracted from each frame using the rolling ball method in ImageJ software (National Institutes of Health, USA).36 Then the first frame of each movie was used for fluorescent spot (regions of interest) selection. Fluorescent spots analysis was done using Speckel Trackerj software.37 Dot-blot Analysis. Dot-blot analysis was carried out to quantitatively estimate the expression level of CXCR4 after induction. 5×105 HeLa, HTC116, HL60 and stably transfected T-REx-293 cells with difference induction time were lysed with PBS buffer containing 2% (w/v) FC14 and 1 mM PMSF for 1 h at 4 ºC. Cell lysates were centrifuged at 13,000 × g for 10 min, supernatant was collected and then 3 µl of each sample was dotted onto nitrocellulose membrane. After air dried for 20 min, the membrane was blocked using 5% (w/v) milk and then incubated with mouse anti-CXCR4 monoclonal antibody (Abcam, USA) for 1 h. After washed 5 times, the membrane was incubated with HRP labeled goat anti-mouse secondary antibody (Tiangen, China) for another 1 h. Finally, the membrane was developed with SuperSignal®West Pico according to manufacturer’s instruction and imaged on a FLA-5100 imaging system (Fuji, Japan). Intensity of dot-blot was analyzed by 8 ACS Paragon Plus Environment

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MultiGauge Ver.3.X software. Chemotaxis Tests of the HeLa and Stably Transfected T-REx-293 Cells. HeLa or stably transfected cells were resuspended (1×104) in DMEM containing 0.5% (v/v) bovine serum albumin. Portions (100 µl) of the cell suspension were placed in the upper wells of Transwell chambers (Corning, USA) (500 µl) containing bare filter with a pore size of 8 µm. The same medium (500 µl) supplemented or not with SDF-1α (100 nM) was placed in the lower chambers. After incubation for 4 h at 37 ºC in a moist atmosphere containing 5% CO2, cells migrated through the filter were stained with crystal violet and counted by ImageJ.

RESULTS 1. Single-Molecule Detection on Living Cells with TIRFM



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Figure 1. Single-molecule imaging of CXCR4-EGFP in living cells using TIRFM. (a) The imaging of stably transfected T-REx-293 cells under laser confocal microcopy after 24 h induction, Green and blue represent CXCR4-EGFP and the Hoechst 33258stained nucleus, respectively (scale bar = 20 µm). (b) A typical single-molecule image of CXCR4-EGFP on the living T-REx-293 cells surface. Stably transfected cells after 4 h Tetracycline induction were imaged with TIRFM. The image is a section (20×20 µm) of the first frame from a stack of images (Movie S2) with background subtracted. The diffraction-limited spots (5×5 pixels， 800×800 nm) enclosed with cyan and yellow circles represented the signals from individual CXCR4-EGFP molecules , and was selected for intensity analysis (scale bar = 2 µm). Cyan circles represent monomers, yellow circles represent dimers. (c) One representative time course of


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GFP emission after background correction showed one-step bleaching. (d) One representative time course of GFP emission after background correction showed two-step bleaching.

To characterize the CXCR4 oligomeric status on cell membrane, the CXCR4-EGFP should be expressed on cell membrane, but not in the cytoplasm. After 24 h tetracycline induction, the constructed stably transfected cell line was imaged under laser confocal microcopy. It is found that most of CXCR4-EGFP molecules were expressed on the surface of cells as shown in Figure 1a. These cells are suitable for following single-molecule TIRFM imaging. Cells expressing CXCR4-EGFP molecules were directly imaged with TIRFM after 4 h induction as shown in Figure S1 and Movie S1. A grey square box of cell-size (20×20 µm) was intercepted from Figure S1 and Movie S1, and the square graph was background subtracted with ImageJ. The results are shown in Figure 1b and Movie S2. It can be seen that expression density of CXCR4-EGFP molecules, corresponding to the number of bright spots, on the surface of cells is less than 80 molecules in a 20×20 µm area. Most of the spots were well dispersed and could be distinguished within the spatial resolution of TIRFM (diffraction-limited fluorescence spots as 5×5 pixels，800×800 nm) as shown in Figure 1b and Movie S2. Fluorescence spots from Movie S2 were then extracted and their photobleaching was analyzed. It can be seen that some spots fit to a one-step bleaching model and others fit to a two-step bleaching model as 11 ACS Paragon Plus Environment


