Research Article www.acsami.org
GPCR Activation and Endocytosis Induced by a 2D Material Agonist Wei-Tao Dou,†,∥ Ya Kong,‡,§,∥ Xiao-Peng He,*,† Guo-Rong Chen,† Yi Zang,*,‡,§ Jia Li,*,‡,§ and He Tian† †
Key Laboratory for Advanced Materials & Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China ‡ National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shoujing Road, Shanghai 201203, P. R. China § University of Chinese Academy of Sciences, UCAS, No. 19A, Yuquan Road, Beijing 100049, P. R. China S Supporting Information *
ABSTRACT: Agonist-induced activation and endocytosis of G protein-coupled receptors (GPCRs) are crucial for a number of physiological and pathological processes. However, tools that are available for probing GPCR endocytosis have been insufficient. Here, we developed a two-dimensional (2D) material agonist by supramolecular self-assembly between an endogenous agonist of κ-opioid receptor (KOR) and 2D molybdenum disulfide. The 2D material agonist has proven to be amenable for eliciting GPCR activation and endocytosis in cells stably expressing KOR rather than in those without KOR expression. Using super-resolution microscopy, we also show that the 2D material agonist colocalizes well with GFPfused KOR intracellularly. Further, the endocytosed 2D material agonist can selectively produce reactive oxygen species in cells that overly express KOR, as controlled by light irradiation. KEYWORDS: 2D material, G protein-coupled receptor, ligand, peptide, fluorescence, imaging, photodynamic therapy, endocytosis
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INTRODUCTION G protein-coupled receptors (GPCRs) are among the largest transmembrane protein families, modulating a number of physiological and pathological processes. The GPCR consists of seven transmembrane helixes that couple with heterotrimeric GTP-binding proteins (G proteins) intracellularly for the initiation of a variety of second messenger cascades (such as the cAMP and phosphatidylinositol signaling pathways).1,2 GPCRs can respond to a diverse range of extracellular ligands, including ions, hormones, lipids, nucleotides, neurotransmitters, and neuropeptides.3,4 In addition, GPCR genes represent about 1% of the total genome of mammals, and almost 30−60% of modern drugs target a GPCR either directly or indirectly.5−8 Similar to that in other types of receptors, conformational change of GPCRs induced by agonist binding leads to their internalization from the plasma membrane to the endosomes. Whereas GPCR internalization was originally thought to be functionally inactive,9 recent evidence suggests that the internalized GPCRs can activate gene transcription by proteolytic fragmentation.10 However, effective tools for the analysis of GPCR endocytosis have been insufficient. The κ-opioid receptor (KOR) is a subtype of opioid receptors that belongs to the class A subfamily of GPCRs. KOR mediates the action of endogenous and exogenous opioids in many physiological processes, including the regulation of pain, consciousness, and mood.11,12 In addition to dynorphin (DYN, a natural endogenous opioid ligand), a © 2017 American Chemical Society
number of natural alkaloids, terpenes, and other synthetic compounds also bind to the receptor. An increasing amount of evidence suggests that dysregulation of the DYN/KOR system contributes to the development and maintenance of psychiatric disorders,13 and KOR expression has been shown to be reduced after chronic agonist treatment. Here, we show that a twodimensional (2D) material agonist formed by the supramolecular assembly between 2D molybdenum disulfide (MoS2) and an effective fragment of DYN A14 is suitable for the fluorogenic investigation of KOR activation and endocytosis. With the advent of graphene, a number of 2D graphene analogues, particularly 2D transition metal dichalcogenides (TMDs), have been discovered and employed in many research fields.15−18 Because of the simplicity in the design and construction of these materials, a diverse range of 2D material bioensembles formed between biomolecular probes and 2D TMDs have been developed for biomedical applications.19−35 Of the 2D TMDs, 2D MoS2 has been used as the material of choice because it has proven to be biocompatible for in vivo theranostics.36,37 Recently, we have shown that 2D MoS2 can enhance the receptor-targeting imaging capacity of glycoligand and peptide-attached fluorescent probes.38,39 With this research, we demonstrate that a 2D MoS2-based material Received: February 24, 2017 Accepted: April 12, 2017 Published: April 12, 2017 14709
DOI: 10.1021/acsami.7b02754 ACS Appl. Mater. Interfaces 2017, 9, 14709−14715
Research Article
ACS Applied Materials & Interfaces
Figure 1. (a) Structure of the fluorescent agonist probe DWT-KY (YGGFLRRIK-5-TAMRA, where TAMRA is 5-carboxytetramethylrhodamine) for KOR binding. (b) Schematic illustration of the formation of a 2D p-Sheet between 2D MoS2 and DWT-KY and the use of the material ensemble for targeted activation of a KOR. This then leads to (1) activation of a downstream signaling pathway to release Ca2+ flux from endoplasmic reticulum and (2) endocytosis of the material that can release ROS intracellularly upon light irradiation. (c−e) High-resolution transmission electron microscopy (HRTEM) of 2D MoS2. (f) Fast Fourier transform pattern of a selected area from HRTEM of 2D MoS2. (g, h) HRTEM image of 2D pSheet (DWT-KY/2D MoS2 = 1 μM/35 μg mL−1) (the dashed circles in (g) highlight several representative 2D p-Sheets, and the arrows in (h) highlight several agonist probe particles adhered to the surface of 2D MoS2).
