Mol # 096636 Native Serotonin 5-HT2C Receptors are Expressed as Homodimers on the Apical ...

56 Pages·2015·1.51 MB·English

Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
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Mol # 096636

Native Serotonin 5-HT Receptors are Expressed as Homodimers
2C
on the Apical Surface of Choroid Plexus Epithelial Cells.

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Katharine Herrick-Davis, Ellinor Grinde, Tara Lindsley, Milt Teitler, n
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Filippo Mancia, Ann Cowan and Joseph E. Mazurkiewicz d fro
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Center for Neuropharmacology & Neuroscience, als
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Albany Medical College, Albany, NY 12208 a
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KHD, EG, TL, MT, JEM E
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Department of Physiology and Cellular Biophysics n
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Columbia University, New York, NY 10032 a
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Center for Cell Analysis and Modeling,
University of Connecticut Health Center,
Farmington, CT 06030
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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
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Mol # 096636

Running title: FCS analysis of native GPCR

Address correspondence to:
Katharine Herrick-Davis, Ph.D.
Professor
Center for Neuropharmacology & Neuroscience
Mail code - 136
Albany Medical College
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47 New Scotland Ave. o
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Albany, NY 12208 lo
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[email protected] d
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Number of pages: 47 rn
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Number of tables: 1 rg
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Number of figures: 9 S
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Number of references: 60 Jo
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Number of words in Abstract: 250 ls o
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Number of words in Introduction: 744 b
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Number of words in Discussion: 1378 1
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Non-standard abbreviations: FCS (fluorescence correlation spectroscopy); PCH (photon 3
counting histogram).

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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
This article has not been copyedited and formatted. The final version may differ from this version.
Mol # 096636

Abstract
G protein-coupled receptors (GPCR) are a prominent class of plasma membrane proteins that
regulate physiological responses to a wide variety of stimuli and therapeutic agents. While
GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain
whether native receptors expressed in their natural cellular environment are monomers, dimers
or oligomers. The goal of this study was to determine the monomer/oligomer status of a native
GPCR endogenously expressed in its natural cellular environment. Native 5-HT receptors in
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choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy lo
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(FCS) with photon counting histogram (PCH). An anti-5HT2C fragment antigen binding protein fro
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was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a olp
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control for decoding the oligomer status of native 5-HT receptors by molecular brightness rm
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analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors tjo
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equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion ls.o
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coefficient of 5 x 10-9 cm2/s and were expressed at 32 receptors/um2 on the apical surface of at A
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choroid plexus epithelial cells. The functional significance and signaling capabilities of the E
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homodimer were investigated in HEK293 cells using agonists that bind in a wash-resistant rn
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manner to one or both protomers of the homodimer. While agonist binding to one protomer F
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resulted in G protein activation, maximal stimulation required occupancy of both protomers. This a
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study demonstrates, for the first time, the homodimeric structure of 5-HT receptors , 2
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endogenously expressed in their native cellular environment and identifies the homodimer as a
functional signaling unit.

