Published as doi: 10.1096/fj.07-8777com.
(The FASEB Journal. 2007;21:3827-3834.)
© 2007 FASEB
A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization
Mark M. Hammer1,
Tom S. Wehrman1,2 and
Helen M. Blau3
Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
3Correspondence: Baxter Laboratory, CCSR 4215, 269 Campus Dr., Stanford, CA 94305-5175, USA. E-mail: hblau{at}stanford.edu
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ABSTRACT
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G-protein-coupled receptor (GPCR) signaling is involved in a wide range of physiological processes and diseases, and around one-half of currently used drugs target GPCRs. Assays for the signaling of GPCRs have suffered from drawbacks, including low signal-to-noise, temporally transient signals, and difficulty in applying a single assay to a wide range of GPCRs. We have developed a set of assays for G-protein-coupled receptor signaling based on β-galactosidase enzyme complementation in live mammalian cells. We previously described an assay for GPCR activation by monitoring the binding of β-arrestin to the receptor. Here we describe a second assay that monitors the internalization of GPCRs to endosomes, an event that follows receptor activation and is critical in desensitizing and resensitizing the receptor. We show that both assays display high signal-to-noise ratios with low variability and are quantitative for a wide range of GPCRs. EC50s obtained with these assays closely match results reported in the literature. Finally, we show that these assays are readily adapted to high-throughput chemical screens. Thus, these two assays for monitoring G-protein-coupled receptor activation and internalization should prove valuable in basic biological studies as well as in high-throughput screens.—Hammer, M. M., Wehrman, T. S., Blau, H. M. A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization.
Key Words: GPCR signaling • ligand binding • high-throughput screening
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INTRODUCTION
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G-PROTEIN-COUPLED RECEPTORS (GPCRs) comprise the largest known family of membrane proteins, functioning as receptors for small molecules or peptide hormones. GPCR signaling is involved in a wide range of physiological processes, including vascular tone, neurotransmission, and immune cell chemotaxis. Because of the central role that GPCRs play in many therapeutically important processes and their amenability to modulation by small molecules, nearly one-half of clinically used drugs currently target GPCRs. Thus, discovering new ways to assess GPCR activity will be a great boon to drug discovery efforts, allowing the identification of novel agonists and antagonists that might be more difficult to detect with existing methodologies.
The molecular mechanisms underlying GPCR signaling have been identified over the past few decades (reviewed in ref. 1
). Briefly, ligand binding induces a conformational change in the transmembrane receptor that is transmitted to its C-terminal cytoplasmic tail. This allows heterotrimeric G-proteins to interact with the receptor’s tail and thereby become activated; different GPCRs couple to different heterotrimeric G-proteins—for example, Gs (which activates cAMP and Ca2+ flux) or Gi (which inactivates cAMP) (reviewed in ref. 2
). The activated cytoplasmic tail is also a substrate of the kinase GRK, and the protein β-arrestin will bind the phosphorylated receptor tail; once β-arrestin has bound, the receptor can no longer activate G-proteins; in addition, β-arrestin recruits clathrin to the receptor, leading to the receptor’s internalization. Once the receptor is internalized, ligand is believed to dissociate, and the receptor can either be recycled back to the cell surface or sent to a lysosome for degradation. Thus, the biology of GPCRs presents some easily assayed activation steps. Classically, the G-protein response was measured (e.g., by monitoring cAMP levels), but this method suffers from the drawback that many receptors will inactivate cAMP or will not signal through cAMP at all. However, the arrestin binding step and the internalization step are both G-protein independent (1)
and therefore should be universal to all GPCRs.
Besides being a "universal" property of GPCRs, receptor internalization is also a biologically significant event in GPCR signaling; as mentioned above, internalization is utilized for down-regulation of receptors as well as receptor recycling to obtain a faster response time (reviewed in ref. 2
). The majority of GPCR internalization assays are currently based on fluorescence microscopy of GFP translocation (reviewed in ref. 3
). Drawbacks of such assays are the time required for data acquisition and the difficulty in analyzing the large quantities of image data generated, a process that is not easily automated. As a result, microscopic methods are used mainly as secondary screening assays. Even in secondary screens, these fluorescence assays suffer from being nonquantitative and yield poor signal-to-noise ratios.