Figure 2. (a) Plot of the fluorescence intensity (F.I.) of DWT-KY (1 μM) as a function of increasing 2D MoS2 concentration. (b) Plot of the F.I. of 2D p-Sheets (DWT-KY/2D MoS2 = 1 μM/35 μg mL−1) as a function of increasing cell concentrations (293/KOR+ = HEK293 cells stably expressing the KOR; 293/KOR− = HEK293 cells without KOR expression). (c) Fluorescence change of the 2D p-Sheet (DWT-KY/2D MoS2 = 1 μM/35 μg mL−1) in the presence of 293/KOR+ (300 000 cells mL−1), 293/KOR− (300 000 cells mL−1), and unselective proteins (BSA = bovine serum albumin; RNase = ribonuclease) (***P < 0.001). All fluorescence measurements were carried out in phosphate-buffered saline (0.01 M, pH 7.4), with an excitation wavelength of 530 nm. (d) Dynamic light scattering (DLS) of 2D MoS2 (35 μg mL−1) and 2D p-Sheets (DWT-KY/2D MoS2 = 1 μM/35 μg mL−1). (e) ζ-potential of 2D MoS2 (35 μg mL−1) and 2D p-Sheets (DWT-KY/2D MoS2 = 1 μM/35 μg mL−1). (f) Raman spectra of 2D MoS2 (70 μg mL−1) and 2D p-Sheets (DWT-KY/2D MoS2 = 2 μM/70 μg mL−1). 14710
DOI: 10.1021/acsami.7b02754 ACS Appl. Mater. Interfaces 2017, 9, 14709−14715
Research Article
ACS Applied Materials & Interfaces
Figure 3. Fluorescence imaging (a) and quantification (b) of HEK293 cells stably expressing the KOR (293/KOR+) and of those without KOR expression (293/KOR−) with 2D p-Sheets (1 μM DWT-KY with increasing 2D MoS2 concentration, as indicated; the scale bar is applicable to all images) (***P < 0.001; 5 μg mL−1 vs 0 μg mL−1 2D MoS2). The images were taken with an Operetta high-content imaging system (excitation channel: 520−550 nm; emission channel: 560−630 nm) and quantified using the Columbus image data analysis system (PerkinElmer). (c) Relative mRNA expression levels of 293/KOR+ and 293/KOR− cells measured by real-time quantitative polymerase chain reaction (***P < 0.001).
(Figure 1g) and (2) particle-like objects, which are probably agonist aggregates, densely adhered to the material surface, forming a 2D bioensemble (Figure 1h). A redshift in UV absorbance was observed for the 2D pSheet with respect to that of DWT-KY alone, suggesting a πstacking between the peptide ligand and material (Figure S1).45 Fluorescence spectroscopy was also used to characterize the material assembly (Figure 2). An increase in the concentration of 2D MoS2 led to concentration-dependent fluorescence quenching of DWT-KY (Figures 2a and S2a). This phenomenon is in agreement with previous investigations as regards the quenching property of 2D MoS2 for fluorophores,19−22 suggesting the attachment of the agonist probe to the material surface. With the fluorescence quenching determined, we then tested whether the presence of cells that express KOR would lead to fluorescence recovery of the material ensemble. We established a human embryonic kidney 293 (HEK293) cell line stably expressing the KOR (293/ KOR+) by transfecting the cells with a cDNA encoding fulllength KOR and used raw HEK293 cells (293/KOR−) as the control. We observed that the addition of 293/KOR+ to the 2D p-Sheet solution led to a concentration-dependent fluorescence recovery (Figures 2b and S2b), whereas the presence of 293/ KOR− caused a much weaker fluorescence enhancement of the system (Figures 2c and S2c). In addition, the presence of several other proteins and enzymes, including pepsin, bovine serum albumin (BSA), ribonuclease, and lysozyme, did not lead to enhancement of the fluorescence of 2D p-Sheets (Figure 2c). These data suggest that the fluorescence enhancement of the 2D material agonist is selective for cells stably expressing KOR. The 2D p-Sheet also showed good photostability under UV irradiation (Figure S3). To further corroborate the material assembly, DLS and Raman spectroscopy were used. DLS analysis showed that the size of the 2D p-Sheet was slightly larger than that of 2D MoS2
agonist (2D p-Sheet) induces KOR endocytosis and that the internalized 2D p-Sheet can produce reactive oxygen species (ROS) in a light-controlled manner (Figure 1).