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shown in Figure 1c, d. It should be mentioned that in each image there are 512×512 pixels and each bright spot occupies a square of 5×5 pixels, which means that each image contains about 100×100 squares. In our experiments, in each image the maximum number of bright spots was less than 80, i.e. the probability to see protein molecules in a square of 5×5 pixels were less than 1%. Under these conditions, the probability to see two free CXCR4 in one square is expected to be less than 0.01% and therefore is negligible. Our control experiment with CD86,38 which can only exist as monomer, also supports that at this expression level multiple photobleaching was not due to the co-localization of free monomers as seen in Figure S2. This control experiment excluded that oligomerization of EGFP-CXCR4 here is induced by EGFP fusion protein. Moreover, other reports on oligomerization of membrane proteins fused with EGFP, such as EGFR,36 Glycine receptor39 and β2-adrenoceptors,40 also suggest that EGFP fusion part does not interfere with membrane protein self-association. 2. Characterization of CXCR4 Oligomeric Status in Resting Cells with Different Expression Level.


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Figure 2. Oligomeric status of CXCR4 with different expression level. Images of CXCR4-EGFP on the stably transfected T-REx-293 cell membrane were obtained under TIRFM at induction time of 1, 4, 8, 12 and 24 h. Cyan circles represent monomers, yellow circles represent dimers and red circles represent oligomers (scale bar = 2 µm).

To characterize the status of CXCR4, the number of photobleaching steps of individual fluorescent spots was counted, through which the stoichiometry of CXCR4 can be determined in detail.30, 41 It has been reported that over-expression of receptors could increase BRET efficiency, caused by energy transfer between molecules that do 13 ACS Paragon Plus Environment


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not form dimer/oligomer but located close enough, and then influence the determination of the oligomeric status of membrane proteins.42-43 To exclude this possibility, the expression level of CXCR4 was firstly quantitatively analyzed using dot-blot analysis at different induction time as seen in Figure S3. As shown in Figure S3, the expression level of CXCR4 in cell lines that natively expressed CXCR4, such as HeLa, HL60 and HCT116 cells, was roughly similar to our stably transfected cells with induction time of 4 h to 8 h. In order to follow how the oligomeric status of CXCR4 changes with expression level, the oligomeric status was characterized at different induction time, ranging from 1 h to 24 h. At the initial stage (especially at 1 h induction), expression level of CXCR4 on the cell surface was very low with no more than 10 fluorescence spots in 20×20 µm area as shown in Figure 2. At this expression level, monomers of CXCR4 were dominant and few dimers or oligomers could be detected as shown in Figure 3a. Dimers of CXCR4 began appear after 2 h induction and oligomers appeared after 8 h induction. These results suggest that CXCR4 was firstly expressed as monomers, and CXCR4 dimers or oligomers did not formed until it achieved high expression level. Interestingly, within these oligomers, trimers and higher order oligomers were also detected as shown in Figure 3b, c. To our knowledge, this is the first time to report the existing of trimers for CXCR4. At induction time of 12 and 24 h, the fluorescence intensity of individual CXCR4-EGFP spot significantly increased, and aggregates appeared with much longer fluorescence dwell time (more than 40 s) than monomers and dimers (data no 14 ACS Paragon Plus Environment

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shown). The expression level of CXCR4-EGFP at 12 h and 24 h induction time was estimated to be about 6- and 8-fold higher compared with that at 8 h induction time as seen in Figure S3. Especially, at induction time of 24 h, individual CXCR4-EGFP complexes could not be distinguished as shown in Movie S4, suggesting that single-molecule detection cannot be realized at such high expression level. To eliminate the influence of over-expression of CXCR4 in the following experiments, cells with induction time of 2 h and 4 h were used, in which the expression level of CXCR4 is even lower than the native expression level of CXCR4 in HeLa, HL60 and HCT116 cells.

Figure 3. Oligomeric status of CXCR4 on the stably transfected T-REx-293 cell membrane with different induction time. (a) Relative amount of monomer, dimer and oligomer of CXCR4-EGFP on the cell membrane at induction time of 1, 2, 4, and 8 h, sampled from more than 200 spots. (b) A typical photobleaching trajectory with three steps. (c) A typical photobleaching trajectory with multiple steps.

3. Chemotaxis Tests of Stably Transfected T-REx-293 Cells.



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Figure 4. Migration index of HeLa and stably transfected T-REx-293 cells at different induction time.

