Unstructured loop is essential for the activation of mGluR2

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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Unstructured loop is essential for the activation of mGluR2 Deo R. Singh a, b, 1, *, Ashish K. Mishra c, 1, Pankaj Pandey d, 1, Kalpana Pandey a, 1, Victor Vivcharuk a, e, Timur Mavlyutov f, ** a

Department of Biochemistry, Weill Cornell Medical College, New York City, NY, USA Department of Cell and Molecular Physiology, Stritch School of Medicine, Maywood, IL, USA c Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA d Department of Zoology, Brahmanand College, Kanpur, UP, India e Department of Physiology and Biophysics, Weill Cornell Medical College, New York City, NY, USA f Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 September 2019 Accepted 28 October 2019 Available online xxx

Metabotropic Glutamate Receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) that are expressed throughout the central nervous system and are involved in several neurological and psychiatric disorders. Although, many studies focused on Glutamate induced activation of mGluR2, however, the role of unstructured loop (or “BC loop”) in activation of metabotropic Glutamate receptors is €rster Resonance Energy Transfer (FRET) based assay in live cells we currently unknown. Here, using Fo show that unstructured loop is required for Glutamate induced conformation and hence the activation of the receptor. © 2019 Elsevier Inc. All rights reserved.

Keywords: €rster resonance energy transfer Fo Metabotropic glutamate receptor 2 G-protein coupled receptor Membrane protein Microscopy Imaging

1. Introduction Metabotropic Glutamate Receptors (mGluRs) are class C Gprotein coupled receptors (GPCRs) that are expressed throughout the central nervous system (CNS) [1]. They play important roles in the modulation of both excitatory and inhibitory synapses in the brain and are also involved in many neurological and psychiatric disorders such as Alzheimer’s disease, Parkinson’s disease, anxiety, depression, and schizophrenia [1,2]. These receptors, therefore, are attractive drug targets to treat these disorders [3]. There are eight metabotropic Glutamate receptors. They are divided into three classes. Class I consists of mGluR1 and 5, Class II consists of mGluR2 and 3, and Class III consists of mGluR4, 6, 7 and 8 [1]. The class I mGluRs couple to Gq/G11 and activate

* Corresponding author. Department of Biochemistry, Weill Cornell Medical College, New York City, NY, USA. ** Corresponding author. Department of Ophthalmology and Visual Sciences, University of Wisconsin- Madison, 1300 University Ave., Madison, WI, 53706, USA. Tel.: 608 262 2437. E-mail addresses: [email protected] (D.R. Singh), tamavlyutov@wisc. edu (T. Mavlyutov). 1 These authors contributed equally.

phospholipase C that leads to calcium mobilization and activation of protein kinase C (PKC) [1]. In contrast to Class I mGluRs, Group II and Group III mGluRs are coupled predominantly to Gi/o proteins. Gi/o linked receptors are involved in inhibition of adenylyl cyclase and directly regulate ion channels and other downstream signaling partners via liberation of GbY subunits [1]. The mGluRs are composed of a large extracellular domain, a heptahelical transmembrane (7 TM) domain, and a short intracellular C-terminal [4]. The extracellular domain consists of a bilobate Venus fly-trap domain (VFT) and a cysteine -rich domain (CRD) [4]. Binding of the Glutamate in a bilobate VFT [5] results in conformational changes in the receptor that propagate from VFT to 7 TMs which in turn activates the G-protein [6]. The 7 TMs are the target for many synthetic molecules that act either as negative or positive allosteric modulators (NAMs and PAMs) [7]. Since, these molecules can fine tune the endogenous signaling, they present an exciting opportunity for drug development. In the solved crystal structures of VFT domain of mGluRs a certain length of amino acids are absent in the structure [5]. Particularly, in the case of mGluR2 the residues that do not appear in the crystal structure range from Leucine 110 to Proline 133 [8]. These missing residues in the crystal structure form a loop between Helix B and Helix C in a single VFT unit in a dimeric VFT domain

https://doi.org/10.1016/j.bbrc.2019.10.187 0006-291X/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: D.R. Singh et al., Unstructured loop is essential for the activation of mGluR2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.187