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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
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Mol # 096636
Introduction
G protein-coupled receptors (GPCR) represent one of the largest families of cell membrane
signaling proteins. They are present on the surface of most all cells and regulate the
physiological functions of all the major organ systems in the human body. GPCR modulate
physiological responses to light, odorants, hormones, neurotransmitters, and therapeutic
agents. While physiological processes regulated by GPCR activation and blockade have been
studied for decades, it is still uncertain whether the functional signaling unit is a monomer, dimer
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or higher order oligomer. o
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Currently, there are hundreds of reports in the published literature describing the dimeric or d
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oligomeric nature of GPCR expressed in recombinant cell systems. Dimer/oligomer formation m
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has been reported to regulate all aspects of GPCR function including synthesis, ligand binding, lph
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G protein coupling, and trafficking (reviewed in Herrick-Davis 2013; Milligan 2013). Even so, the .as
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presence and functional relevance of GPCR dimerization in vivo is widely debated (reviewed in urn
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Ferré et.al., 2014). Concerns have been raised about the functional relevance of GPCR .org
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dimerization as monomeric receptors have been reported to activate G proteins in reconstituted S
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systems (Bayburt et.al., 2007; Whorton et.al., 2007) and the crystal structure of the agonist Jo
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occupied beta -adrenergic receptor revealed a monomeric receptor in complex with a single G ls
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protein (Rasmussen et.al., 2011). On the other hand, structural studies of native rhodopsin eb
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receptors have revealed their dimeric organization in rod outer segments (Liang et.al., 2003) 1
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and their association with G transducin (Jastrzebska et.al., 2013a). Additionally, leukotriene, 2
3
dopamine, and serotonin receptors have been solubilized as homodimers in complex with a
single G protein (Banères and Parello, 2003; Han et.al., 2009; Pellissier et.al., 2011).
The presence of GPCR dimers or oligomers in intact native tissues and in vivo is inferred
from studies employing indirect biophysical methods, immunofluorescence in fixed tissue
sections, and functional studies in transgenic animals. For example, ligand-based FRET studies
identified oxytocin homodimers in rat mammary gland tissue (Albizu et.al., 2010), and
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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
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heterodimers of D and D dopamine receptors in striatal neurons are inferred from antibody-
1 2
based FRET in fixed brain slices (Hasbi et.la., 2011). Supporting evidence is provided by
studies using heterodimer selective antibodies (Berg et.al., 2012) and transgenic mice
expressing mutant receptors that restore (Rivero-Müller et.al., 2010) or inhibit (González et.al.,
2012) normal receptor function. Pharmacological approaches have used heterodimer selective
agonists (Waldhoer et.al., 2005; Fujita et.al., 2014) or antagonists that bind in a pseudo-
irreversible manner to one protomer within the homodimer (Smith et.al., 2011). However,
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studies employing direct biophysical methods capable of monitoring protein interactions in living n
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tissues are needed to substantiate these findings. d
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While recent studies have employed direct methods with near single molecule sensitivity, m
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such as total internal reflection fluorescence (Calebiro et.al., 2013; Kasai and Kusumi 2013, arm
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Teichmann et.al., 2014) and fluorescence correlation spectroscopy, FCS (Briddon et.al., 2008; e
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Malengo et.al., 2008; Ganguly and Chattopadhyay, 2010; Herrick-Davis et.al., 2012, 2013; rna
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Corriden et.al., 2014), these studies were performed using recombinant cell lines. The purpose a
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of the present study was to determine the monomer/oligomer status of a native GPCR PE
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endogenously expressed in its natural cellular environment. The serotonin 5-HT receptor was u
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selected because it is highly expressed in choroid plexus epithelial cells, an established primary on
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cell culture for studying native 5-HT receptors (Esterle and Sanders-Bush 1992), and ru
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monoclonal antibodies recognizing the native conformation of the 5-HT2C receptor have already 0, 2
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been developed (Mancia et.al., 2007). Previously, we demonstrated the application of FCS with
photon counting histogram (PCH), a sensitive method for the direct quantification of photons
emitted from individual fluorescent molecules, for determining the oligomeric status of GPCR
expressed in recombinant cell lines (Herrick-Davis et.al., 2012, 2013). In FCS, fluorescence-
tagged proteins that associate with one another or are part of a larger protein complex will co-
diffuse with one another. Autocorrelation analysis reveals the diffusion characteristics of
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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
This article has not been copyedited and formatted. The final version may differ from this version.
Mol # 096636
fluorescence-tagged proteins in the sample and provides an estimate of the number of
fluorescent proteins co-diffusing together as a complex within the plasma membrane.
In the present study, a monoclonal anti-5HT fragment antigen binding protein was used as
2C
a probe for labeling native 5-HT receptors in choroid plexus epithelial cells. FCS and PCH
2C
