i

Assay and array technologies for G-protein coupled receptors

Kelly Bailey BSc. Nanotech (Hons)

A thesis submitted in fulfillment of the requirements for the degree of Doctor of

Philosophy

Biochemistry Discipline

School of Molecular and Biomedical Sciences

The University of Adelaide

Adelaide, Australia

and

CSIRO, Division of Molecular and Health Technologies

Adelaide, Australia

December 2008

138

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Zhou, J.Y., Toth, P.T. & Miller, R.J. 2003, "Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3", The Journal of Pharmacology and Experimental Therapeutics, vol. 305, no. 2, pp. 460-466.

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6 Appendices

6.1 No changes in TR-FRET indicate little non-specific interactions

In order to determine the specificity of the interaction observed between the

fluorescently labelled G-protein subunits monitored using the TR-FRET assay, proteins

such as BSA (Figure 6.1.1a) and lysozyme (Figure 6.1.1b), which were not expected to

specifically interact with either of the subunits were added to the assays. In the presence

of a competing unlabelled G-protein subunit, labelled heterotrimer separation was

observed by a decrease in the TR-FRET fluorescence measured at the emission

wavelength of the acceptor fluorophore (Alexa) (Figure 6.1.1a). Addition of this non-

competing protein did not show the same decrease in TR-FRET fluorescence (Figure

6.1.1a and b).

Figure 6.1.1 TR-FRET of heterotrimer formation and ββββγγγγ subunit specific competition. a) Steady state TR-FRET between 20 nM Gαi1HisTb and 20nM β4γ2Alexa in 100 μl assay volume. Arrow indicates addition of buffer (�); 300 nM BSA (�); 300 nM β4γ2 (�). Excitation = 340 nm; Emission = 572 nm, 50 μs delay and a 900 μs count time. Data shown are mean ± SEM (n = 3). b) Steady state FRET (% maximum @ 572 nm) between 2.5 nM Gαi1HisTb + 3.75 nM β4γ2Alexa in 100 μl volume. Incubation with buffer or lysozyme with G-protein heterotrimer does not display a decrease in TR-FRET fluorescence at 572nm indicating that no separation of the subunits has occurred.

6.2 Extrusion changes turbidity of membrane extracts

Extruding the lysed cell membrane extracts through polycarbonate filters decreased the turbidity

of the sample as can be seen in Figure 6.2.1. This was a sign of decreased particle size within

the mixture.

Buffer Lysozyme0

25

50

75

100

125

% m

axim

um F

RE

Tflu

ores

cenc

e57

2nm

a) b)

0 10 200

500

1000

1500

Time (min)

Fluo

resc

ence

572n

m(a

.u)

167

Figure 6.2.1 Extruding membrane extract. Left-hand side is membrane extract after first passage through the polycarbonate filter (1.0 μm, millipore track-etch), right-hand side is extract prior to extrusion. The increased turbidity of the sample on the right-hand side is apparent due to limited visibility of measurement ticks on farthest side of glass syringe, whereas words on farthest side of syringe on left-hand side can be seen through the clearer sample.

6.3 Phase diagrams of monoolein and phytantriol

Lipid phase behaviours (polymorphisms) are commonly mapped out via phase

diagrams, the simplest of which varies water content and temperature. These are an

important reference to obtain the correct temperature and water content to achieve the

desired phase (such as cubic phase) of the lipid mixture.

Figure 6.3.1 Phase diagrams of (a) monoolein (Qiu & Caffrey 2000) and (b) phytantriol (Barauskas & Landh 2003). Diagrams are both interpretations of x-ray diffraction data. Lα - lamellar; Lc- lamellar crystal; L2 – reversed micellar; FI- fluid isotropic phase; Q230 or Ia3d – reversed cubic of crystallographic space group Ia3d; Q224 or Pn3m – reversed cubic of space group Pn3m; HII – reversed hexagonal.

a)

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6.4 Multiplexing arrays

In order to avoid any background binding of functionalised cubosomes, a strategy was

employed to backfill the non-arrayed area with PLL-g-PEG. This strategy began with

the direct spotting of the PLL-g-PEG-biotin onto the clean glass surface, followed by a

backfill of PLL-g-PEG polymer, then the slides were re-aligned for a second spotting of

the neutravidin/biotinylated oligo complex directly ontop of the biotinylated polymer.

Figure 6.4.1 Example of multiplexed array strategy. The wells of cel-line slides were spotted with pattern of PLL-g-PEG-biotin (blue spots) using the peizoarrayer (Perkin-Elmer). Wells were then back-filled with PLL-g-PEG (yellow fill). PLL-g-PEG-biotin spots were then overlayed with neutravidin/biotinylated oligo spots (green).

6.5 Calculating total lipid concentration in membrane extracts

The total protein concentration in the membrane extracts is known in mg/ml from the

Bradford protein assay. The lipid mass within the same volume of extract was

calculated using lipid extraction. The ratio of protein mass to lipid mass within a sample

was used here to estimate the concentration (in molar) of the lipids within the sample.

This was important to determine the lipid:cholesterol ratio used in the tagging of the

vesicles.

Baculovirus infected Sf9 cells (e.g.H1-histamine receptor)

PLL-g-PEG-biotin

PLL-g-PEG

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Protein:Lipid ratio = 1.16:1

Therefore, in the same volume 750 μg protein (from Bradford protein assay) = 647 μg

lipid

Lipid composition (Marheineke et al. 1998)

Note: Molecular weights obtained from Avanti polar lipids and based on acyl fatty acid

composition (e.g. 16:0) which predominates within particular phospholipid class

(Marheineke et al. 1998)

23% phosphatidylinositol

nPI = (23/100 x 647μg)/880.15 = 1.7 x 10-7

32% phosphatidylethanolamine

nPE = (32/100 x 647 μg)/1301.73 = 1.6 x 10-7

42 % phosphatidylcholine

nPC = (42/100 x 647 μg)/ 986.15 = 2.8 x 10-7

3.7% cardiolipid

nC = (3.7/100 x 647 μg)/1493.92 = 1.6 x 10-8

Total n = 6.26 x 10-7

C total = 62.6 μM

Non- infected Sf9 cells

Protein:Lipid ratio = 1.24:1

Therefore, 750 μg protein (from Bradford protein assay) = 605 μg lipid

Lipid composition (Marheineke et al. 1998)

Note: Molecular weights obtained from Avanti polar lipids and based on fatty acid

composition which predominates particular phospholipid class (Marheineke et al. 1998)

23% phosphatidylinositol

nPI = (23/100 x 605μg)/880.15 = 1.6 x 10-7

36% phosphatidylethanolamine

nPE = (36/100 x 605 μg)/1301.73 = 1.7 x 10-7

35 % phosphatidylcholine

nPC = (35/100 x 605 μg)/ 986.15 = 2.2 x 10-7

4.6 % cardiolipid

nC = (4.6/100 x 605 μg)/1493.92 = 1.9 x 10-8

Total n = 5.69 x 10-7

C total = 56.9 μM

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6.6 Bradford protein assay of extruded membrane extracts

The total protein concentration as measured by Bradford protein assay decreased post

extrusion.