To develop a quantitative assay for GPCR internalization, we adapted a recently described method of monitoring intracellular protein translocation using low-affinity 
-complementation of β-galactosidase (4)
. Historically, in -complementation of Escherichia coli β-galactosidase, a short peptide from the N-terminal region of β-galactosidase (designated ) spontaneously complements a large fragment that is an N-terminally deleted version of the intact enzyme (designated 
) (5)
. In our earlier work, we adapted this complementation system to eukaryotic cells, which was highly successful for monitoring cell fusion (6
7
8
9)
but relatively limited in studies designed to monitor protein interactions (10
, 11)
. As a result, we recently developed novel β-galactosidase mutant fragments (4)
. We now routinely assess protein interactions using a truncated low-affinity -peptide selected specifically because it weakly complements the fragment. Since the interaction of the two enzyme portions is insufficient to maintain a complemented enzyme, reversible interactions can be monitored, and the activity of the enzyme is directly related to the local concentration of the enzyme fragments. By fusing proteins to the enzyme fragments, this assay of β-galactosidase activity can be used to quantitatively monitor interactions of the two proteins. Using β-galactosidase complementation, we have previously assayed translocation of proteins into and out of the nucleus (4)
, defined a role for herceptin in modulating ErbB2 receptor interactions (12)
, and investigated the effects of nerve growth factor on TrkA and p75 receptors (13)
. Here we apply this complementation approach to the study of GPCR activation and internalization in response to agonists and antagonists.
To monitor internalization, we fused the fragment to a protein "probe" for the cytoplasmic face of endosomes and the fragment to various GPCRs. When the GPCR is activated and internalized, the concentration of the fragment on the surface of endosomal vesicles increases dramatically, resulting in a robust increase in complementation as measured by β-galactosidase activity. Using an enzyme-based assay, protein interactions could be readily assayed with a chemiluminescent substrate, an assay easily adapted to high-throughput applications.
Further, we characterized the system in comparison to the recently described GPCR activation assay, which measures the interaction at the plasma membrane between an activated GPCR and β-arrestin using β-gal complementation (12
, 14)
. These two assays were extended to four different G-protein-coupled receptors and were shown to give similarly robust results. We observed quantitative modulations of receptor activation and internalization in response to a range of agonists, antagonists, and temperature changes. The robustness of these assays allowed them to be easily applied to a small-molecule screening platform.
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MATERIALS AND METHODS
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Plasmids
Plasmids encoding fusion proteins were generated by subcloning PCR products into MFG-eYFP-(H31R)-IRES-CD8 (abbreviated eYFP-) and pWZL--IRES-neo (abbreviated ) plasmids, which were designed as described previously (4)
. The FYVE domain of endofin was generated by RT-PCR from a mouse cell line using primers 5'-atcgacggatccATGCAGAAACAACCTACATGGG and 5'-ctgagtcaatgTTTATTTATAGTCTCATAGC; this PCR product was cloned N-terminal to eYFP- or using BamHI and MfeI restriction sites. Other FYVE domains were generated similarly by RT-PCR. The β2AR- construct was used as described (15)
. To generate a β2AR-eYFP- construct, the eYFP- fragment was subcloned into the β2AR- plasmid using XhoI and HindIII. The angiotensin receptor 1a was derived by RT-PCR from C57/Bl6 mouse brain RNA using primers for the entire coding sequence minus the stop codon; it was cloned N-terminally to eYFP- or with MfeI and XhoI. Human arginine vasopressin receptor 1a cDNA was purchased from the UMR cDNA Resource Center; PCR products were generated and cloned similarly to the angiotensin receptor 1a. Finally, to generate fusion proteins with β-arrestin, the full coding sequence of human β-arrestin B2 was PCR amplified from a cDNA clone and inserted into the MfeI-XhoI sites of the and eYFP- vectors.