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RESULTS AND DISCUSSION We first synthesized the fluorescent agonist probe by coupling a peptide fragment (YGGFLRRI) of DYN A14 to 5-carboxytetramethylrhodamine (5-TAMRA) through lysine (K) as a linker (Figure 1a). The probe (DWT-KY) produced consists of two adjacent, positively charged L-arginine groups (R), which are envisioned to have strong electrostatic interactions with the negatively charged 2D MoS2 surface. The 2D p-Sheet formed by the supramolecular self-assembly between DWT-KY and 2D MoS2 has been shown to be capable of activating the Ca2+-flux downstream signaling pathway of KOR while inducing KOR endocytosis, which further leads to the selective intracellular production of ROS upon light irradiation (Figure 1b). 2D MoS2 sheets were produced by the established liquidexfoliation method.40 The 2D p-Sheet was then formed by mixing the DWT-KY probe with an aqueous solution of 2D MoS2 overnight. The presence of L-arginine in a peptide sequence has been shown to facilitate electrostatic interactions with negatively charged materials.41 In addition to the electrostatic forces between L-argenine and the S layer of MoS2, the van der Waals force between peptide/TAMRA and the material might also contribute to the supramolecular assembly.42 With the p-Sheet in hand, a variety of techniques were used for material characterization. HRTEM was first employed. The objects shown in Figure 1c,d appear to be thin flakes, which are morphologically similar to thin-layer MoS2 produced in previous studies.38−40 Further, the hexagonal symmetry of the 2D material is illustrated in Figure 1e,f (applying the fast Fourier transform filtering of HRTEM images).43,44 After assembly with the agonist probe, we observed that (1) the morphology of 2D MoS2 did not change 14711
DOI: 10.1021/acsami.7b02754 ACS Appl. Mater. Interfaces 2017, 9, 14709−14715
Research Article
ACS Applied Materials & Interfaces
Figure 4. (a) Time-dependent fluorescence imaging of HEK293T cells expressing the KOR (293T/KOR+) fused with GFP with 2D p-Sheets (DWT-KY/2D MoS2 = 1 μM/5 μg mL−1) by confocal laser-scanning microscopy (the scale bar is applicable to all images). The excitation channels for the nucleus (stained with Hoechst), GFP, and DWT-KY are 360−400, 460−490, and 520−550 nm, and the emission channels for the nucleus, GFP, and DWT-KY are 410−480, 500−550, and 560−630 nm, respectively. (b) High-resolution imaging of 293T/KOR+ fused with GFP with 2D pSheets (DWT-KY/2D MoS2 = 1 μM/5 μg mL−1) by stimulated emission depletion (STED) laser-scanning confocal microscopy (the dashed circles highlight the endocytosed peptide probes as well-localized with GFP; Pearson’s correlation coefficient = 0.699; the scale bar is applicable to all images). The excitation channels for the nucleus (stained with Hoechst stain), GFP, and DWT-KY are 360−400, 460−490, and 520−550 nm, and the emission channels for the nucleus, GFP, and DWT-KY are 410−480, 500−550, and 560−630 nm, respectively.