were used to monitor the diffusion characteristics and oligomer status of 5-HT receptors
2C
expressed in their native cellular environment. The functional significance was investigated in
HEK293 cells using ergots that bind in a wash-resistant manner to one protomer (cabergoline)
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or both protomers (ergotamine) of the homodimer. These studies provide the first
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pharmacological and functional evidence for the presence of 5-HT2C receptor homodimers and ed fro
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highlight the signaling capabilities of the individual protomers. m
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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
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Materials and Methods
Cell culture and transfection HEK293 cells (ATCC) were cultured in Dulbecco’s Minimal
Essential Medium (DMEM, Cellgro) with 10% FBS (HyClone) in a humidified incubator at 370C,
5% CO . For the confocal microscopy and FCS experiments, HEK293 cells were plated in 6 well
2
plates fitted with 25mm poly-D-lysine coated glass coverslips (Fisher) at a density of 4 x 105
cells per coverslip and transfected with 200ng of the indicated plasmid DNA using lipofectamine
reagent (Invitrogen) for five hours. Following transfection, cells were cultured in MEM (without
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phenol red) with 10% charcoal stripped serum (Gibco) for 20 hours at 370C, 5% CO . Prior to o
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FCS analysis, coverslips were washed twice in HEPES-buffered Krebs-Ringer solution. d
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Plasmid: cDNAs encoding the 5-HT receptor and the VSV isoform of the 5-HT receptor were m
2A 2C m
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cloned into pEGFP-N1, pECFP-N1 and pmCherry-N1 vectors (Clonetech) to create chimeric lph
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receptors with fluorescent tags on the C-terminus of each receptor. CD-86/GFP and CD- .as
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86/GFP-GFP were generously provided by G. Milligan (University of Glasgow). Site-directed urn
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mutagenesis (Stratagene) was used to create an A206K mutation in all GFP constructs to .org
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eliminate potential aggregation (Zacharias et.al., 2002). S
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GFP-tagged, anti-5-HT2C fragment antigen binding protein (2C-Fab-GFP) A monoclonal Jou
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antibody recognizing an external domain of the 5-HT receptor was previously developed and ls
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shown to label 5-HT receptors in transfected HEK293 cells (Mancia et.al., 2007). The cDNA eb
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sequences encoding the heavy and light chains of the monoclonal anti-5-HT fragment antigen 1
2C 0
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binding protein (Fab) were sub-cloned into mammalian expression vectors (Assur et.al., 2007) 2
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and plasmids containing clone 1C4 (Mancia et.al., 2007) were used in the present study. The
light chain cDNA sequence was cloned into an expression vector upstream of an internal
ribosomal entry site (IRES) followed by red fluorescent protein as a selection marker (Assur
et.al., 2007). For the present study, the heavy chain cDNA sequence was modified as follows: a
His 6 tag was added in place of the stop codon and the cDNA was ligated into the pEGFP-N1
expression vector (Clontech) for direct labeling of the heavy chain C-terminus with GFP. Site-
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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
This article has not been copyedited and formatted. The final version may differ from this version.
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directed mutagenesis (Stratagene) was used to create an A206K mutation in the GFP tag to
eliminate potential aggregation (Zacharias et.al., 2002). HEK293 cells were transfected by
calcium phosphate precipitation with a mixture of the light and heavy chain plasmids. A stable
cell line expressing both light and heavy chains was identified by fluorescence microscopy and
Western blot. Stably transfected cells were plated at a density of 5 x 106 cells/100mm dish and
cultured (370C, 5 % CO ) for one week in DMEM with 10% low IgG FBS (HyClone). 2C-Fab-
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GFP was purified from the culture media on nickel resin columns (Thermo Scientific) according
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to the manufacture’s protocol. Following column purification, samples were dialyzed in HEPES-
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buffered Krebs-Ringer (pH7.4), and concentrated to 0.1μM in spin columns (30K NMW cut-off; ed
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Millipore). m
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Western blot Intact choroid plexus tissue isolated from the third and lateral ventricles of an adult a
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female Sprague-Dawley rat was placed in a microcentrifuge tube containing 50ul of cold RIPA p
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buffer with protease inhibitor cocktail (Sigma). The tube was placed on ice for 15 minutes, then rn
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sonicated in an ice cold water bath for 15 minutes, and placed on ice again for 15 minutes prior arg
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to centrifugation at 30,000g for 30 minutes at 40C. Ten microliters of the supernatant was diluted P
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1:1 in non-reducing Laemmli sample buffer. Purified 2C-Fab-GFP and pre-stained protein u
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standards (BIORAD) were also diluted in non-reducing Laemmli sample buffer. The samples o
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were heated at 700C for 15 minutes and run on a 10% Tris-HCl BioRad Ready Gel at 95 V for ru
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70 min. Gel proteins were transferred to nitrocellulose (Hybond ECL, Amersham) and probed 0
, 2
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with 2C-Fab-GFP overnight at 40C in 1%milk/1%BSA blocking solution. Following incubation 3
with GFP(B-2)-HRP (1:3000; Santa Cruz), proteins were visualized using enhanced
chemiluminescence (Amersham).
Choroid plexus epithelial cell culture and immunostaining Primary choroid plexus epithelial cells