Cell culture and viral transduction
Virus was produced in the 
NX ecotropic packaging cell line (P. L. Achacoso and G. P. Nolan, unpublished data) by transient transfection of plasmids using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Supernatant was applied to C2C12 myoblasts after 12–72 h; cells were then centrifuged for 30 min at 2000 rpm in the presence of 8 µg/ml polybrene (Sigma, St. Louis, MO, USA) after a 15 min incubation at 37°C. Transduced cells were selected either by antibiotic resistance using 1 µg/ml Geneticin (Invitrogen) or by flow cytometric sorting for eYFP, using a FACSTAR flow cytometer with MoFlo electronics. C2C12 myoblasts were grown in DMEM/20% FBS.
Assays and cell preparation
Isoproterenol, propranolol, angiotensin II, [Arg8]-vasopressin, and somatostatin (somatostatin-14) were purchased from Sigma. Vasopressin units were converted from I.U. to molar using data obtained from Raggenbass et al. (16)
. LysoTracker Red and Texas Red-labeled transferrin were purchased from Molecular Probes (Carlsbad, CA, USA). The kinase/phosphatase inhibitor library was purchased from BIOMOL (Plymouth Meeting, PA, USA). Cells were plated and grown to confluency in 96-well white tissue culture dishes (Corning Costar, Acton, MA, USA) in 100 µl of growth media. Dose response curves were generated by adding the respective ligand to the media and incubating for 2 h at 37°C. Compound library screening was performed by replicating the compound library into 96-well or 384-well plates using disposable 96- or 384-pin replicators (Genetix, New Milton, Hampshire, UK) and incubating for 30 min at 37°C before stimulating cells with ligand for 1 h at 37°C. At the end of the incubation time, Gal-Screen reagent (buffer B formulation; Applied Biosystems, Foster City, CA, USA) was used to assay for β-galactosidase activity according to the manufacturer’s protocol; media was aspirated from the plate, and 50 µl (or 25 µl for 384-well plates, without prior media aspiration) of Gal-Screen substrate diluted in lysis buffer was added to each well. Plates were incubated at room temperature for 40 min to 1 h before reading in a TR717 luminometer (Tropix, Bedford, MA, USA). EC50 values were calculated by applying nonlinear regression (fitting to the logistic, or Hill, curve) using Prism (GraphPad Software, San Diego, CA, USA).
Fluorescence microscopy
Cells were grown on 4-well chamber slides (BD Biosciences, Bedford, MA, USA) or in tissue culture plates (Corning Costar). Texas Red-labeled transferrin was purchased from Molecular Probes and used at 50 µg/ml; cells were incubated in DMEM/transferrin for 30–45 min at 37°C, then fixed in 4% paraformaldehyde before mounting in Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Fixed or live cells were imaged using a Zeiss laser scanning confocal microscope (LSM510 on an Axiovert 100M; Carl Zeiss, Thornwood, NY, USA).
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RESULTS
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Design of receptor internalization and receptor activation assays
To design an internalization assay for GPCR activation, we exploited the low-affinity enzyme complementation technology, which relies on protein proximity to yield complementation of two fragments of β-galactosidase (4)
. For the design of the internalization assay, we utilized an endosomal probe (one enzyme fragment localized to the cytosolic face of endosomes) and a tag on the GPCR of interest (the other fragment on the GPCR’s cytoplasmic tail); this paradigm is shown diagrammatically in Fig. 1
a.