agonist probe by the KOR+ cells. This is in agreement with our previous observations that 2D MoS2 is capable of enhancing the interaction between ligand probes and transmembrane receptor proteins.38,39,48 This observation also represents, to the best of our knowledge, the first example that suggests the role of a 2D material in enhancing agonist internalization through GPCR endocytosis. With a subsequent polymerase chain reaction assay, we determined that the expression level of KOR in 293/ KOR+ was much higher than that in 293/KOR− cells (Figure 3c). This further corroborates that cell imaging by 2D p-Sheets was KOR-selective. Because agonist binding can activate downstream signaling pathways of KOR such as calcium (Ca2+) mobilization, we used a Ca2+ mobilization assay to analyze the Ca2+ responses of the HEK293 cells treated with the agonist probe and 2D p-Sheet. We determined that Ca2+ responses were elicited by the agonist probe (DWT-KY) in 293/KOR+ rather than in 293/KOR− cells (Figure S6a). Meanwhile, a similar Ca2+ response was observed on treating the 2D material agonist with 293/KOR− cells (Figure S6b). This suggests that the presence of the 2D MoS2 substrate did not affect the activation of the downstream signaling pathway of KOR. We further used confocal microscopy to illustrate endocytosis of the KOR with the 2D p-Sheet (Figure 4). We established a HEK293T cell line (293T/KOR+) that transiently expresses KORs fused with
alone, ranging from 100 to 400 nm in diameter (Figure 2d). The ζ-potential of the negatively charged 2D MoS2 was increased after assembly with the positively charged DWT-KY (Figure 2e). Typical Raman shifts at 404 and 378 cm−1 were observed for 2D MoS2, characteristic of the out-of-plane vibration of S (A1g) and in-plane relative motion between the S and Mo (E12g) modes of the MoS2 crystal, respectively (Figure 2f).46 As a consequence, the increased E12g/A1g ratio of the 2D p-Sheet (0.62) with respect to that of the 2D MoS2 (0.57) suggests a perturbed in-plane motion between S and Mo as a result of the surface coating of DWT-KY.47 Next, we tested the imaging ability of the 2D p-Sheet for HEK293 cells using fluorescence microscopy. We first observed that the fluorescence of the agonist probe (DWT-KY) was much stronger in 293/KOR+ than that in 293/KOR− cells (Figure S4) and that the presence of a free KOR agonist (YGGFLRR) caused concentration-dependent suppression of the fluorescence in 293/KOR+ cells (Figure S5). These preliminary results suggest that the agonist probe can image HEK293 cells in a KOR-dependent manner. We then evaluated the interaction between the 2D material agonist and cells. With an increase in 2D MoS2, a gradual fluorescence enhancement of DWT-KY was detected in 293/KOR+ rather than in 293/ KOR− cells (Figure 3a,b). This suggests that the presence of the 2D material substrate enhanced the internalization of the 14712
DOI: 10.1021/acsami.7b02754 ACS Appl. Mater. Interfaces 2017, 9, 14709−14715
Research Article
ACS Applied Materials & Interfaces
Figure 5. (a) Time-dependent determination of the ROS released from HEK293 cells stably expressing the KOR (293/KOR+) and those without KOR expression (293/KOR−) treated with 2D MoS2 (5 μg mL−1) or 2D p-Sheets (DWT-KY/2D MoS2 = 3.5 μM/5 μg mL−1) with (light+) or without (light−) light irradiation. ROS was quantified with dihydrorhodamine 123 (a fluorogenic ROS probe, 4 μM), with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. (b) Flow cytometry of 293/KOR+ and 293/KOR− cells treated with 2D MoS2 (5 μg mL−1) or 2D p-Sheets (DWT-KY/2D MoS2 = 6 μM/5 μg mL−1) with (light+) or without (light−) white-light irradiation (where Q1, Q2, Q3, and Q4 represent necrotic, late apoptotic, live, and early apoptotic cells, respectively). The black dashed circle highlights the necrotic and slightly late apoptotic 293/ KOR+ cells after treatment with 2D p-Sheets and light irradiation.
observed for both 293/KOR+ and 293/KOR− cells without light irradiation, irrespective of the presence of the materials, a gradually enhanced ROS signal was observed for the cells with time upon light irradiation (Figure 5a). For the light-positive (light+) group, we also observed that (1) more ROS was produced in 293/KOR+ cells pretreated with p-Sheets than that in those pretreated with 2D MoS2 and (2) more ROS was produced in 293/KOR+ cells than that in 293/KOR− cells treated with 2D p-Sheets and light irradiation. A subsequent cell-counting assay suggested that (1) the viability of 293/ KOR+ cells rather than that of 293/KOR− cells pretreated with 2D p-Sheets decreased with an increase in irradiation time (Figure S9) and (2) the viability decrease of 293/KOR+ cells was also dependent on the 2D MoS2 concentration (Figure S10). Finally, we determined by flow cytometry that only the treatment with 293/KOR+ cells rather than that with 293/ KOR− cells with 2D p-Sheets, followed by light irradiation, caused evident production of necrotic cells (Figure 5b). These results suggest that the 2D material agonist was endocytosed by the KOR-expressing cells and that the 2D material can produce ROS intracellularly in a light-controlled manner.