R exrac

t prior t

o extru

sion

1HR

extra

ct post

extru

sion

1H R extra

ct prio

r to e

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tr. (1

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1HR ex

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prior e

xtrusio

n

2MR ex

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post ex

trusio

n

2M

0.000.050.100.150.200.250.300.350.400.450.500.550.600.65

[Tot

al p

rote

in] (

mg/

ml)

Figure 6.6.1 Bradford protein assay results prior to and post extrusion of membrane extracts. Total protein of membrane extracts was calculated by reading adsorption of Bradford reagent at 595nm in the presence of protein within membrane extracts, and comparing to a BSA standard curve. Membrane extracts prior to extrusion displayed lower levels of protein than before extrusion according to results from this assay system. Set 1 and 2 separated by dotted verticle line show two different H1R extract protein concentrations, while set 3 shows an M2R extract protein concentration, prior to, and post-extrusion.

6.7 TAC-Alexa binding to M2-muscarinic receptor vesicles

M2-muscarinic receptor functionality was investigated using a TAC ligand conjugated

to an Alexa546 fluorophore. For the immobilization studies shown here, an internal

reference for total vesicle binding was determined by capturing fluorescently labelled

M2R and non-infected (NI) vesicles. Labelling was carried out for each preparation at

identical molar ratios with a lipophilic dye, octadecyl rhodamine (Invitrogen). Vesicles

were captured using the same complementary oligonucleotides on different isolated

areas of the spotted slide. The absence of the M2R in the immobilised vesicles resulted

in a 35% decrease in relative fluorescence intensity (RFI). TAC-Alexa was used in these

studies due to the discontinuation of the BODIPY-pirenzepine product from the

171

commercial suppliers, Invitrogen.

Figure 6.7.1 Specific binding of TAC-Alexa to vesicles containing the M2-muscarinic receptor. a) M2R vesicles immobilized via complementary oligos, incubated with TAC-Alexa (10 μM). b) Non-infected vesicles immobilized via complementary oligos, incubated with TAC-Alexa (10 μM). c) M2R vesicles labelled with lipid-like octadecyl rhodamine as reference of total vesicles bound; d) Non-infected vesicles labelled with lipid-like octadecyl rhodamine as reference of total vesicles bound. Scale bar represents approximately 2 mm, spots are 120-150μm in diameter.

M2R NI0

102030405060708090

100110

***

% M

axim

um R

FI

Figure 6.7.2 Relative fluorescence intensity of specific immobilisation of liposomes containing the M2-muscarinic receptor as measured by TAC-Alexa binding. RFI was determined by measurement of the fluorescence of the TAC-Alexa bound to captured vesicles (570nm) in relation to the total amount of vesicles bound (as determined by rhodamine labelled vesicle immobilisation measured at 570nm on a separate array area). Data represents mean ± SEM, n = 10. Statistical analysis was determined using unpaired t-test, *** P< 0.0001.

a) b)

c) d)

172

6.8 Patterned oligo arrays

The printing technique was capable of preparing various patterns.

Figure 6.8.1 Site-specific rhodamine cubosome binding on array pattern. a) Array pattern consisted of left-hand side of the christmas tree spotted with neutravidin oregon green + BA1 oligo and right-hand side spotted with neutravidin oregon green neutravidin + BB1 oligo on PLL-g-PEG-biotin background. b) 546 nm excitation and 570 nm emission of rhodamine cubosomes tagged with CA1 cholesterol oligo tag; c) 546 nm excitation and 570 nm emission of rhodamine cubosomes tagged with CB1 cholesterol oligo tag; d) image b overlayed with neutravidin green fluorescence monitored at 530 nm after excitation at 480 nm which shows overlay of rhodamine and oregon green in yellow, and oregon green alone in green. e) image c overlayed with neutravidin green fluorescence monitored at 530 nm after excitation at 480 nm which shows overlay of rhodamine and oregon green in yellow, and oregon green alone in green.

6.9 Quantitative analysis of arrays

The relative fluorescence intensity of array spots which contain the complementary

oligo sequence to those which contain a non-complementary sequence are demonstrated

here. The fluorescence intensities of the rhodamine labelled cubosomes are normalised

against a streptavidin647 fluorophore reference spot.

173

Figure 6.9.1 Relative fluorescence intensity of site specific cubosome binding Site directed immobilisation of cubosomes onto oligo array surface. CA1` is the cholesterol tagged oligonucleotide with a complementary sequence to BA1, the surface bound biotinylated oligonucleotide. CB1` is the cholesterol tagged oligonucleotide with a complementary sequence to BB1, the surface bound biotinylated oligonucleotide. a) Cubosomes were tagged using a single cholesterol per oligonucleotide; b) Site directed immobilisation of cubosomes onto oligo array surface tagged with two cholesterols per oligonucleotide. RFI- relative fluorescence intensity of fluorescence intensity at 590 nm (rhodamine)/ fluorescence intensity at 685nm (streptavidin647 reference). Data represents mean ± SEM, n = 15.

The fluorescence intensities of the rhodamine labelled vesicles are normalised against a

neutravidin labelled OG fluorophore reference spot.

NeutO

G- R

h M2R C

B1

NeutO

G- R

h M2R

CA1

NeutO

G - Rh M

2R

NeutO

GBB1 -

Rh M

2R C

B1

NeutO

GBB1-

Rh M2R C

A1

NeutO

GBB1 -

Rh M2R

NeutO

G BA1 -

Rh M2R

CB1

NeutO

G BA1-

Rh M2R

CA1

NeutO

G BA1 -

Rh M

2R0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

RFI

(590

nm/5

30nm

) (a.

u)

Figure 6.9.2 Relative fluorescence intensity of site specific vesicle binding a) Site directed immobilisation of vesicles containing M2R labelled with octadecyl rhodamine, onto oligo array surface. Vesicles were tagged using a single cholesterol per oligonucleotide. RFI- relative fluorescence intensity of fluorescence at 590 nm (rhodamine)/ fluorescence at 530nm (neutravidin oregon green reference). Data represents mean ± SEM, n = 25.