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Figure 1. a) β-Galactosidase complementation assay schemes. Top scheme shows the internalization assay, where the receptor (GPCR) is coupled to the fragment and the FYVE domain of endofin is coupled to the fragment; upon internalization, the receptor colocalizes with PI-3P-positive endosomes coated with FYVE domains. Bottom scheme shows the activation assay, where the receptor is coupled to the fragment and β-arrestin is coupled to the fragment; upon ligand binding, β-arrestin is recruited to the receptor, reconstituting β-galactosidase. "Enzyme fragment" refers to either or eYFP-. b) Localization of various protein-YFP- peptide fusions; endofin FYVE, EEA1 FYVE, and fens FYVE refer to the isolated FYVE domains of the respective protein. Of the FYVE domains, only that of endofin was sufficient to cause endosomal localization. c) Time course of β2-adrenergic receptor stimulation with 10 µM isoproterenol: top panel shows internalization assay as in panel a; bottom panel shows fluorescence of β2AR-eYFP-; note the punctuate appearance of internalized β2AR. d) Dose response of β2-adrenergic receptor in the internalization assay (FYVE-+GPCR-eYFP-, open circles) and activation assay (β-arrestin-+GPCR-eYFP-, filled circles) at 1 h. Also shown are the best-fit sigmoidal dose response curves. Error bars represent SD of 2–4 biological replicates.
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To design the endosomal probe, we took advantage of the fact that the cytosolic face of endosomes is enriched in the phospholipid phosphatidylinositol-3-phosphate (PI-3P) to which FYVE domain-containing proteins bind (17)
. We screened several FYVE domains for this purpose, that of Fens, EEA1, and endofin; the localization of FYVE domain fusions to YFP- peptide constructs is shown in Fig. 1b
along with several endosomally localized rab GTPases for comparison. Several FYVE domains show punctuate staining indicative of endosomal localization. The endofin FYVE domain showed the clearest localization, as expected based on previous studies (18)
. The endosomal label transferrin was used to validate that the punctuate staining pattern exhibited by the endofin FYVE-eYFP fusion specifically labeled endosomes; extensive colocalization of the red transferrin signal and green eYFP signal confirmed the almost complete localization of endofin-FYVE to endosomes (Supplemental Fig. 1).
As a first test of the system, C2C12 cells expressing the endofin FYVE-eYFP- construct were transduced with a construct encoding a C-terminal fusion of β2AR with the fragment (β2AR-). The resulting assay cell line was treated with isoproterenol, and β-galactosidase activity was measured in 96-well dishes using a chemiluminescent substrate. Agonist treatment resulted in a 6-fold induction of β-gal activity by 1 h of stimulation (Fig. 1c
). These results demonstrate that our complementation-based internalization assay could measure GPCR internalization. The time course of increased β-gal activity shows that this process reaches a plateau by 1 h of stimulation. The internalization of β2AR to endosomes was confirmed by intracellular localization and colocalization with transferrin (Fig. 1c
and data not shown). As a second test of the system, we generated a dose response curve by treating the cells with isoproterenol and measuring β-gal activity at 60 min (see Figs. 1d
and 3a
). We obtained an EC50 of 20 nM, which is in line with previous studies of β2AR activation (19)
showing that the assay can be used as a quantitative measure of GPCR activation/internalization.
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Figure 2. Characterization of β2-adrenergic receptor (β2AR) assays. a) Inhibition of β2AR activation and internalization by the competitive antagonist propranolol in the presence of 10 µM isoproterenol. b) Low-temperature selective inhibition of β2AR internalization: top panel shows complementation assay results; bottom panel shows confirmation by fluorescence of YFP fusion proteins. Error bars represent SD of biological replicates, and curves show best-fit sigmoidal dose response curves.
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The canonical pathway for GPCR internalization involves binding of a β-arrestin molecule that bridges the GPCR and the internalization machinery. We earlier developed a complementation assay for the interaction of a GPCR and β-arrestin (4
, 14)
. The development of the internalization assay affords the ability to quantitatively compare GPCR internalization and β-arrestin binding. To compare results of the internalization assay and the GPCR activation assay, we measured the dose response of the two assays in parallel; the dose response curves yielded EC50s of 14 nM (activation) and 20 nM (internalization), in agreement with a previously reported value of 14 nM (see Figs. 1d
and 3a
) (18)
.
