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Am J Physiol Cell Physiol 290: C728-C732, 2006. First published October 19, 2005; doi:10.1152/ajpcell.00310.2005
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METHODS IN CELL PHYSIOLOGY

Qdot Nanocrystal Conjugates conjugated to bombesin or ANG II label the cognate G protein-coupled receptor in living cells

Steven H. Young and Enrique Rozengurt

Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, CURE: Digestive Diseases Research Center and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California

Submitted 28 June 2005 ; accepted in final form 15 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Quantum dots (Qdot Nanocrystal Conjugates; Quantum Dot, Hayward, CA) exhibit high fluorescence and low photobleaching compared with organic dyes, properties that should enhance their detection at low densities. In view of the properties of Qdots and the biological and pharmaceutical importance of G protein-coupled receptors (GPCRs), we attempted to use Qdots to label GPCRs in a variety of live cell types. An agonist consisting of biotinylated bombesin or ANG II was conjugated to Qdot Nanocrystal Conjugates coated with streptavidin through a biotin-streptavidin linkage (Qdot agonist). Herein we demonstrate that Qdot-bombesin conjugate can label the bombesin-preferring GPCR in living mouse Swiss 3T3 cells and in Rat-1 cells. Similarly, we used the Qdot-ANG II conjugate to label GPCR in intact rat intestinal epithelial cells (IEC)-18 and in a human pancreatic adenocarcinoma cell line of ductal origin, HPAF-II cells. We demonstrate that Qdot-ANG II is brighter and more photostable than agonist labeled with the organic dye Cy3. Our results demonstrate that Qdot technology can be adapted to monitor ligand binding to GPCRs. Combined with the narrow and symmetric emission profile of Qdot Nanocrystal Conjugates, this information suggests the potential for a new multiplex strategy to determine the effect of agonists and/or antagonists on agonist binding to several GPCRs simultaneously in living cells.

3T3 cells; intestinal epithelial cells; human pancreatic adenocarcinoma cells of ductal origin

G PROTEIN-COUPLED RECEPTORS (GPCRs) are the largest family of cell-surface molecules involved in signal transduction (10, 16). These receptors are activated by a wide variety of agonists, including peptide and nonpeptide neurotransmitters, hormones, growth factors, odorant and tastant molecules, and photons (9, 10, 16). GPCRs are characterized by an extracellular NH2-terminal segment, seven transmembrane {alpha}-helices, three extracellular loops, three cytoplasmic loops, and a COOH-terminal segment (9). The large number of GPCRs and the importance of their roles in the regulation of multiple fundamental physiological functions (1, 16, 18, 22), which is directly supported by studies performed with GPCR-knockout mice (13), have made the search for novel therapeutic drugs targeting GPCRs an important, constantly expanding activity in the pharmaceutical industry (10). Consequently, novel approaches to evaluate agonist binding to GPCRs in intact and living cells could offer new avenues for the identification of agonists and/or antagonists of investigative and/or therapeutic value.

Qdot Nanocrystal Conjugates (Qdots; Quantum Dot, Hayward, CA) are nanocrystals with unique optical properties, including bright fluorescence, resistance to photobleaching, and a narrow emission bandwidth (2). These qualities, along with the availability of high-sensitivity charge-coupled device (CCD) television cameras, make Qdots attractive reagents with which to encode individual cells with unique fluorescent properties (11) for the detection of potentially low-density cell surface receptors on living cells using videomicroscopy (12). Recently, Qdots have been used to track individual glycine receptors and analyze their lateral mobility in the neuronal membranes of living cells (8), as well as to label the breast cancer marker Her2 on the surface of fixed and live cancer cells (23). In view of the highly promising properties of Qdots for cellular imaging and the biological and pharmaceutical importance of GPCRs, we attempted to use Qdots to label GPCRs in a variety of cell types.

The amphibian tetradecapeptide bombesin and mammalian peptides structurally related to bombesin, including gastrin-releasing peptide (GRP), bind to a high-affinity GPCR and elicit a complex network of signaling pathways and biological responses, including cell proliferation, in a variety of normal and cancer cells (14–17). Herein we report the use of Qdots to label the bombesin-preferring GPCR in living Swiss 3T3 cells and Rat-1 cells. Similarly, we used Qdots to label the ANG II GPCR in epithelial cells. An agonist consisting of biotinylated bombesin or ANG II was conjugated to Qdot 655-streptavidin through a biotin-streptavidin linkage to generate a fluorescent agonist that binds to live cells in a displaceable manner. Our results demonstrate that Qdot technology can be adapted to monitor ligand binding to GPCRs and suggest a new multiplex strategy for the determination of the effect of agonists and/or antagonists on agonist binding to several GPCRs simultaneously.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Stock cultures of mouse Swiss 3T3 cells, Rat-1 cells, rat intestinal epithelial cells (IEC-18 cells), and a human pancreatic adenocarcinoma cell line of ductal origin (HPAF-II cells) were maintained in DMEM supplemented with 10% FBS in a humidified atmosphere containing 10% CO2-90% air at 37°C. For experimental purposes, cells were subcultured onto small glass coverslips that could be mounted onto a chamber placed on the stage of a fluorescence microscope (Axioskop 2; Carl Zeiss, Thornwood, NY). Filter sets for imaging Cy3 and Qdot 655 were purchased from Chroma Technology (Brattleboro, VT).

