doctoral thesis corrected - Medizinischen Universität Wien · LL '(&/$5$7,21 The work presented in this thesis was performed at the Center for Physiology and Pharmacology, Medical

Doctoral thesis at the Medical University of Vienna

for obtaining the academic degree

Submitted by

Supervisor: Univ.-Prof. Dr.med.univ. Michael Freissmuth

Institute of Pharmacology

Center for Physiology and Pharmacology Medical University of Vienna

Währingerstraße 13A, 1090 Vienna, Austria

Vienna, 10/2014

The work presented in this thesis was performed at the Center for Physiology and

Pharmacology, Medical University of Vienna, Schwarzspanierstraße 17, 1090 Vienna,

Austria. It is my original and own work.

The contributions from co-authors and collaborators are stated at the beginning of the

results chapter.

DECLARATION

TABLE OF CONTENTS

LIST OF FIGURES

ABSTRACT

ZUSAMMENFASSUNG

PUBLICATIONS ARISING FROM THIS THESIS

ABBREVATIONS

ACKNOWLEDGEMENTS

1. INTRODUCTION

1.1 G-protein coupled receptors – introduction

1.2 GPCR trafficking

1.2.1 Folding in the endoplasmic reticulum

1.2.2 Misfolding diseases

1.2.3 Pharmacological chaperones

1.2.4 ER export

1.2.5 Turnover

1.3 Adenosine as a retaliatory metabolite

1.3.1 Sites of adenosine production

1.3.2 Signaling via adenosine receptors

1.3.3 Effects elicited by adenosine

1.4 Aims of this thesis:

2. RESULTS

2.1 Publication

3. DISCUSSION

3.1 Endogenous pharmacological chaperones

3.2 Extension of the retaliatory metabolite concept

3.3 Possible mechanism of adenosine entry into endoplasmic reticulum

3.4 Conclusions and future prospects

5. REFERENCES

6. CURRICULUM VITAE

Figure 1: Major pathways determining intra- and extracellular adenosine levels.

Figure 2: Extension of the retaliatory metabolite concept.

The density of G-protein coupled receptors at the cell surface can be increased by

treatment with cell-permeable orthosteric ligands, called pharmacological chaperones.

These compounds act in the endoplasmic reticulum, where they facilitate folding and

stabilize the correct conformation of maturating proteins. In this work, we wanted to

explore if endogenously generated adenosine can chaperone its cognate receptor. To test

this hypothesis: (i) we used an ER-retained, folding-defective mutant of the human A1-

adenosine receptor as a sensor and (ii) we raised the cellular levels of endogenous

adenosine by simultaneous inhibition of adenosine kinase, adenosine deaminase and

equilibrative nucleoside transporters or by hypoxia. The applied inhibitors restored the

surface expression of the A1-Y288A-receptor in stably transfected HEK 293 cells,

similarly to the A1-antagonist DPCPX. The upregulated receptor had mature

glycosylation pattern and was binding-competent, with a radioligand affinity

indistinguishable form the wild type A1-receptor. The effect of the inhibitors was

specific, as it did not enhance the cell surface expression of the folding-deficient V2-

vasopressin receptor, which in contrast responded to pharmacochaperoning by their

cognate antagonist. Application of hypoxia phenocopied the effect of the inhibitors;

endogenous adenosine increased the number of the A1-Y288A-receptors at the plasma

membrane within 1 hour. These results were recapitulated for the wild type A1-receptor.

Taken together, our work documents that endogenous adenosine may act as a ligand

chaperone and thus counteract the damaging effects of hypoxia via increased A1-

receptor signaling. This mechanism can be seen as a ramification of the retaliatory

metabolite concept of adenosine and a new twist in understanding the role of the

endoplasmic reticulum.

Die Dichte von G-Protein-gekoppelten Rezeptoren auf der Zelloberfläche kann durch

Behandlung mit zellgängigen orthosterischen Liganden (d.h. mit pharmakologischen

Chaperons) erhöht werden. Diese Stoffe wirken im endoplasmatischen Retikulum, wo

sie die Faltung der Proteine fördern und ihre korrekte Konformation stabilisieren. Das

Ziel der vorliegenden Arbeit war es zu prüfen, ob endogen erzeugtes Adenosin an

seinem zugehörigen Rezeptor als Chaperon wirken kann. Um diese Hypothese zu

testen, wurde (i) eine ER-retinierte Mutante des humanen A1-Adenosinrezeptor (A1-