Quantitative modulation of β2AR activation and internalization
We monitored the time responsiveness of both β2AR assays. Internalization of the β2AR began to plateau at 1 h whereas receptor activation continued to increase for up to 2 h (Fig. 1c
and data not shown). This time course was confirmed by imaging fluorescent receptor fusions, which showed translocation of the receptor from the plasma membrane to punctate intracellular vesicles (Fig. 1c
). Both assays could be inhibited by the β2AR antagonist propranolol (Fig. 2
a), with an IC50 value of 38 nM in the presence of 10 µM isoproterenol, in agreement with published reports (20)
. These results show that both the internalization and activation assays can be used to assay modulators of GPCR activity.
To determine whether the activation and internalization assays could be uncoupled, we tested the effects of temperature on the internalization and activation of the β2AR. It has been described that low temperatures inhibit internalization (21)
, and detailed studies have demonstrated that a drop to 16°C is sufficient to prevent internalization (22)
. In agreement with these findings, β2AR internalization was inhibited at temperatures below 16°C relative to activation (Fig. 2b
). Fluorescent imaging confirmed these results, showing no effect on FYVE domain localization but a lack of β2AR internalization at 16°C (Fig. 2b
, bottom panels). As expected, the activation assay continued to function at temperatures as low as 0°C, although the magnitude of induction was reduced.
Generalization of receptor activation and internalization assays
Upon ligand binding, most classes of GPCRs are internalized. To determine the applicability of the assay to different GPCRs, we tested whether the internalization assay could be used to detect the internalization of angiotensin II 1a (AT1a), arginine-vasopressin 1a (AVPR1a), and somatostatin 2 receptors. Using the angiotensin II receptor 1a (AT1a), we observed a 4-fold increase in activity with the internalization assay, with an EC50 of 3.3 nM, close to the value of 1.2 nM reported in the literature for IP3 production (23)
(Fig. 3
b). When we used the arrestin binding activation assay for this receptor, we obtained an 8.5-fold increase in enzyme activity and an EC50 of 2.4 nM. Similarly robust results were obtained with the arginine-vasopressin receptor 1a (AVPR1a) in response to vasopressin: the internalization assay resulted in an 
4-fold induction with an EC50 of 4.4 nM, and the arrestin assay yielded 9-fold induction with an EC50 of 6 nM (activation) (Fig. 3c
). With the somatostatin 2 receptor in response to somatostatin-14, a 3-fold induction and 7 nM EC50 were observed using the internalization assay and a 14-fold induction and 3 nM EC50 with the arrestin assay (Fig. 3d
). These results show that the internalization assay yields 3-fold or better inducibility, with relatively low error.
High-throughput screen proof-of-principle
Given that our set of assays displayed a robust response with low noise, we proceeded to test them in a high-throughput setting. For this trial, we selected a kinase/phosphatase inhibitor library (BIOMOL International) and performed the assay in a 384-well plate in duplicate. Figure 4
a shows the results of the screen, with assays yielding a Z-factor of 0.6 in this setting; horizontal lines show twice the SD away from the mean. The screen yielded wortmannin, a phosphatidylinositol 3-kinase inhibitor, as a significant hit (arrows in Fig. 4a
), which was confirmed by a separate dose response curve (Fig. 4b
). As expected, wortmannin’s inhibition of PI-3-kinase depleted the PI-3P levels on endosomes; as FYVE domains use this phospholipid for their localization, we would expect a loss of specific endosomal localization. Indeed, we did observe that the endofin FYVE-GFP- probe relocalized to the cytoplasm upon treatment of cells with wortmannin (Fig. 4c
and ref. 22
). Thus, wortmannin would be expected to prevent colocalization, and therefore complementation, of the endosomal marker and internalized receptors to yield a consequent decrease in the internalization assay signal. This expectation is in good agreement with our findings. However, we noticed that, unexpectedly, wortmannin also appeared to inhibit the activation (β-arrestin binding) assay by 50%, although the effects on the internalization assay were more pronounced with an 90% inhibition. We discovered two more inhibitors of internalization in addition to wortmannin; these were staurosporine and erbstatin, both kinase inhibitors. The fact that kinase inhibitors would affect GPCR internalization poses several interesting novel hypotheses about the mechanism of internalization, which will be the subject of further study.