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crystal structure. This loop is called unstructured loop or “BC loop”. The Cysteine residues in the unstructured loop form intersubunit disulfide bridge between the VFT domains [9]. This disulfide bond partially mediates dimerization of full length mGluRs [9]. In the present study, we examined the role of unstructured loop €rster Resonance Energy Transfer in activation of mGluR2 using Fo (FRET) in live cells. We show that upon binding to Glutamate the FRET between two subunits changes while we do not see any change in FRET when BC loop is absent. 2. Result and discussion To understand the role of BC loop in the activation process we used FRET based assay in live cells. The AAV 293 cells were transfected with plasmids that encode the fusion proteins SNAP-mGluR2 or SNAP-mGluR2DBC. mGluR2DBC is the full length mGluR2 in which the BC loop (the residues from 109 to 135 from full length receptor (Fig. 1A)) has been deleted (Fig. 1B). Following protein expression, the cells were reseeded with very low density in poly-Dlysine coated 4 chamber glass bottom dishes so that cells are well separated. When the cells started adhering to the surface, the cells were incubated for 45 min with Alexa 488. Thus, Alexa 488 got conjugated to SNAP tagged receptors on the membrane of the cells. After incubation, the cells were washed with 1XPBS three to four times to remove unconjugated Alexa 488 and imaged in PBS buffer (with Mgþ2) using an inverted microscope equipped with a blackthinned EMCCD camera. The images of AAV 293 cells expressing mGluR2 and mGluR2DBC suggest that deletion of BC loop does not prevent the receptor to reach on the surface (Fig. 1C). To perform FRET experiment, the cells were incubated for 45 min with a mixture of Alexa 488 and Alexa 595 in 1e5 ratios. Since the SNAP tagged receptors were incubated in 1e5 ratios of Alexa 488 and Alexa 595, therefore, the cells express Alexa 488 and Alexa 595 conjugated SNAP tagged receptors in 1e5 ratios. After incubation, the cells were washed with 1XPBS three to four times to remove unconjugated Alexa 488 and Alexa 595 and imaged in PBS buffer (with Mgþ2). The image acquisition of a field of view for each sample was performed for Alexa 488, Alexa 595, and FRET (Alexa 488 excitation, Alexa 595 emission) channels (Fig. 1D). During FRET process Alexa 488 conjugated SNAP tagged receptors were donor of energy and Alexa 595 conjugated SNAP tagged receptors were

acceptor of energy. Metamorph software was used to quantify fluorescent intensity for the images collected in each channel. Using this data, the FRET efficiency, donor fluorescence and acceptor fluorescence for each cell were calculated. It has been shown that conformational change induced in mGluR2 due to binding of the Glutamate is responsible for the activation of the receptor [10]. Therefore, we titrated FRET as the function of Glutamate concentration for both mGluR2 and mGluR2DBC. The raw FRET data for mGluR2 and mGluR2DBC in the presence of different concentrations of Glutamate are shown in Fig. 2A and Fig. 2B, respectively. As the concentration of Glutamate is increasing the average FRET value keeps on decreasing and after a certain concentration of Glutamate when all the receptors are bound to Glutamate the FRET does not decrease any more in the case of mGluR2. For mGluR2DBC, we do not see any change in FRET value as a function of Glutamate concentration. We normalized the change in FRET efficiency and plotted it as a function of Glutamate concentration for mGluR2 (Fig. 2C) and mGluR2DBC (Fig. 2D). We see that normalized FRET increased and stabilized after a certain concentration of Glutamate for mGluR2. However, normalized FRET is almost flat for mGluR2DBC. This suggests that when BC loop is removed, Glutamate binding to mGluR2DBC does not induce conformational change. Thus, BC loop is required for Glutamate induced conformational changes and hence activation. Since, upon binding to the Glutamate mGluR2 undergoes from relaxed conformation to active conformation this suggests that BC loop acts as a hinge through disulfide bond within a dimer. Deletion of BC loop means removing the hinge. Therefore, mGluR2DBC does not respond to Glutamate. The Hill fit of titration of mGluR2 with Glutamate gives the hill coefficient equal to 1, suggesting that binding of Glutamate to mGluR2 does not show co-operativity. This is in contrast to the observation reported in the literature [10]. The titration curve of mGluR2 lacking BC loop remains flat, signature of non-responsiveness to Glutamate. 3. Materials and methods 3.1. Plasmid constructs We used N-terminal SNAP-mGluR2 construct in our experiments as described in Doumazane et al., 2011 [11]. To obtain the