0.08

0.09

0.10

0.11

0.12

0.13

0.14

0.15 ***

*

BB1 surface

CB1`(comp)

CA1`(non-comp)

BA1 surface

CB1`(non-comp)

CA1`(comp)

RFI

0.10

0.15

0.20

0.25 ***

BB1 surface

CB1`(comp)

CA1`(non-comp)

RFI

b) a)

174

6.10 Manuscript

The following manuscript has been published in the journal, PROTEOMICS (Bailey et

al. 2009). This is a pre-published version.

G-protein coupled receptor array technologies: site directed immobilisation of

liposomes containing the H1-histamine or M2-muscarinic receptors

Kelly Bailey1,2 *, Marta Bally3 *, Wayne Leifert1,4, Janos Vörös3 and Ted McMurchie1

1 CSIRO Molecular and Health Technologies, Adelaide, SA, Australia 2 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA, Australia 3 Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH and University Zurich, Switzerland 4 CSIRO Human Nutrition, Adelaide, SA, Australia

* These authors contributed equally to this work

Correspondence:

Laboratory for Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH and University Zurich

Gloriastr. 35 8092 Zurich (Switzerland)

Tel: +41 43 633 65 99. Fax: +41 44 633 45 11. E-mail: bally@biomed.ee.ethz.ch

Abbreviations:

GPCR, G-protein coupled receptor; sf9, Spodoptera frugiperda; Oligo, oligonucleotide; QCM-D, quartz crystal

microbalance with dissipation; GDP, guanosine diphosphate; GTP, guanosine triphosphate; H1R, H1-histamine

receptor; M2R, M2-muscarinic receptor; PLL-g-PEG, poly(L-lysine)-grafted-poly(ethylene glycol); PEG-biotin,

biotinylated polyethylene glycol; NA, neutravidin; SA633, streptavidin conjugated to Alexa fluor633; SA532,

streptavidin conjugated to Alexa fluor532; AMP-PNP, adenylylimidodiphosphate; [35S]-GTPγS, radiolabeled isotope 35S-conjugated to 5`-O-(3-thiophosphate); Δf, frequency shift; D, dissipation factor; BODIPY, dipyrromethane boron

difluoride; bDNA, biotinylated oligonucleotide; cDNA, cholesterol modified oligonucleotide; cryo-TEM, cryo

transmission electron microscopy; Bmax, maximum number of binding sites.

Keywords:

G-protein coupled receptors / Liposomes / Membrane protein microarray / Oligonucleotide directed immobilisation /

Spotted arrays

Abstract

This paper describes a novel strategy to create a microarray of G-protein Coupled Receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H1-histamine receptor and the M2-muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for

over-expression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol-modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a Quartz Crystal Microbalance with Dissipation (QCM-D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs

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captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self-sorting liposome array of GPCRs which would underpin a variety of future novel applications.

Introduction

GPCRs are cell surface receptors which have 7 membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 40% of the currently available prescription drugs [1]. This is likely to increase with high-resolution structural data of such proteins now beginning to be published [2]. GPCRs bind to a wide variety of ligands ranging from volatile odorant molecules to biogenic amines and peptides. The list of known ligands to date is not comprehensive and there still exists more than 100 “orphan” receptor, to which the endogenous ligand is yet to be identified. The signalling pathway of the GPCR begins with a ligand binding event which usually occurs within the extracellular domain of the GPCR. This binding event activates the GPCR which in turn activates the coupled messengers termed G-proteins. The G-proteins consisting of 3 dissimilar proteins, the Gα subunit and the Gβγ dimer. The G-proteins couple to the intracellular region (carboxy terminus) of the GPCR. Once the GPCR is activated the G-proteins undergo conformational changes which result in the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on the Gα subunit. Once activated, the Gα and Gβγ dimmer also alter their proximity to one another either by separation [3-5] or rearrangement [6].

There are a number of GPCRs that have been used as models for receptor immobilisation onto solid supports. These include the β2-adrenergic receptor [7], the neurokinin-1 receptor (NK1R) [8], chemokine receptor CCR5 and CXCR4 [9], the human delta opioid receptor [10], and the α2a-adrenergic receptor [11]. However in most instances, bovine rhodopsin has been used as it is one of the only GPCRs which has high natural abundance and for which the crystal structure is known. [12-15]

There is substantial interest in establishing solid supports capable of functional immobilisation of membrane proteins such as GPCRs. One

motivating factor is the belief that microarrays of membrane proteins would constitute a major advantage in the area of drug discovery by allowing parallel and high-throughput ligand screening. The extensive number of known and potential GPCRs encoded in the human genome together with the even larger number of potential ligands, establishes a basis for the advantages lying in such an approach, (reviewed in [16]). However, difficulties arise from the fact that proteins, in general, are chemically and structurally very diverse, often not very robust and subject to denaturation [17]. Membrane proteins, such as GPCRs, are particularly susceptible to denaturation because they are composed of large regions of hydrophobic residues, the structure of which, are destabilised in the aqueous environment. Due to these inherent micro-environmental requirements of membrane proteins, technologies establishing supported [18, 19] and free-spanning lipid bilayers [20-22], immobilised lipid vesicles [23-27], nanodiscs [28] or high-density lipoprotein particles [29] offer favourable interfaces to create arrays of membrane proteins. Many of these techniques, however, require the solubilisation and purification of the receptor prior to incorporation into prepared synthetic lipids. However, GPCRs are particularly difficult to isolate from their native membrane environment and the strategy of using crude membrane extracts might be advantageous.

Solubilised and purified receptors reconstituted into a lipid environment [7, 8] as well as receptors maintained in cell membrane extracts [30-33] have been used in attempts to attach these membrane proteins to a surface. Methods to control orientation of the membrane proteins involve the direct attachment of the C- or N-terminus of the receptor onto the surface via a affinity tag such as a biotin group [7, 8, 12, 34], a histidine tag [29, 35], or GPCR-directed antibodies [7]. While evidence shows that in solution, receptors which are engineered with a tag for immobilisation retain G-protein coupling activity [29, 36, 37], there is little evidence to show G-protein activity whilst the receptors are attached to the surface, with the exception of surface plasmon resonance studies conducted using bovine rhodopsin [12].