In order to further explore the relationship between CXCR4 oligomeric status and their chemotaxis function, chemotaxis tests of HeLa and our stably transfected cells at different induction time were investigated. It is found that migration index of stably transfected T-REx-293 cells changed with induction time. As shown in Figure 4, the migration index increased with induction time up to 8 h and then decreased. It is worth noting that migration index of HeLa, a cell line with CXCR4 natively expressed, is roughly equivalent to that of the stably transfected cell with induction time of 4 h. 4. Influence of Agonists on the Oligomerization of CXCR4. In order to investigate how agonists affect the oligomerization of CXCR4 on the cell membrane, the oligomeric status of CXCR4 treated with different agonists were systematically characterized with single-molecule TIRFM at induction time of 1 h, 2 h and 4 h, respectively. Three common agonists, including SDF-1α (full agonist), AMD3100 (partial antagonist) and TC14012 (inverse agonist), were selected for investigation. It


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is noticed that for cells treated with SDF-1α and AMD3100 only monomers were found at induction time of 1 h, and no dimers and oligomers were detected even stimulated with high concentration of agonists (data not shown). From induction time of 2 h, the addition of SDF-1α or AMD3100 could slightly increase the number of CXCR4 dimers, but oligomers were still missing as shown in Figure 5a, c. Higher concentration of SDF-1α and AMD3100 did not further promote the dimerization of CXCR4. With induction time of 4 h, the expression level of CXCR4 is similar to the native expression level of CXCR4 in tumour cells, and at this stage, the addition of SDF-1α or AMD3100 could induce more CXCR4 dimers and even oligomers with an obvious concentration dependent manner as shown in Figure 5b, d, where the full agonist SDF-1α was much more efficient than the antagonist AMD3100 at the same concentration. With the stimulation of 1 µM SDF-1α, the number of CXCR4 monomers, dimers and oligomers are almost equal on the cell membrane, however, for cells stimulated with even 2 µM AMD3100, CXCR4 monomers were still dominant as shown in Figure 5d. As a CXCR4 inverse agonist, TC14012 has been reported to induce a decrease of the BRET efficiency in CXCR4 dimerization studies.27 However, it is controversial whether this is caused by conformational changes within CXCR4 dimers or equilibrium shift of CXCR4 dimers/oligomers and monomers.27 We then studied the changes of CXCR4 oligomeric status before and after stimulation with TC14012 at induction time of 4 h and 8 h. Unlike full agonist SDF-1α and antagonist AMD3100, 17 ACS Paragon Plus Environment


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which can improve the dimerization/oligomerization, the inverse agonist TC14012 showed weak inhibition on the dimerization and oligomerization of CXCR4. As can be seen in Figure 5e, f, the addition of TC14012 caused increase of monomer CXCR4 and corresponding decrease of dimers 4 h after induction. Concentration dependence was also observed, where the monomers increased from 80% (240 of 303 spots from 11 cells) at the absence of TC14012 to 93% (259 of 280 spots from 10 cells) at 1 µM TC14012. Experiments carried out at 8 h after induction showed that with the increase of TC14012 concentration, the relative amount of monomers increased and the oligomers decreased, however the amount of dimers kept almost constant. Our experiments cannot exclude the existence of conformational change of CXCR4 upon the addition of TC14012, but it clearly demonstrates that the inverse agonist can regulate the oligomerization status of CXCR4, in a reverse way as the full agonist SDF-1α and antagonist AMD3100.


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Figure 5. Influence of different agonists on oligomeric status of CXCR4 on the stably transfected T-REx-293 cell membrane, sampled from more than 200 spots. (a) and (b) Influence of SDF-1α on oligomeric status of CXCR4 at induction time of 2 h and 4 h, respectively. (c) and (d) Influence of AMD3100 on oligomeric status of CXCR4 at induction time of 2 h and 4 h, respectively. (e) and (f) Influence of TC14012 on oligomeric states of CXCR4 at induction time of 4 h and 8 h, respectively. 19 ACS Paragon Plus Environment


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Taken together, with direct observation, our results clearly suggest that agonists can regulate the oligomeric status of CXCR4 on cell membrane, but different type of agonists work differently. This kind of agonist-induced regulation may be closely related

with

the

activation

mechanism

of

GPCRs

through

dimerization/oligomerization.