Rezeptor-Y288A) als Faltungssensor verwendet und (ii) die zelluläre Konzentration von

endogenem Adenosin durch gleichzeitige Hemmung der Adenosinkinase,

Adenosindeaminase und äquilibrierenden Nucleosid-Transporter oder durch Hypoxie

erhöht. Die eingesetzten verwendeten Inhibitoren erhöhten die Expression des A1-

Rezeptoren-Y288A auf der Zelloberfläche in stabil transfizierten HEK 293-Zellen in

einem Ausmaß, das dem Effekt des A1-Antagonisten DPCPX entsprach. Die vermehrt

exprimierten Rezeptoren hatten ein reifes Glykosylierungsmuster und banden einen

spezifischen Radioliganden mit ähnlicher Affinität wie der Wildtyp A1-Rezeptor. Die

Wirkung der Inhibitoren war spezifisch, da sie nicht die Zelloberflächenexpression des

faltungsdefizienten V2-Vasopressinrezeptors verbesserten; im Gegensatz dazu wurde

dieser V2-Vasopressinrezeptor durch einen entsprechenden Antagonisten an die

Zelloberfläche gebracht. Unter Hypoxie wurde endogenes Adenosin in den Zellen

gebildet. Das führte innerhalb von 1 Stunde zur Erhöhung der Anzahl von A1-

Rezeptoren-Y288A in der Plasmamembran. Diese Ergebnisse wurden auch für den

Wildtyp A1-Rezeptor rekapituliert. In ihrer Gesamtschau zeigen diese Befunde, dass

endogenes Adenosin als Ligand-Chaperon wirken kann. Die damit verbundene

Erhöhung von A1-Rezeptoren an der Zelloberfläche verstärkt den protektiven Effekt von

Adenosin und schützt damit die Zellen vor den schädlichen Auswirkungen der Hypoxie.

Dieser Mechanismus kann als eine Ausweitung des „retaliatory metabolite concept“ für

Adenosin betrachtet werden. Darüber hinaus weisen die Beobachtungen auf eine

regulatorische Rolle des endoplasmatischen Retikulums hin, das ein Reservoir von

Rezeptoren bereit stellt, die bei einem äußeren Stimulus an die Zelloberfläche gebracht

werden.

Kusek J, Yang Q, Witek M, Gruber CW, Nanoff C, Freissmuth M (2014) Chaperoning

of the A1-adenosine Receptor by Endogenous Adenosine - An Extension of the

Retaliatory Metabolite Concept Mol Pharmacol mol.114.094045; published ahead of

print October 29, 2014, doi:10.1124/mol.114.094045

A1R A1-adenosine receptor

ATP adenosine triphosphate

BFA Brefeldin A

Bmax maximal binding capacity

BSA bovine serum albumin

C-terminus carboxyl-terminus

cAMP cyclic adenosine monophosphate

CFTR cystic fibrosis transmembrane conductance regulator

CMV cytomegalovirus

CNT concentrative nucleoside transporter

COPII coat protein complex II

DDM n-dodecyl- -D-maltoside

DIP dipyridamole

DMEM Dulbecco’s Modified Eagle Medium

DMSO dimethyl sulfoxide

DPCPX 8-Cyclopentyl-1, 3-dipropylxanthine

EC50 half maximal effective concentration

EDEM ER degradation-enhancing mannosidase-like

EHNA erythro-9-(2-hydroxy-3-nonyl) adenine

Endo H Endo- -N-acetylglucosaminidase H

ENT1,2,3 equilibrative nucleoside transporter 1, 2, 3

ER endoplasmic reticulum

ERAD ER-associated degradation

ERES ER exit sites

ERGIC ER-Golgi intermediate compartment

FBS fetal bovine serum

FFA1 free fatty acid receptor 1

GABA gamma-aminobutyric acid

GalNAc N-acetylgalactosamine

GAT GABA transporter

GDP guanosine diphosphate

GFP green fluorescent protein

Glc glucose

GlcNAc N-acetylglucosamine

GnRHR gonadotropin-releasing hormone receptor

GPCR G-protein-coupled receptor

GRK G-protein-coupled receptor kinase

GTP guanosine triphosphate

HCA1,2 hydroxy-carboxylic acid receptor 1, 2

HEK293 human embryonic kidney 293 cell line

HPLC high performance liquid chromatography

HRP horseradish peroxidise

HSP heat shock protein

IC50 half maximal inhibitory concentration

IDO 5-iodotubercidin

IP3 inositol triphosphate

Man mannose

MAPK mitogen-activated protein kinase

N-terminus amino-terminus

NBMPR nitrobenzylthioinosine

NMDA N-Methyl-D-aspartate

PBS phosphate-buffered saline

PC-12 rat adrenal pheochromocytoma cell line

PCR polymerase chain reaction

PDI protein disulfide isomerase

PKA protein kinase A

PNGase F peptide-N-glycosidase F

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SF-TAP Strep/FLAG-tandem affinity purification