Previous studies on which this work expands have demonstrated sequence specific and site-selective immobilization of synthetic phospholipid vesicles onto a non-fouling and vesicle resistant poly(ethylene glycol) layer [23, 38]. Using complementary oligonucleotide sequences, the vesicles have been captured on surfaces constructed using Molecular Assembly Patterning

176

by Lift-off (MAPL) [38] or by spotting functional complexes [23]. While these studies have demonstrated the feasibility on model membranes and proteins, here we adapted the concept for the creation of functional GPCR arrays. In this study, Sf9 cell membrane extracts containing the H1-histamine (H1R) or M2-muscarinic (M2R) receptors have been prepared by differential centrifugation and attached to specific regions on a surface using complementary oligo sequences.

Materials and Methods

Materials Baculovirus stocks of G-protein subunits Gαi1His, Gγ2 were obtained from Prof. Richard Neubig, University of Michigan, USA, while Gβ4baculovirus stocks were obtained from Prof. James Garrison, University of Virginia, USA. GPCR baculovirus stock of the M2-muscarinic receptor was obtained from Prof. Alfred Gilman, University of Texas, USA, and H1-histamine receptor baculovirus was obtained from Prof. Willem J. DeGrip, University Medical Center, Nijmegen, Holland. Ratnala et al. details further information regarding the generation of GPCR baculovirus stocks [39]. 3H-Scopolamine radioligand was purchased from Amersham Pharmacia Biotech. [35S]-GTPγS and 3H-pyrilamine were purchased from Perkin Elmer Life Sciences. Fluorescent ligands BODIPY-pirenzepine (Excitation/Emission) 558/568 nm (Muscarinic antagonist) was purchased from Invitrogen. GTPγS, and all other reagents (unless otherwise stated) of the highest quality grade were purchased from Sigma-Aldrich, Switzerland.

The graft copolymers poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) and its biotinylated version PLL-g-PEG/PEG-biotin were purchased from SuSoS AG, Switzerland. PLL-g-PEG consists in a Poly-L-lysine backbone [20kDa] grafted with PEG side chains [2kDa] in a grafting ratio of 3.5. The biotinylated PLL-g-PEG/PEG-biotin had a similar architecture with 50% of the side chains consisting of biotinylated polyethylene glycol [3.4 kDa]. Oligonucleotide sequences were purchased from Eurogentec, Belgium and were as follows: - bDNA1: 5'- CCC CCA TGG AAT CGT AA -3'; bDNA2: 5’- CCC CCT TCA GAG CAT AT -3’ both with a 5’ biotin modification; cDNA1: 5'- CCC CCT AGT TGT GAC GTA CAT TAC GAT TCC AT-3' ; cDNA2: 5’- CCC CCT-AGT TGT GAC GTA CAA TAT GCT CTG AA -3’ both with a 5’ tri-ethylene-glycol (TEG) cholesterol modification. Neutravidin (NA), streptavidin Alexa Fluor633 (SA633) and streptavidin Alexa Fluor532 (SA532) were purchased from Invitrogen,

Switzerland. In all cases ultrapure water (MilliQ Gradient, A 10 system, resistance of 18 M�/cm, total 4 ppb, Millipore Corporation, Switzerland) was used.

Cell culture, GPCR expression and preparation Sf9 (Spodoptera frugiperda) cells in SF900II media (Invitrogen, Australia) were grown in suspension culture at 28˚C with shaking (138 rpm in an orbital shaker). Baculovirus stocks were filtered (using 0.2μm syringe filter or by vacuum filtration using a Millipore Stericup) and added to Sf9 cells at 1-2 x 106 cells/ml to infect at a multiplicity of infection of 2. Infected cells were grown for 48-72 hours at 28˚C with shaking (138 rpm) prior to harvesting. Sf9 cell membranes containing either the H1R or M2R were prepared using a modified method to remove endogenously expressed G-proteins [40] as previously described [3].

Liposome preparation Sf9 membrane preparations were resuspended to a total protein concentration of 0.75 mg/ml. Following this, some preparations were then extruded through a 1 �m polycarbonate membrane (Avestin, Germany) followed by a 400 nm polycarbonate membrane (Avestin, Germany) at room temperature. In some cases this was followed again by a 200 nm polycarbonate membrane (Avestin, Germany). Total protein within the membrane extract was measured using the Bradford Protein Assay [41]. The membrane preparations were tagged with an oligo sequence by incubation (RT, 10 minutes) in a solution containing oligos modified with a cholesterol moiety (cDNA1 and cDNA2, see section 2.1 for description) at a concentration of 0.35μM.

Confirmation of GPCR Expression and Functionality 3H-Ligand binding To determine receptor-ligand binding specificity, saturation curves were generated using 3H-scopolamine for the M2R preparation, and 3H-pyrilamine for the H1R preparation. Receptor preparations, at a total protein concentration of 1µg, were incubated with various concentrations of the appropriate 3H ligand in TMN buffer (50 mM Tris, 100 mM NaCl, 10 mM MgCl2, pH 7.6) in a final assay volume of 100 µl. To determine non-specific binding, an additional concentration curve was generated in the presence of the appropriate receptor antagonists (100 μM Atropine or 100 μM Triprolidine were used as antagonists for the M2R and H1R respectively to block the 3H-ligand binding site). Specific binding was calculated by the subtraction of non-specific

177

binding from total binding. Samples were incubated for 90 minutes with gentle mixing at 27°C then filtered over glass microfiber filter papers (GF/C filters) (Filtech, Australia). Filters were washed 3 times with 4 ml of TMN buffer. Liquid Scintillant was added to the filters and a scintillation counter (Wallac 1410 liquid scintillation counter) was used to determine the amount of 3H-ligand bound. Kd values for saturation curves were determined by non-linear least-squares analysis fitted to a single hyperbolic binding function (GraphPad Prism V4.0, san Diego, CA, USA).