Qdots emitting fluorescence at 655 nm (Qdot 655) and coupled to streptavidin were purchased from Quantum Dot. It is estimated that there are 5–10 streptavidin molecules per Qdot and that each streptavidin molecule has multiple binding sites, indicating that there may be 15–30 available biotin-binding sites per Qdot. To construct a Qdot agonist, Qdot 655-streptavidin was first diluted into Ca2+- and Mg2+-free PBS with 1 mg/ml BSA to an 80 nM concentration. Biotinylated LC-LC bombesin or biotinylated LC-ANG II, purchased from Anaspec (San Jose, CA), was diluted in Ca2+- and Mg2+-free PBS with 1 mg/ml BSA to a 400 nM concentration. Subsequently, equal volumes of Qdot 655-streptavidin were mixed with either biotinylated bombesin or biotinylated ANG II and maintained at 4°C with constant mixing for 30–45 min (1:5 Qdot-to-agonist molar ratio). This resulted in approximately five peptide molecules per Qdot, on average, with few unconjugated peptide molecules. Before cells were labeled, the mixture was diluted in HBSS (Invitrogen, Carlsbad, CA) to a final 2–10 nM Qdot concentration.

Photostability experiments. Red-emitting Cy3-streptavidin (Amersham Biosciences, Piscataway, NJ) was coupled to agonist after we had performed the procedures stated above. Cells were incubated with Qdot agonist for 10 min at room temperature and then rinsed with HBSS. Images of Qdot fluorescence were obtained using a cooled CCD camera (SPOT 2; Diagnostic Instruments, Sterling Heights, MI) and stored on a computer disk for later analysis. When imaging a series of slides for agonist competition experiments, the excitation intensity and camera exposure times were kept constant for all slides in the series. An image analysis program (NIH Image software; National Institutes of Health, Bethesda, MD) was used to measure the average fluorescence intensity in selected regions of interest covering areas comprising several cells (n = 6–10). The intensities of three or more regions were averaged. For demonstration purposes, the level and contrast of an experimental series was adjusted. All images in a given series were adjusted equally. No such adjustment was made for images used in the intensity analysis.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
To determine whether Qdot-ligand conjugates can be used to label endogenously expressed GPCRs, cultures of confluent and quiescent Swiss 3T3 cells were exposed for 10 min to Qdot 655-streptavidin (i.e., Qdots emitting fluorescence at 655 nm and coupled to streptavidin) conjugated to biotinylated bombesin. Cultures were incubated at 22°C, a condition that slows ligand-induced receptor internalization of bombesin GPCR in Swiss 3T3 cells (26). We verified that biotinylated bombesin bound to the bombesin receptor in Swiss 3T3 cells as judged by the ability of this derivative peptide to induce a rapid increase in the intracellular Ca2+ concentration in these cells (data not shown), a known early event induced by bombesin in Swiss 3T3 cells as well as in many other cell types. As shown in Fig. 1, cell-associated fluorescence in cells labeled with 2 nM Qdot-bombesin (Fig. 1, center) was strikingly higher than that observed in cells exposed for 10 min to non-agonist-coupled 2 nM Qdot-streptavidin (Fig. 1, left). Cell-associated fluorescence of Swiss 3T3 cells incubated with 2 nM Qdot-bombesin was markedly reduced by incubation in the presence of increasing concentrations of unconjugated bombesin (Fig. 1, right). As summarized in Fig. 1, bottom left, a 1,000-fold excess of unlabeled bombesin reduced Qdot-bombesin label intensity to 2% compared with Qdot-bombesin alone. In contrast, the level of Qdot-bombesin fluorescence associated with Swiss 3T3 cells was not substantially affected by addition of 100-fold excess of ANG II, neurotensin, or EGF (Fig. 1, bottom right). In the presence of these biologically active peptides, Qdot-bombesin labeling remained at >85% of control values. This result is in accord with the fact that the binding of 125I-GRP to intact 3T3 cells (24) as well as the cross-linking of this peptide to its GPCR in these cells (25) is not inhibited by multiple structurally unrelated peptides and growth factors. These results indicate that biotinylated bombesin conjugated to Qdot 655-streptavidin can label the bombesin GPCR endogenously expressed by Swiss 3T3 cells.