DISCUSSION

Increasing evidence suggests that GPCRs can form homo- and hetero-dimerization and oligomerization. It has been believed that dimerization and oligomerization are key processes regulating GPCRs activity. However, quantitative information on the oligomeric status of GPCRs on the membrane of living cells and the regulation by binding

ligands

is

very

limited.

Most

of

the

reports

on

GPCR

dimerization/oligomerization were based on co-immunoprecipitation,44 FRET45-46 and BRET47 experiments. These experiments have provided very important information to demonstrate the existence of dimers or oligomers of GPCRs. However, with these techniques, it is hard to distinguish monomers, dimers, trimers and even higher-order oligomers, not to mention quantifying each state. On the other hand, these experiments are normally carried out under over-expression conditions so that results may not reflect the real status of GPCRs under physiological conditions.48-49 To gain better understanding on the oligomeric status of GPCRs and their regulation, 20 ACS Paragon Plus Environment

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alternative techniques are required. Single-molecule fluorescence imaging has been applied to directly observe the dimers and oligomers of membrane proteins and study their dynamic behavior on the membrane of living cells under physiological conditions.31-33 This method allows oligomeric status characterized by counting the number of monomers in a complex directly, through photobleaching of single molecules. Statistical analysis of the monomers, dimers and oligomers can further provide quantitative information on the oligomeric status of membrane proteins. Single-molecule fluorescence imaging study on the dynamic behavior of β1-/β2-adrenoceptors and GABAB receptors on the living cell membrane has afforded deep insight on their dimerization property.41 CXCR4 is an important GPCR molecule, which related with the migration of 23 cancers and HIV virus entry, thus is valuable drug target. However, the oligomeric status of CXCR4 and its regulation by binding ligands are still highly controversial. In this work, we take the advantages of single-molecule fluorescence imaging to directly characterize the oligomeric status of CXCR4 on living cell membrane. Interestingly, our results show that at low expression level CXCR4 exists mainly as monomers. Dimers can be observed with less than 10% abundance at low expression level. Higher-order oligomers can only be detected at higher expression level. This observation suggests that CXCR4 has an appropriate dissociation constant fitting well with its expression level under physiological conditions so that the oligomeric status of CXCR4 can be regulated by its expression level. According to the 21 ACS Paragon Plus Environment


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immune-densitometry experiments, it is noticed that the expression level of CXCR4 after 4 h induction is roughly equivalent to that of tumor cells. At this expression level, CXCR4 exists as mixture of monomers and dimers on cell membranes, of which dimers are about 20%. It is therefore reasonable to speculate that CXCR4 in tumor cells is mainly in monomeric and dimeric status. Interestingly, trimers can also be detected at induction time of 8 h. Although GPCRs’ analogue bacteriorhodopsin forms trimers,50-51 GPCRs existing as trimers were seldomly reported. Further investigation will be required to reveal the functions of trimers and single-molecule techniques, which can detect different states individually on living cell membrane, is expected to be a good measure for this purpose. β1- and β2-adrenergic receptors are also found to exist as a mixture of monomers, dimers, trimers and oligomers on cell membrane. The β2-adrenergic receptor mainly exists as dimers and oligomers even at low receptor density, while for β1-adrenergic receptor monomers is dominant at different expression levels. Family C GPCRs, such as GABAB receptor, mainly exist as constituted dimers and tetramers at low density with few monomers could be detected. According to the efficacy for G protein activation, drugs targeting GPCR are traditionally classified as agonists that promote G-protein signaling, antagonist or inverse agonists that inhibit agonist with no impact, or a negative impact, on the receptor basal activity.52 The effect of agonists and antagonists on GPCR oligomerization is another highly controversial topic on GPCR dimerization 22 ACS Paragon Plus Environment