[35S]-GTPγγγγS binding assay Functional activity of the receptors was measured by monitoring the activity of the second messenger G-proteins. Specifically, receptor induced activation of the Gα subunit was demonstrated using a radioactive GTP binding assay as previously described [3]. Purified Gαi1

and β4γ2 G-protein subunits at a concentration of 20 nM were incubated with receptor membrane preparations. Agonist stimulated [35S]-GTPγS binding studies were performed using a modification of published techniques [42]. A reconstitution mix consisting of 0.05 mg/ml M2R or H1R native membrane extracts, 5 μM GDP, 10 μM AMP-PNP, 20 nM G-proteins and 0.25 nM [35S]-GTPγS were prepared in a final volume of 100 μl in TMND buffer (50 mM Tris, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, pH 7.6) on ice. The reactions were initiated with either buffer alone (basal) or in the presence of receptor agonist (1 mM histamine for the H1R and 120 mM Carbachol for the M2R). Specificity of agonist stimulation was determined by introducing a receptor antagonist (100 μM pyrilamine for the H1R or 100 μM atropine for the M2R) to block the agonist binding site and hence further signalling through the G-proteins. Samples were incubated for 90 minutes with gentle mixing at 27°C then filtered over GF/C filters. Filters were washed 3 times with 3 ml washes of TMN buffer. Liquid Scintillant was added to the filters and a scintillation counter (Wallac 1410 liquid scintillation counter) was used to determine the amount of [35S]-GTPγS bound.

Electron Microscopy Membrane preparations were applied to lacey carbon grids which had been glow discharged under vacuum (30 seconds). The lacey carbon provides a mesh support comprised of holes of various sizes, which allow for viewing of the sample through the thin vitreous ice formed within the holes, during transmission electron microscopy (TEM). Excess solution was blotted for 10-30 seconds using filter paper. The grid was

immediately plunged into liquid ethane to form a thin layer of vitreous ice containing the liposomes. Images were taken using low dose settings with an electron dose of less than 10 electrons per square Angstrom to minimise damage to the sample by the electron beam, on a FEI Technai 12 TEM operating at 120 kV and a magnification of 67,000x or 110,000x.

Quartz Crystal Microbalance with Dissipation Studies The QCM-D instrument (Q-Sense E4, Sweden) was used to measure in situ changes in mass (measured by the frequency shift Δf) and viscoelasticity (measured by the dissipation factor, D) achieved through the adsorption of layers onto the surface of an oscillating crystal. The quartz crystal was coated with a 6 nm thin layer of Nb2O5 applied at the Paul Scherrer Institute (Switzerland). Prior to mounting in the liquid-exchange cell of the instrument, the substrates were cleaned by immersion in 2 % (w/w) sodium dodecyl sulphate for a minimum of 30 minutes, then rinsed in Milli-Q water and dried under a stream of nitrogen. This was followed by a UV/ozone treatment (30 minutes). Resonance frequencies were measured at several harmonic overtones (with overtone numbers of 3,5 and 7). The surfaces were prepared in temperature stabilized (21-22°C) degassed HEPES buffer (10 mM 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid and 150 mM NaCl, pH 7.4) using modifications of published protocols [38]. A schematic of the surface modification process is shown in figure 1. The surfaces were first coated with a monolayer of biotinylated PLL-g-PEG (0.1 mg/ml) by adsorption from solution via electrostatic interactions between the positively charged PLL backbone and the negatively charged metal oxide surface. The biotin surface density could be varied using mixtures of functionalized and non-functionalized polymers as described previously [43]. If not stated otherwise, a mixture of 25% (w/w) PLL-g-PEG/PEG-biotin and 75% (w/w) non-functionalized PLL-g-PEG was used (12.5% of the PEG side chains biotinylated). Subsequently, the high affinity interaction of biotin and neutravidin was utilised to tag the biotinylated PEG chains with a biotinylated oligo (bDNA1 or bDNA2), 17 bases in length. The neutravidin (0.33 μM) and biotinylated oligonucleotides (0.35 μM) were pre-incubated for 10 minutes in HEPES buffer prior to exposure to the biotinylated surface. Finally, liposomes (either extruded or non-extruded preparations) pre-incubated with a cholesterol modified oligo for at least 10 minutes (0.35 μM unless stated otherwise) in TMN buffer, were introduced into the QCM-D chamber. Prior to the injection of the

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GPCR membrane preparations, the buffer was exchanged with TMN. The Δf and ΔD for each modification step were monitored in situ and gave qualitative information on the mass and on the viscoelastic properties of the adsorbed layers, respectively.

Microarray Preparation and Imaging Glass slides or Ta2O5 waveguides (Zeptosens, a division of Bayer (Schweiz) AG, Switzerland) were cleaned by ultrasonication for 10 min in 2-propanol, rinsed with ultrapure water, blow-dryed with nitrogen, followed by an O2 plasma treatment (2 minutes). Immediately after cleaning, the slides were coated with 100% (w/w) PLL-g-PEG/PEG-biotin (50% of the PEG chains biotinylated) by dipping the slides in the polymer solution (0.1 mg/ml, 40 minutes), followed by a rinse with Milli-Q water and blow-drying with nitrogen.

A pin and ring spotter (GMS 417 arrayer, Affymetrix, USA) was used for the contact printing of oliguonucleotide spots (~150 μm in diameter) according to a previously published protocol [23]. Briefly, the array was created by spotting complexes of neutravidin (4.2 μM) and biotinylated oligos bDNA1 or bDNA2 (both at 4.4 μM) diluted in 50% zeptoMARK spotting buffer (v/v) (Zeptosens, Switzerland) onto the biotinylated polymer surface. Streptavidin Alexa fluor532 (2.5 μg/ml) was spotted as reference spots in the array.

Fluorescence experiments were performed in the zeptoCARRIER flow cell (Zeptosens, Switzerland). The slide was pre-wet with 200 μl TMN buffer after mounting. The membrane extracts were prepared as for the QCM-D experiment (section 2.6) and added in a volume of at least 50 μl. After a 4 hour incubation, the slides were rinsed twice with 200 μl buffer. The fluorescent GPCR ligand BODIPY-pirenzepine (muscarinic receptor) was used to monitor GPCR liposome immobilisation and added in the appropriate concentration after the membrane extract had been captured on the surface and unbound extract washed away. Fluorescent experiments were performed using either a Confocal Laser Scanning Microscope (ZeissLSM 510, Germany) or the ZeptoREADER (Zeptosens, a division of Bayer Schweiz, AG, Switzerland). The confocal microscopy images were taken with a diode pumped solid state (DPSS) laser (10 mW, 561 nm) using a 40x (LD, NA 0.7) objective. Microarray imaging was performed with the ZeptoREADER (Zeptosens, a division of Bayer Schweiz AG, Switzerland), a highly sensitive microarray reader based on waveguiding technology. Briefly, a laser light was coupled into

and out from a Ta2O5 thin film deposited onto a glass substrate via an optical grating etched into the substrate. Surface-bound fluorophores detected by CCD camera were excited by the evanescent field resulting from total internal reflection of the laser beam making this technique extremely surface sensitive. The device was equipped with two lasers (532 nm and 635 nm) and a CCD camera for fluorescence detection, as further detailed in other publications [44-46].