Figure 1
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  		Fig. 1. Qdot Nanocrystal Conjugate (Qdot)-bombesin conjugate labels bombesin receptors on living Swiss 3T3 cells. The labeling of confluent cultures of Swiss 3T3 cells was performed at 2 nM Qdot-bombesin conjugate for 10 min at 22°C and then washed with saline solution before capturing the image (top center). As a control, similar incubations were performed with either unconjugated 2 nM Qdot (top left) and/or with 2 nM Qdot-bombesin conjugate in the presence of 200 nM bombesin (top right). Increasing the concentration of unconjugated bombesin displaced Qdot-bombesin binding in a dose-dependent manner (bottom left). The displacement was specific: 200 nM ANG II, neurotensin, or EGF did not displace 2 nM Qdot-bombesin (bottom right). Scale bar in top center image, 15 µm.

 
To substantiate further the specificity with which Qdot-ligand conjugates can be used to label GPCRs, we also used wild-type Rat-1 cells, which do not express bombesin GPCR, and Rat-1 cells stably transfected with the bombesin GPCR. These transfected cells were generated and characterized previously in our laboratory (3, 4, 20). Cultures of Rat-1 cells expressing the bombesin GPCR were exposed for 10 min to biotinylated bombesin conjugated to Qdot 655-streptavidin. As shown in Fig. 2 , center, cell-associated fluorescence was strikingly higher than that observed in parallel cultures incubated with Qdot-bombesin in the presence of excess unconjugated bombesin (Fig. 2, right) or in cultures of wild-type Rat-1 cells that do not naturally express bombesin GPCR (Fig. 2, left). These results with Rat-1 cells expressing or not expressing the bombesin GPCR reinforce the notion that Qdot-ligand conjugates can be used to label GPCRs.


Figure 2
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  		Fig. 2. Qdot-bombesin conjugate labels bombesin receptors on living Rat-1 cells stably expressing the bombesin receptor. The labeling of confluent cultures of Rat-1 cells was performed at 2 nM Qdot-bombesin conjugate for 10 min at 22°C and then washed with saline solution before the image was captured (center). As a control, similar incubations were performed with either wild-type Rat-1 cells, which do not express the bombesin receptor (left), and/or with 2 nM Qdot-bombesin conjugate in the presence of 200 nM bombesin (right). Scale bar in top center image, 15 µm.

 
To extend the results obtained with Qdot-bombesin conjugates to other GPCR agonists, we produced a different conjugate using biotinylated ANG II and Qdot 655-streptavidin conjugate. As shown in Fig. 3, the resulting Qdot-ANG II conjugate labeled endogenously expressed ANG II receptors in cultures of either rat IEC-18 cells (Fig. 3, left) or HPAF-II cells (Fig. 3, bottom right). In the presence of excess unconjugated ANG II, Qdot-ANG II binding is greatly reduced (Fig. 3, top right). Previous work documented the expression of functional subtype 1 ANG II receptors in both IEC-18 and HPAF-II cells (5–7, 19).


Figure 3
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  		Fig. 3. Qdot-ANG II labels rat intestinal epithelial cells (IEC-18 cells) and a human pancreatic adenocarcinoma cell line of ductal origin (HPAF-II cells). The labeling of sparse cultures of IEC-18 cells was performed at 10 nM Qdot-ANG II conjugate for 10 min at 22°C and then washed with saline solution before the image was captured (left). Labeling of IEC-18 cells was strikingly reduced in the presence of 100x (1,000 nM) unconjugated ANG II (top right). Similarly, individual HPAF-II cells were scanned after 10-min incubation with 10 nM Qdot-ANG II, followed by saline wash (bottom right). Scale bar in left image, 15 µm.

 
Next, we determined whether Qdot-agonist conjugates provide imaging advantages compared with agonists labeled with organic dyes such as Cy3. Specifically, we compared the strength of the signal (i.e., brightness) and its capacity to retain signal strength after multiple light exposures (photostability) of Qdot 655-ANG II with Cy3-ANG II. As shown in Fig. 4, under equal labeling and imaging (Cy3 optics) conditions, Qdot 655-ANG II (Fig. 4, top row) is not only initially brighter than Cy3-ANG II (Fig. 4, bottom row) but also retains signal strength after 10 exposures, whereas the signal strength of Cy3-ANG II is greatly reduced. When viewed with Qdot 655 optics, Qdot 655 is considerably brighter than Cy3. Figure 4 (top row, right) shows the same cell cluster after 20 exposures under Cy3 optics but imaged with Qdot 655 optics and with the exposure time reduced fourfold (1/4) below that used with Cy3.