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DISCUSSION
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Endocytosis plays a critical role in cell function, regulating the amount of receptor on the cell surface as well as helping to transduce signals from the receptor and controlling the recycling or degradation of the receptor molecules themselves. Quantitative measurements of receptor internalization are difficult to accomplish; classical biochemical methods (i.e., cellular fractionation) suffer from low purity and are not amenable to high-throughput paradigms. Alternative methods, such as use of radiolabeled or fluorescently labeled ligands, require the availability of such reagents and may be cumbersome to implement; finally, monitoring the internalization of GFP-tagged receptors, while experimentally far easier, is inherently nonquantitative and also requires high-throughput microscopic imaging apparatus and sophisticated computer analysis software.
In this report, we show that by using localized β-galactosidase complementation, the concentration of a protein in a specific intracellular compartment can be assayed in live cells. This assay yields quantitative results in a high-throughput setting and requires relatively simple assay reagents and instrumentation. By using the β-galactosidase complementation system, we were able to develop a robust quantitative assay for GPCR internalization.
G-protein-coupled receptors undergo regulated internalization as part of their desensitization process, a critical component of GPCR signaling in physiological pathways. There is much interest in finding novel activators and inhibitors of GPCR signaling because of the role of GPCRs in a wide range of biological functions, including cardiovascular tone and immune function; the advent of β-arrestin-based assays for GPCR activation is poised to revolutionize these screening attempts by providing a universal method for assaying GPCR activation instead of traditional second messenger assays. Indeed, we have shown that such GPCR assays can be carried out not only in cultured cells, but also in live animals (14)
. However, although β-arrestin assays function well and are quite promising, there are reasons to look elsewhere in GPCR signaling for additional assays. In particular, some specificity exists with regard to which arrestins bind to a GPCR (25
, 26)
. Many reports in the literature show arrestin-independent GPCR internalization (reviewed in ref. 27
). Finally, in many cases the effects of a ligand on receptor internalization are of primary interest; for example, the addictiveness of morphine has been attributed in part to the fact that, although it is a strong agonist, it fails to internalize the µ opioid receptor (28)
. This finding highlights the potency of drugs that activate, but do not lead to internalization of, a receptor and suggests the importance of finding such agonists in certain applications.
In this paper we show that, by using localized β-galactosidase complementation to study GPCRs, we can develop assays that yield correct pharmacological values for agonists and antagonists in a wide range of receptors. The internalization assay compares favorably to the arrestin-based activation assay and therefore should be useful in general, as well as specifically for assaying arrestin-independent GPCRs. Finally, we show that the internalization assay is specific, since it can be inhibited by antagonists and low temperature.
Although GPCR desensitization often correlates with arrestin binding, the two events are temporally and functionally distinct. Chemical library screens that use either assay should yield novel drugs that modify the behavior of the target GPCR. Moreover, although applied to GPCRs in the present work, localized complementation assays should be readily adaptable to quantitative measurement of the endocytosis of various other cell surface molecules. Finally, the development of this system provides proof that positional complementation can be used to identify relative levels of proteins in subcellular compartments that are not readily assayed using other techniques, and thus it should be broadly applicable to further define the trafficking of proteins throughout the cell.
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ACKNOWLEDGMENTS
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The authors thank B. Raab for technical assistance and A. Palermo and J. Pomerantz for helpful discussions and editorial comments on the manuscript. T.S.W. was supported by National Institutes of Health (NIH) biotechnology training grant T32 GM08412, NIH aging training grant T32 AG0259, and a Genentech fellowship; M.H. was supported by a Stanford Undergraduate Research Opportunities grant; H.M.B. was supported by NIH grants HD018179, AG009521, AG024987, and AG020961 and by the Baxter Foundation.
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FOOTNOTES
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1 These authors contributed equally to this work. 
2 Current address: DiscoveRx Corp., Fremont, CA, USA.
Received for publication May 23, 2007.
Accepted for publication July 5, 2007.
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ABSTRACT
INTRODUCTION