Results GPCR and G-protein expression and functionality Sf9 cell expression of the M2R and the H1R was confirmed by the production of saturation binding curves using 3H-scopolamine (figure 2a) and 3H-pyrilamine (figure 2b), respectively. The ligand binding properties of the H1R or the M2R preparations did not change once the preparation had been extruded through a 400 nm polycarbonate filter (figure 2b). The apparent dissociation constant, Kd, for 3H-scopolamine binding to the M2R was 0.62 ± 0.075 nM, which is in a similar range to previously published results [47]. The Kd value for 3H-pyrilamine binding to the H1R was approximately 1.3 nM, which also agrees with published Kd results [48]. Saturation binding curves indicated that the M2R had a Bmax value of approximately 6.1 pmoles/mg, similar to published expression levels [47] and the H1R a value of 10.9 and 12.6 pmoles/mg prior to and post extrusion respectively (figure 2).

The membrane preparation carrying the H1R (figure 3a) or M2R (figure 3b) also retained its ability to couple to the heterotrimeric G-protein (Gαi1β4γ2) once reconstituted in the [35S]-GTPγS binding assay. Figure 3a shows the activity of the H1R by displaying agonist (1mM histamine) induced [35S]-GTPγS binding to the receptor-associated Gαi1 G-protein. [35S]-GTPγS binding can be prevented by the introduction of an excess of antagonist (100 μM pyrilamine) to block the receptor’s orthosteric agonist binding site. This is shown by the decrease of [35S]-GTPγS binding to the level of basal activity (i.e. the level of binding that occurs when no agonist is present). The receptor activity is retained both prior to and post extrusion through 400nm polycarbonate membranes. Similar results were obtained for the M2R (figure 3b) using carbachol as agonist and atropine as antagonist.

Electron Microscopy The membrane extracts contained liposome-type particles prior to extrusion (figure 4a & b). The solutions contained structures of various shapes

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and sizes (from 50nm to ≥1 μm), with an outer layer approximately 5nm wide. The size and shape of the structures which were generally spherical and fully enclosed indicated that the extracts were unilamellar liposomes. Once the preparation was extruded through a 400 nm polycarbonate filter, the liposomes appeared as a more homogeneous mix of spherical shapes and generally ranged in size from 50-400 nm (figure 4c).

Specific Site-directed immobilisation of GPCR liposomes QCM-D The Nb2O5 surface of the quartz crystal was modified over a number of additions to the QCM-D flow cell. Frequency and dissipation shifts were monitored over each step (figure 5a & b). The first step involved the adsorption of the PLL-g-PEG/PEG-biotin onto the Nb2O5 surface. Surface saturation of the adsorbed PLL-g-PEG/PEG-biotin polymer was achieved within 40 minutes and resulted in a normalized change in the third frequency overtone (Δf3/3) of -37 ± 1 Hz and a dissipation (ΔD3) of 2.2 ± 0.1 x 10-6. The second step of the surface modification was the introduction of the neutravidin/oligonucleotide complexes. Neutravidin/oligo complex binding to the biotinylated surface reached equilibrium within 1 hour. The adsorption resulted in a Δf3/3 = -30.7 ± 2 Hz and ΔD = 3.0 ± 0.3 x 10-6. Both values are the mean ± SEM, n = 10 and are in agreement with previous reports [38]. Before injecting the liposomes, the buffer was exchanged from HEPES to TMN, a common assay buffer for the GPCR liposomes. This resulted in a small frequency change (<2 Hz) which stabilised before introduction of the GPCR liposomes.

The GPCR liposome preparations carrying the M2R, of <400 nm in diameter were immobilised onto the surface within 1.5 hours (figure 5 a & b). A significant decrease in frequency (figure 5a) and an increase in dissipation (figure 5b) values indicated the successful immobilisation of GPCR liposomes carrying the oligo tag complementary to the one on the sensor surface. When the tag was present within the liposomes, the measured values were Δf 3/3= -208 Hz and ΔD = 41 x 10-6 (values are mean, n = 2). Surface immobilization was oligonucleotide specific with low binding of untagged liposomes (ΔF3/3= -1.2 Hz) (figure 5a & b) or liposomes carrying a non-complementary oligonucleotide tag (< 8 Hz) (data not shown). There was little non-specific binding of liposomes to the surfaces at each step of the surface preparation (data not shown).

Specific liposome immobilization was also achieved with H1R and non-infected membrane extracts, and immobilisation was achieved for all three extracts in the extruded and non-extruded state. However, the total adsorbed mass for non-extruded liposomes varied from cell extract batch to batch and could be adjusted by varying the amount of cholesterol DNA added to the membrane extract preparations (results not shown). Furthermore, it was observed that for non-extruded liposomes, smaller changes in frequency and dissipation were observed and longer incubation times were required to reach binding saturation (>90% of the binding took place within 3 hours), indicating that there was a lower binding efficiency with these preparations (figure 5c).

Spotted Arrays GPCR liposomes were monitored using the fluorescent-labelled ligand BODIPY-pirenzepine (M2R) (figure 6a). Sequence specific immobilisation of the GPCR liposomes onto the oligo arrays was further demonstrated using arrays composed of spots functionalised with two different oligo sequences (bDNA1 and bDNA2). Low non-specific binding to the pegylated background was observed. Spots functionalised with the non-complementary oligo sequence displayed less than 20% of the fluorescence of the spots carrying the complementary oligonucleotide (figure 6b).

For the M2R, ligand binding was found to be specific as shown by the zeptoREADER images of spots presenting either GPCR-free liposomes (Figure 7a) or M2R containing liposomes (Figure 7b) after exposure to BODIPY-pirenzepine. However, as measured with the ZeptoREADER, 30% of the signal observed resulted from non-specific ligand binding to the liposomes so that the signal measured for immobilized M2R membrane preparations was about 3.3-fold higher compared to liposomes isolated from non-infected cells (pirenzepine concentration: 1.25 μM) (Figure 7c). It should be noted that for this experiment, the immobilised liposome mass, measured in a control experiment performed with QCM-D, was similar.