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studies.53-55 Previous experiments based on FRET or BRET indicate that agonist and antagonist have little effect on CXCR4 dimerization either in whole cells27 or cell lysis solution26. Although obvious BRET efficiency changes induced by agonist binding were observed, this was attributed to conformational change rather than shift of oligomeric equilibrium of CXCR4. Our results suggest that SDF-1α and AMD3100 can obviously shift the equilibrium and induce the formation of dimers, trimers and even higher-order oligomers. However, this effect is highly concentration dependent. When the concentration or expression level of CXCR4 is low, the addition of SDF-1α and AMD3100 cannot induce oligomerization of CXCR4, which may be controlled by the dissociation constant of CXCR4 (Kd). Differently, as an inverse agonist of CXCR4, TC14012 could induce the dissociation of CXCR4 dimers and oligomers. It is noticed that the evidence shown here is not consistent with previous report by Percherancier et al,27 where the effect of agonist binding on the oligomerization of CXCR4 was studied with BRET and the change of BRET efficiency was attributed to conformational change instead of dissociation of CXCR4 dimers. The reason for this inconsistency is not clear, but it should be noticed that 1) due to its intrinsic limitation, BRET cannot distinguish dimers and oligomers and both dissociation/association and conformational change can cause BRET efficiency change; and 2) although single-molecule fluorescence imaging method applied in this work can clearly distinguish monomers, dimers and oligomers and directly observe the shift of oligomeric status, it cannot detect conformational changes of CXCR4. Our results 23 ACS Paragon Plus Environment


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clearly show that agonist/antagonist can regulate the oligomeric status of CXCR4, which is similar to the results for sigma-1 receptor,12,16 but cannot exclude the possibility of conformational change upon ligand binding. Further study is required to reveal whether both dissociation and conformational change without dissociation can be induced by ligand binding. To date, although more and more GPCRs have been reported existing as dimers or even oligomers, why GPCRs dimerize and the relationship between dimerization and their signal transduction function are not yet clear. Previous reports suggest that dimerization of CXCR4 can regulate functions.56-57 We also notice that full agonist SDF-1α, which can activate G protein and result in complete downstream signal,58 can induce significant oligomerization of CXCR4. Antagonist AMD3100, which is also reported as partial agonist59 and can induce partial activation of G protein, can also slightly promote oligomerization of CXCR4. Differently, inverse agonist TC14012, which is reported to reduce G protein activity and inhibit downstream signal transduction,58 can decrease oligomerization of CXCR4. Taken together, our results strongly suggest that dimerization or oligomerization may play a key role in signal transduction of CXCR4, and different agonists may modulate activation of CXCR4 through regulation of oligomeric status. This is also consistent with previous reports that cognate ligands could regulate the activity of their receptor by altering the oligomeric/monomeric receptor ratio.12 To further explore the possible relationship of oligomeric status of CXCR4 and 24 ACS Paragon Plus Environment

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their function in signal transduction, chemotaxis of stably transfected T-REx-293 cells at different induction time was evaluated. It is noticed that the migration index of cells increases with induction time up to 8 h and decreases then. Very interestingly, our results based on single-molecule imaging show that the amount of dimers also increase with induction time within 8 h after induction. The amounts of dimers could not be determined with induction time longer than 8 h due to the too high expression of CXCR4 in our experiments but is not expected to increase with induction time due to the formation of higher order oligomers. These results indicate a close correlation between the chemotaxis ability and oligomeric status of CXCR4 in living cells. The equilibration of monomers, dimers and oligomers may be one of the regulators for their signal transduction function. In conclusion, by using real-time imaging of CXCR4 receptors on the living cell membrane at single molecule level, we find that CXCR4 exists as a mixture of monomers and dimers at the expression level similar to that in native tumor cells. Oligomeric status of CXCR4 regulates chemotaxis of the stably transfected cells. Both ligand SDF-1α and antagonist AMD3100 can enhance dimerization and oligomerization at certain CXCR4 expression level, and the efficacy of full agonist SDF-1α is much higher than antagonist AMD3100 at the same concentration. However, inverse agonist TC14012 shows clear inhibition on the dimerization and oligomerization of CXCR4. The regulation of agonist and antagonist on the oligomeric status of CXCR4 may be an important way to regulate CXCR4 activity. 25 ACS Paragon Plus Environment


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ACKNOWLEDGMENTS

This work was financially supported in part by the National Key Basic Research Program of China (2012CB518000), the National Natural Science Foundation of China (No. 21273287, 21373271, 21673294 and 21573289), the National High Technology Research and Development Program of China (2014AA093505) and the Fundamental Research Funds for the Central Universities. ASSOCIATED CONTENT Supporting Information We provide further single-molecule imaging and movie of CXCR4 and CD86, and dot-blot of CXCR4 density in HeLa, HTC116, HL60 and stably transfected cells with different induction time.

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Single-Molecule Imaging Demonstrates Ligand Regulation of

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