Fig. 1. Localization of mGluR2 and mGluR2DBC in AAV293 cells. (A) Cartoon representation of mGluR2. Different domains of mGluR2 highlighted. SP: Signal Peptide, VFT: Venus Fly-Trap Domain, CRD: Cysteine Rich Domain, TMD: Trans Membrane Domain, IC: Intra Cellular tail. The highlighted position of residues 109 to 135 represent the unstructured loop (BC loop). (B) Crystal structure of mGluR1 VFT domain in dimeric form in the absence Glutamate (PDB ID 1EWT) [5]. The dotted lines connect the helix B and helix C in each VFT domain. The loop connecting the two helices in each VFT domain represents the unstructured loop or BC loop. The blue oval between two loops represents the disulfide bond formed between two units of VFT. Green and red ovals show the approximate positions of SNAP tag conjugated with Alexa 488 and Alexa 595, respectively. (C) Representative images of AAV 293 cells expressing mGluR2 and mGluR2DBC. Proper localization of mGluR2DBC in the membrane suggests that deletion of BC loop does not misfold the protein. (D) Representative images of AAV293 cells in donor, acceptor, and FRET channels, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Please cite this article as: D.R. Singh et al., Unstructured loop is essential for the activation of mGluR2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.187

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Fig. 2. Dose response activation of mGluR2 and mGluR2DBC due to Glutamate: (A) FRET efficiency distribution and box plot (average value shown as mid-line joining the box from left to right) for mGluR2 in the presence of different concentrations of Glutamate. (B) FRET efficiency distribution and box plot (average value shown as mid-line joining the box from left to right) for mGluR2DBC in the presence of different concentrations of Glutamate. (C) Dose response data for mGluR2. The fitting of this data with Hill function suggests no co-operativity as Hill fit gives the Hill coefficient nH ¼ 1. The kd value obtained from this fitting was 23 mM similar to obtained by Vafabaksh et al. [10]. (D). Dose response data for mGluR2DBC. It remains flat suggesting that mGluR2DBC does not respond to Glutamate.

SNAP-mGluR2DBC, we deleted the segment of residues from Serine 109 to Alanine 135 in full length mGluR2 (PDB ID: 5CNI) [8]. 3.2. Cell culture and transfection AAV 293 cells were cultured in DMEM cell culture medium supplemented with 10% fetal bovine serum (FBS) (Thermo Scientific, Waltham, MA). Twenty-four hours after culture, the cells were transiently transfected using MBS mammalian transfection kit (Agilent Technologies, Stratagene, La Jolla, CA) as per instructions provided with the kit. 24 h post-transfection the cells were trypsinized (Thermo Scientific, Waltham, MA) and replated onto polyD-lysine coated glass bottom chambers and allowed to settle down for 1 h. Following that the cells were incubated in 1e5 mixture of Alexa 488 and Alexa 595 for 45 min. The cells were washed three times before imaging.

FAlexa488 are the matching fluorescence intensity from FRET, Alexa 595, and Alexa 488 images, respectively, and G represents FRET intensity corrected for the bleed-through of the channels. The parameters a and d are bleed-through constants calculated as a ¼ FFRET/FAlexa488 for a control sample transfected with only Alexa 595 and d ¼ FFRET/FAlexa595 for a control sample transfected with only Alexa 488. These values were determined to be a ¼ 0.013 and d ¼ 0.075. 3.4. Glutamate titration The imaging medium with various Glutamate concentration was prepared. The imaging medium was replaced with medium containing Glutamate. The cells were imaged after 5 min after replacing the medium with medium containing Glutamate. Declaration of competing interest

3.3. Acceptor sensitization fluorescence resonance energy transfer The authors declare no conflict of interest. Imaging was performed using a wide-field fluorescent microscope as described previously [12]. Cells were imaged in 1 X PBS (with Mgþ2) (Hyclone Laboratories, Logan, Utah) using an inverted microscope (Nikon Eclipse Ti) equipped with a black-thinned EMCCD camera (iXon 887, Andor Technology, Belfast, Northern Ireland). The image acquisition of a field of view for each sample was performed with a 400.75 NA objective and 50 ms exposure time for Alexa 488, Alexa 595, and FRET (Alexa 488 excitation, Alexa 595 emission) channels. Metamorph software (Molecular Devices, Sunnyvale, CA) was used to quantify fluorescent intensity for the images collected in each channel. FRET efficiency was calculated according to E ¼ G/(G þ 0.25  FAlexa488) where G ¼ FFRETaFAlexa595dFAlexa488 [10], where FFRET, FAlexa595, and

Acknowledgement We acknowledge Seth L. Robia from Department of Cell and Molecular Physiology at Stritch School of Medicine for lab facilities. There was no specific funding to support this work. References [1] C.M. Niswender, P.J. Conn, Metabotropic glutamate receptors: physiology, pharmacology, and disease, Annu. Rev. Pharmacol. Toxicol. 50 (2010) 295e322. [2] C.J. Swanson, M. Bures, M.P. Johnson, A.-M. Linden, J.A. Monn, D.D. Schoepp, Metabotropic glutamate receptors as novel targets for anxiety and stress

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Please cite this article as: D.R. Singh et al., Unstructured loop is essential for the activation of mGluR2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.187