Figure 4
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  		Fig. 4. Qdot 655 is more photostable and brighter than Cy3. Top: cluster of IEC-18 cells labeled with 10 nM Qdot 655-ANG II conjugate for 10 min at 22°C and then washed with saline solution. After 10 exposures using Cy3 optics, image intensity was not reduced. After 10 more exposures, an image was obtained with Qdot 655 optics but with exposure time reduced fourfold (1/4 previous exposure time). Bottom: IEC-18 cell cluster labeled with 10 nM Cy3-ANG II conjugate for 10 min at 22°C and then washed with saline solution. Optics and exposure were identical to that of Qdot 655 (top), but image intensity was lower than that obtained with Qdot 655. After 10 exposures, image intensity was greatly reduced. Scale bar in top left image, 25 µm.

 
These results demonstrate that the Qdot-agonist conjugate is brighter and more photostable than agonist labeled with Cy3. These are important advantages when cells are imaged multiple times, such as in experiments in which the fate of ligand-receptor complexes are followed as a function of time.

Many GPCRs undergo ligand-induced endocytosis, a process that contributes to regulate the number and functional activity of receptors present in the plasma membrane through receptor sequestration and downregulation (21). Emerging evidence suggests additional functions of endocytosis in mediating GPCR signaling via certain effector pathways, including MAPK modules. Herein we have determined whether the Qdot-ANG conjugate can be used to monitor ligand internalization in IEC-18 cells by incubating the cells under conditions that either blocked (4°C) or allowed (37°C) agonist-induced receptor internalization. As shown in Fig. 5, when IEC-18 cells were incubated with 10 nM Qdot-ANG II for 1 h at 4°C, most of the label was retained at the cell surface, indicating that little ligand-induced internalization occurred. In contrast, when parallel cultures of IEC-18 cells were exposed to 10 nM Qdot-ANG II for 0.5 h at 37°C, most of the Qdots were observed in vesicles (e.g., endosomes, lysosomes) throughout the cell, indicating striking ligand-induced internalization. Polyvalent ligands may exhibit receptor tracking patterns that differ from those induced by univalent ligands. To examine Qdot-agonist internalization using a largely univalent ligand, Qdot-ANG II was formed with a 1.0:0.1 Qdot-to-ANG II ratio. As shown in Fig. 5 (right), this procedure also resulted in ligand-induced internalization. Although specific differences between univalent and polyvalent Qdot-ANG II in IEC-18 cells remain to be studied, these results demonstrate that Qdot-agonist conjugates, with their signal strength and photostability, provide a novel approach to monitor agonist-induced GPCR internalization.


Figure 5
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  		Fig. 5. Qdot-ANG II was rapidly internalized in IEC-18 cells at 37°C. Left: sparse cultures of IEC-18 cells were labeled with 10 nM Qdot-ANG II (formed with 1:5 Qdot-to-ANG II ratio) for 1 h at 4°C. Representative cell demonstrating both diffuse and punctate distribution restricted to plasma membrane is shown. Center: sparse cultures of IEC-18 cells labeled with 10 nM Qdot-ANG II for 0.5 h at 37°C. Representative cell is shown demonstrating extensive internalization of Qdots in vesicular compartments throughout the cell. Right: to produce largely univalent binding Qdot-ANG II, sparse cultures of IEC-18 cells were labeled with 10 nM biotin-ANG II formed with a 1.0:0.1 Qdot-to-ANG II ratio at 37°C for 0.5 h. Representative cell demonstrating internalization of Qdots in the cell is shown. Scale bar in left image, 15 µm.

 
The optical properties of Qdots make them ideal fluorophores for ultrasensitive multicolor multiplexing applications in molecular biotechnology and bioengineering (2, 12). In the present study, we have shown that Qdot-labeled bombesin or ANG II can be used to detect the cognate GPCRs expressed either endogenously or ectopically in mouse (Swiss 3T3), rat (Rat-1 and IEC-18), or human cells (HPAF-II). Many agonists can be biotinylated, and when required, "spacer arms" can be included between the biotin and the residues necessary for receptor binding to reduce steric hindrance. The availability of Qdots emitting light at several distinct wavelengths, coupled with a narrow emission profile, paves the way for detecting and labeling several GPCRs simultaneously.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-55003 and DK-56930 and by the W. M. Keck Foundation.


   ACKNOWLEDGMENTS
 
We thank Jim Sinnett-Smith for advice on experimental design and critical review of the manuscript.

E. Rozengurt is the Ronald S. Hirshberg Professor of Translational Pancreatic Cancer Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Rozengurt, Div. of Digestive Diseases, Dept. of Medicine, David Geffen School of Medicine, Univ. of California at Los Angeles, 900 Veteran Ave., Warren Hall, Rm. 11-124, Los Angeles, CA 90095-1786 (e-mail: erozengurt{at}mednet.ucla.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
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