Discussion Results in this paper describe a method by which crude membrane preparations containing a specific membrane protein of interest, such as a GPCR, can be immobilised onto a specified site on a surface. This process is also conducive to enabling the immobilisation of a functional signalling complex consisting of the receptor and its second messengers, the G-proteins. This approach makes use of a standard oligonucleotide

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array which is converted into a heterogeneous membrane protein array, a strategy similar to that proposed by Niemeyer and coworkers for a variety of biomolecules and nano-objects [49-51]. The following benefits of the methodology described should be highlighted: Firstly, nucleotide arrays have nowadays become a golden standard for several biological and biomedical applications [52, 53]; A great variety of production protocols have been proposed and several oligonucleotide arrays are commercially available for various biological and biomedical applications [52, 53]. Due to the large number of possible oligonucleotide sequences, it may be possible to specifically immobilize a large number of various membrane proteins in a microarray format. Furthermore, difficulties in the production of protein and membrane protein arrays which usually arise from the fact that proteins are chemically and structurally very diverse, less robust than oligonucleotides and generally subject to denaturation, may be overcome with this approach: the receptor’s natural environment is maintained, and harmful drying steps are avoided, minimising the risk of functional alterations to the protein.

This immobilization strategy was shown to be well-suited for the immobilization of membrane extracts from non-infected cells as well as from cells over-expressing different GPCRs while preserving ligand binding specificity. This illustrates the flexibility and versatility of our approach. Furthermore, both extruded and non-extruded extracts could be immobilized with high-specificity.

The small batch-to-batch variations observed in the liposome binding properties measured by QCM-D could be the result of different DNA per liposome numbers for each batch which could lead to different binding efficiencies or liposome flattening as previously observed with synthetic vesicles [38], or different cholesterol incorporation efficiencies due to slight variation of the lipid composition between infected and non-infected Sf9 cells [54]. The presence of polysaccharides on the cell membrane surface with limited permeability [55] could also affect DNA hybridization and explain differences in binding efficiency between extruded and non-extruded extracts. Preliminary results showed that the mass binding to the QCM sensor could be influenced by varying the concentration of cDNA added to the membrane extracts.

Both extruded and non-extruded extracts could be immobilized with high-specificity. The membrane extracts were extruded in an attempt to homogenize the solution and minimize the

variability that might be associated with a very heterogeneous mixture of liposome sizes. Characterisation of the extruded preparations using cryo-TEM indicated predominantly spherical, enclosed, unilamellar structures of sizes 50-400nm in diameter. Images of non-extruded preparations, despite showing greater variation in size and shape, indicated that the extracts prior to extrusion, were also composed of liposomes. Nevertheless, to ensure maintenance of all components within the membrane necessary for native receptor function, and to minimise steps within the immobilisation protocol, membrane extracts which had not been extruded were used for work with the spotted arrays shown in this paper.

The use of crude membrane extracts without solubilisation and purification techniques results in limited control over issues such as the orientation of the receptor proteins within the liposomes. However the [35S]-GTPγS binding assay indicates that the receptor protein orientation may not be a limiting factor. Results indicate a degree of “leakiness” of these liposomes as both the intracellular carboxy terminus (G-protein binding site) and extracellular amino terminus (involved in ligand binding) must be accessible to enable both the agonist and [35S]-GTPγS to bind. It is worth noting that most studies involving H1R signalling, report of interactions with the Gαq subunit, however, in this in vitro assay system it has also shown to signal through the Gαi1 subunit. We have not determined the relevance of this interaction with in vivosignalling as it is not within the scope of this paper, however, it has previously been reported that a functional H1R-Gαi1 interaction does occur in vivo [56]. The Gαi1 subunit was used in this study because it binds [35S]-GTPγS readily and specifically in this assay system.

In this paper, a single cholesterol moiety was used to anchor the liposome to the surface displaying the complimentary oligos. This single cholesterol interaction with the lipid bilayer would, however, most likely prove inadequate for a self sorting array since DNA exchange between liposomes has been reported previously [26]. Therefore the method presented here enables only for the creation of heterogeneous arrays by a sequential immobilization of the different liposome populations. An answer to this problem can potentially be found in the work of Pfeiffer and Höök who have demonstrated that multiple cholesterol anchoring can provide a much stronger bilayer anchoring to the bilayer [26]. Although not shown in this paper, preliminary data indicated that this double cholesterol/oligo tagging of the liposomes also resulted in the successful

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immobilisation of the GPCR containing liposomes onto a complementary surface. For mammalian cells, covalent coupling to azido groups could also provide an alternative [57].

Array images indicate some (30%) non-specific ligand adsorption to the liposomes regardless of receptor presence (figure 7) when the fluorescent pirenzepine was present at micro-molar concentrations. This concentration range was primarily used to ensure complete saturation of the receptors within the liposomes and to ensure clear visualisation. Higher concentrations were also used because pirenzepine has a lower affinity for the M2 subtype of the muscarinic receptor family (Ki [equilibrium dissociation constant] = 303 nM) than it does for the M1 subtype (Ki = 7.1nM) [47] . This non-specific binding indicates that the BODIPY-pirenzepine conjugate could be lipophilic, a feature of fluorescent conjugates of GPCR ligands that has been described previously [58, 59]. ZeptoREADER results, using fluorescent ligand concentrations in the lower micro-molar range (2.5 and 1.25μM) displayed approximately four times lower non-specific binding on non-GPCR containing liposomes than images obtained using 100 μM of the fluorescent ligand (data not shown).

We have shown that this strategy provides the opportunity to immobilise a G-protein coupled receptor without the need to modify the receptor itself or to solubilise it from its native environment. The ability for the GPCR to retain its G-protein coupling capability may enable the production of a GPCR array which is less dependent on monitoring fluorescent ligand binding but relies more on true functionality. Limitations which lie in arrays reliant on fluorescent ligands include the availability of the ligand for a wide range of receptors, and the difficulty in labelling small molecule ligands without compromising functional activity and binding efficacy. An array format such as this which allows interaction freedoms for the receptor, including the ability to bind to various binding partners and undergo required conformational changes consistent with their physiological response, may provide the opportunity to monitor ligand activation through changes in associated proteins, such as G-protein conformational changes. This could be achieved by using a technique such as Fluorescence Resonance Energy Transfer (FRET) [60].

To conclude, we have demonstrated the use of surface immobilised oligos as a method of capturing membrane proteins. The liposomes used in the study were composed of native Sf9 cell membranes. These liposomes contained GPCRs

expressed using the baculovirus/Sf9 expression system. The receptors were functional as demonstrated using ligand binding and receptor/G-protein signalling assays. These assays also indicate that both the intra- and extracellular domains of the receptor are available for small molecule binding. During surface immobilisation studies, the PLL-g-PEG polymer background showed little non-specific binding of these crude membrane preparations. QCM-D data and fluorescent array images captured by confocal microscopy and the zeptoREADER microarray scanner, demonstrate that capture of these liposomes occurs in a manner dependent on the oligo complementarities between those tagging the liposomes and those displayed on the surface. Such technologies will be useful in the field of membrane proteins, especially these proteins which are difficult to isolate from their native environment such as GPCRs.