were prepared as described by Esterle and Sanders-Bush, 1992. Briefly, choroid plexi were
dissected from the third and lateral ventricles of fetuses of timed-pregnant Sprague-Dawley rats
at 19 days of gestation. The choroids were placed in digest buffer containing phosphate-
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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
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buffered saline, pH 7.4, with 0.33mg/ml pronase (Sigma) and 0.25mg/ml DNase 1 (Sigma) for
25 minutes at 370C, washed twice, then dissociated by trituration in DMEM/F12 (1:1) (Cellgro)
with 0.13mg/ml DNase 1. The supernatant containing dissociated epithelial cells was
centrifuged at 1100 rpm for 3 minutes. Epithelial cells were resuspended in culture media
containing DMEM/F12 (1:1) with 10% FBS (Gibco), 1% N2 supplement (Invitrogen), 10ng/ml
epidermal growth factor (Invitrogen), and 1% PenStrep (Invitrogen), then transferred to a 60mm
dish and incubated (370C, 5% CO ) for two hours to allow fibroblasts to adhere to the dish. The
2
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culture medium containing unattached epithelial cells was removed from the dish and the
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epithelial cells were plated on 25mm glass coverslips (Fisher) coated with laminin (Gibco). After ed
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three weeks in culture, primary choroid epithelial cells were labeled with the monoclonal anti-5- m
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HT -Fab-GFP antibody (2C-Fab-GFP, diluted 1:3 in HEPES-buffered Krebs-Ringer) for 40 a
2C rm
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minutes at 230C immediately prior to FCS recording. For co-staining with anti-transthyretin sp
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(Bioss), three week old cultures of choroid epithelial cells were fixed in phosphate-buffered 3.7% rn
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paraformaldehyde (10 minutes at room temperature), permeabilized with 0.1% triton, blocked arg
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with 4% donkey serum, and stained overnight at 40C with 2C-Fab-GFP (diluted 1:10) and rabbit P
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anti-transthyretin (diluted 1:100) in HEPES-buffered Krebs-Ringer, pH7.4 with 1% donkey u
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serum. The rabbit anti-transthyretin was visualized with an Alexa 488 conjugated donkey anti- o
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rabbit IgG (diluted 1:1000; Invitrogen). ru
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Inositol phosphate (IP) assay HEK 293 cells were seeded at 2x105 cells/well, in 24 well plates, 10
, 2
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in DMEM with 10% FBS. Cells were transfected with 100ng plasmid containing cDNA encoding 3
the VSV isoform of the 5-HT receptor using lipofectamine reagent for five hours at 370C.
2C
Following transfection, cells were cultured in DMEM with 10% FBS for 24 hours, then washed
and labeled overnight in inositol-free, serum-free DMEM with 0.5uCi of 3H-myoinositol. At the
start of the experiment, cells were washed twice with PBS and were pretreated with serum-free
DMEM in the absence or presence of drug for 60 minutes (or as indicated), followed by drug
wash-out. The wash-out period consisted of four washes with PBS over a 30 minute period: one
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Molecular Pharmacology Fast Forward. Published on January 21, 2015 as DOI: 10.1124/mol.114.096636
This article has not been copyedited and formatted. The final version may differ from this version.
Mol # 096636
wash immediately following the end of drug pretreatment, followed by three subsequent media
changes at 10 minute intervals wherein the cells were returned to the incubator. Following drug
wash-out, the IP assay was initiated by adding lithium chloride to the assay media, with or
without drugs as indicated, for an additional 60 minutes. Total 3H-IP production was measured
by anion exchange chromatography as previously described (Berridge et.al., 1983). Data were
analyzed using GraphPad Prism software.
Whole cell radioligand binding assay HEK 293 cells were seeded at 4x105 cells/well, in 12 well
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plates, in DMEM with 10% FBS. Cells were transfected with 200ng plasmid containing cDNA
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encoding the VSV isoform of the 5-HT2C receptor in serum-free media using lipofectamine ed fro
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reagent for five hours at 370C. Following transfection, cells were cultured in DMEM with 10% m
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FBS for 24 hours, then washed and incubated overnight in serum-free DMEM. Cells were a
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pretreated with the indicated drugs for 60 minutes followed by four washes with PBS over a 30 p
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minute period. Washed cells were labeled with 3H-mesulergine (2.5nM) in 0.5ml of serum-free rn
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DMEM, in the absence and presence of 1uM 5-HT to define specific binding to cell surface 5- arg
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HT receptors, for 20 minutes at 370C. Cells were washed with PBS to remove unbound P
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radioligand and 0.5 ml of ice-cold 3% TCA was added to each well. The plate was incubated on u
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ice for 20 minutes to release surface bound ligand from the cells without cell lysis. Following o
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incubation, 450µl of the TCA solution was removed from each well, mixed with 5mls of Ecoscint ru
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cocktail, and counted in a Beckman liquid scintillation counter. 0
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Fluorescence correlation spectroscopy (FCS) For FCS measurements, cells were washed twice 3
with HEPES-buffered Krebs-ringer (without glucose) and the coverslip was placed in an
Attofluor Cell Chamber (molecular Probes, Inc.) with 1ml of HEPES-buffered Minimal Essential
Medium (MEM, without phenol red). FCS measurements were made using a Zeiss LSM-780
confocal microscope (Zen 2010 software; Carl Zeiss, Germany) equipped with a 32 element
gallium arsenide phosphide (GaAsP) linear array detector, as described (Herrick-Davis and
Mazurkiewicz 2013). One-photon excitation with a continuous wave argon ion laser was
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