green fluorescence protein (GFP) for the visualization of the receptor and agonist probe colocalization. Shown in Figure 4a is a time-dependent fluorescence image of 293T/KOR+ cells with 2D p-Sheets. We observed that both the p-Sheet and GFP fluorescence translocated from the plasma membrane to the cytoplasm with time. The fluorescence of both fluorescent species merged well in these images. To further confirm the KOR endocytosis pathway, we used a known KOR inhibitor (GNTI, an arrestin recruitment inhibitor that inhibits the internalization of KOR) and promotor (U50488, a selective KOR agonist that promotes the internalization of KOR) to treat 293/KOR+ cells prior to incubation with 2D p-Sheets. The results shown in Figure S7 suggest that the presence of the inhibitor and promotor suppressed and enhanced the fluorescence of 2D p-Sheets, respectively. This confirms that the p-Sheet was internalized through the KOR endocytosis pathway. To better illustrate the fluorescence colocalization, we used high-resolution STED laser-scanning confocal microscopy to analyze KOR endocytosis (Figure 4b). In the merged image, we observed the isolated agonist and GFP fluorescence intracellularly, which might be a result of receptor−ligand dissociation in endosomes after endocytosis. However, the colocalized GFP and peptide agonist fluorescence was predominant (Pearson’s correlation coefficient = 0.699), highlighting the strong binding between the two species. Eventually, to demonstrate the KOR-selective endocytosis of the 2D material, we measured the ROS production of the 293/ KOR+ and 293/KOR− cells after treatment with 2D p-Sheets because of the ROS-producing ability of 2D MoS2 upon light irradiation.19−22 We first determined that the formation of 2D p-Sheets did not compromise the ROS-producing property of 2D MoS2 with a fluorogenic ROS probe (Figure S8). Subsequently, we measured the light-controlled ROS production of the materials intracellularly. Whereas no ROS signal was
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CONCLUSIONS To summarize, we have developed a 2D p-Sheet as a unique type of material agonist for targeted KOR imaging and activation. Without affecting the downstream signaling pathway, the material agonist induced KOR endocytosis, which further led to the intracellular production of ROS upon light irradiation. The GFP-fused KOR was well colocalized with the agonist fluorescence, as determined by super-resolution STED microscopy. To the best of our knowledge, this research unprecedentedly demonstrated the possibility of using 2D material ensembles for GPCR targeting and activation. Considering the pivotal role of GPCR in modern drug discovery, here, we offer new insights into GPCR-targeted 14713
DOI: 10.1021/acsami.7b02754 ACS Appl. Mater. Interfaces 2017, 9, 14709−14715
Research Article
ACS Applied Materials & Interfaces
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delivery of therapeutic materials for photodynamic therapy. Further, the 2D material agonist developed also represents a powerful tool for the analysis of GPCR endocytosis and related biological events because the material seemed not to interfere with the physiological function of GPCRs.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b02754. Additional figures and additional experimental sections including details of spectroscopic analyses and cell imaging experiments (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (X.-P.H.). *E-mail:
[email protected] (Y.Z.). *E-mail:
[email protected] (J.L.). ORCID
Xiao-Peng He: 0000-0002-8736-3511 He Tian: 0000-0003-3547-7485 Author Contributions ∥
W.-T.D. and Y.K. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research is supported by the 973 project (2013CB733700), the Natural Science Foundation of China (21572058, 21576088, and 81673489), the Shanghai Science and Technology Development Funds (16430724100), the Fundamental Research Funds for Central Universities (222201717003), and the Shanghai Rising-Star Program (16QA1401400) (X.-P.H.). Prof. Xin Xie at SIMM is warmly thanked for kindly gifting the 293/KOR+ cells. Cells were obtained from American Type Culture Collection (Manassas, VA).
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DOI: 10.1021/acsami.7b02754 ACS Appl. Mater. Interfaces 2017, 9, 14709−14715
Research Article
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DOI: 10.1021/acsami.7b02754 ACS Appl. Mater. Interfaces 2017, 9, 14709−14715