Acknowledgements

We thank Dr. Brigitte Städler for providing her expert assistance with regard to vesicle arrays, Dr. Connie Darmanin and Ms Lynne Waddington (CSIRO, Australia) for their assistance with the cryo-TEM, Ms Amanda Aloia for her guidance with the use of the H1-histamine receptor. We would also like to thank Prof. Richard Neubig, University of Michigan, USA for the provision of Gαi1His, Gγ2; Prof. James Garrison, University of Virginia, USA, for the provision of Gβ4baculovirus stocks. For the provision of GPCR baculovirus stocks we would like to thank Prof. Alfred Gilman, University of Texas, USA for the M2-muscarinic receptor and Prof. Willem J. DeGrip, University Medical Centre, Nijmegen, Holland for the H1-histamine receptor. Funding contributions for this work were provided by the CSIRO’s Emerging Science Area for Nanotechnology funding scheme and the OzNano2Life/DEST and the CTI (Project Nr. 7241.1 NMPP-NM).

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Figure 1: Schematic representation of the immobilisation protocol: (1) Clean surfaces were modified by (2)

adsorption of PLL-g-PEG with 12.5% or 50% of the PEG chains terminated by biotin molecules (for QCM-D or

array spotting respectively). (3) Further modification included immobilisation of the pre-formed

neutravidin/biotinylated oligonucleotide complex. (4) Liposomes containing GPCRs that were tagged with

complementary oligonucleotides modified with a cholesterol moiety were captured onto the oligonucleotide surface.

Figure 2: Specific binding of receptor ligands to Sf9 membrane preparations (a) (�) Specific M2-muscarinic

receptor binding (3H-Scopolamine) in Sf9 membranes. Non-specific binding was measured in the presence of 100μM

atropine (antagonist) and subtracted from total binding in the absence of the competitor (3H-Scopolamine); Bmax =

6.1 ± 0.23 pmoles/mg; Kd = 0.62 ± 0.075nM, mean ± SEM, n = 5. (b) Specific H1-histamine receptor binding (3H-

Pyrilamine) in Sf9 membranes. Non-specific binding was measured in the presence of 10 μM triprolidine (antagonist)

and subtracted from total binding in the absence of the competitor (3H-pyrilamine). (•) Solid line, non-extruded

membranes; Bmax =10.9 pmoles/mg; Kd = 1.3nM, n = 2; (�) dotted line membrane preparation extruded through

400nm polycarbonate membrane, Bmax = 12.6 pmoles/mg; Kd = 1.6nM, (n = 2, data shown is the mean and error

bars represent the range of the duplicates).

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Figure 3: Assay of receptor signalling activity for reconstituted membranes containing M2R or H1R. 20nM G-

protein subunits, Gαi1 and Gβ4γ2, were combined with 0.05mg (total membrane protein)/ml receptor preparation; 0.25

nM [35S]-GTPγS; 10 μM AMP-PNP; 5 μM GDP. This reconstitution mix was then incubated with either buffer alone

(basal, black chequered column), an agonist (horizontal striped column), or an agonist in the presence of excess

antagonist (diagonally striped column). a) H1R, stimulated with 1mM histamine (agonist) and blocked with 100μM

pyrilamine (antagonist); .b) M2R, stimulated with 120mM carbachol (agonist) and blocked with 100μM atropine

(antagonist). Data shown are mean ± SEM (n = 9).

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Figure 4: Transmission electron microscopy images of liposomes: cryo-TEM images of crude Sf9 cell membrane

preparations containing the M2R a) & b) non-extruded at 110,000x magnification and 120 kV or c) extruded through

400nm polycarbonate membrane at 67,000x, in a thin layer of vitreous ice over a lacey carbon grid. Scale bars in all

images represent 200nm. Lacey carbon grids can be seen in dark grey in top left of image (a) and surrounding central

vesicles in (c).

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Figure 5: Sf9 cell membrane liposome immobilisation. (a) Changes in frequency of overtone 3 and (b) changes in

dissipation as measured on a QCM-D upon immobilisation of <200nm liposomes composed of preparations of Sf9

cell membranes containing over-expressed M2R. (c) Changes in frequency of overtone 3 upon immobilisation of non-

extruded extracts of Sf9 cell membranes over-expressing M2R (grey spotted line), H1R (black solid line). The

following steps refer to all graphs a, b and c unless otherwise stated: 1) Adsorption of PLL-g-PEG (12.5%

biotinylated) onto a Nb2O5 coated quartz crystal; 2) Immobilisation of biotin-oligo complexed to neutravidin; 3a) &

3b) Surfaces are exposed to GPCR liposomes (extruded through 200 nm membranes) which carry no oligonucleotide

sequence (black solid line), or to GPCR liposomes tagged with a complementary oligonucleotide sequence (grey

spotted line); 3c) Surfaces are exposed to non-extruded extracts tagged with complementary oligonucleotide

sequence; 4) Unbound liposomes are washed away with buffer. Unmarked peaks in the data sets indicate a buffer

wash.

Figure 6: Site specific immobilisation of liposomes containing GPCRs shown using (a) confocal microscopy

image of oligonucleotide-tagged GPCR liposomes (M2R) immobilized on a spot presenting the complementary

oligonucleotide cDNA1 after incubation with BODIPY-pirenzepine (100μM). (b) ZeptoREADER image of M2R

membrane extract tagged with cDNA1 attached to an oligonucleotide array presenting the oligonucleotide sequence

bDNA1 and the oligonucleotide sequence bDNA2. ref represents a SA532 reference row (2.5 μg/ml).

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Figure 7: Fluorescent ligand binding to liposomes containing the M2-muscarinic receptor. ZeptoREADER

images of images show liposomes composed of native Sf9 lipids a) not expressing the M2R, and b) over-expressing

the M2R receptor captured via a complementary DNA pair. c) Data obtained from a ZeptoREADER image of

sequence specific oligonucleotide attachment. Fluorescence intensity normalized to the reference (reference is a

SA532 reference row (2.5 μg/ml)) resulting from the binding of BODIPY-pirenzepine for 2 ligand concentrations.

Black columns are the M2R containing liposomes and the grey columns the non-infected liposomes.