SLC solute carrier

SUCNR1 succinate receptor 1

TGR5 G-protein-coupled bile acid receptor

TNF- tumor necrosis factor alpha

UDP uridin diphosphate

UGGT UDP-glucose glycoprotein glucosyltransferase

VEGF vascular endothelial growth factor

V2R V2-vasopressin receptor

YFP yellow fluorescent protein

First and foremost, I would like to thank my supervisor Misha Freissmuth, who guided

me through the meanders of my PhD thesis. Without his positive energy, support and

freedom that he gave me, I would have not been able to go so far in my doctoral studies.

He has the encyclopedic knowledge, intuition in motivating people and a generous

heart. Using analogy, I would compare him to a master chaperone protein which assists

the folding of immature, incorrectly folded junior-proteins. I believe that he equipped

me with the necessary knowledge and skills to be successful in my professional life. I

will always be grateful for his help and unshaken faith in a positive outcome.

Secondly, I like to thank my co-supervisor Christian Gruber for his support and

guidance. He has a real passion for science. But most importantly, he created a great,

open atmosphere in our group. We worked, learned, and celebrated together.

I also want to thank Christian Nanoff who patiently and gently explained the scientific

problems and was always ready to solve experimental difficulties. I was always

impressed by his teaching qualities.

I truly enjoyed the time spent with “The Gruber Group”: Erjola, Esther, Giulia, Jessi,

Kathrin, Mina, Zita, Chris, Huang, Johannes, Marcus, Roland and visiting students Meri

Emili and Alfred. You were my “little” international family. Big thanks for the

professional, as well as the emotional support!

I am grateful to all the members of the Center of Pharmacology and Physiology who

helped me during my doctoral studies: especially to Qiong for the help with FACS

experiments and Martin for having patience with the adenosine analysis, but also to

Hend, Marion, Sonia, Azmat, Florian, Helmut, Oliver and Simon K – all of them make

the world a bit better place.

I would like to thank my colleagues from the CCHD program: students and supervisors

for their valuable advice and the happy time we spent together.

Lastly, I want to thank my family, friends and Taki. I am so grateful that you believed

in me.

G-protein coupled receptors (GPCRs) form a large and diverse superfamily of integral

membrane proteins. They evolved to sense the extracellular environment and respond to

a plethora of different stimuli: neurotransmitters, hormones, pheromones, photons,

lipids, peptides, proteins etc. (Pierce et al., 2002). Upon activation by an agonist, the

receptor undergoes conformational changes, which allow it to bind its cognate

heterotrimeric G protein(s). This event triggers intracellular signaling cascades that are

translated into cellular responses. GPCRs are expressed in all organs and control an

array of physiological functions. Accordingly, they are at the centre of the drug

development – it has been claimed that nearly half of the modern pharmaceuticals target

GPCRs (Lefkowitz, 2004). However, this number is misleading, because only a small

fraction (i.e., about 70) of all human GPCRs (>800) are in fact addressed by currently

approved drugs (Gruber et al., 2010).

G-protein receptors share a common structure. They comprise an extracellular N-

terminus, seven conserved transmembrane -helices, three loops on both sides of the

plasma membrane and cytosolic C-terminus. The first segment of the C-terminus forms

an additional -helix (helix 8), which runs perpendicular to the transmembrane

hydrophobic core and parallel to the plan of the lipid bilayer. Post-translational

modifications of GPCRs include: (i) palmitoylation of one or two cysteine residues at

the end of helix 8, (ii) N-linked glycosylation of one or several asparagine residues

either in the N-terminus or in one of the extracellular loops and (iii) formation of

disulphide bonds between cysteine residues in the extracellular loops, which stabilize

the receptor. Based on the structural similarity and amino acid sequence, GPCRs are

classified into five families: glutamate, rhodopsin, adhesion, frizzled/taste2 and secretin

receptor subfamily; this classification is referred to by its acronym as the GRAFS

system. The overwhelming majority of human receptors belong to the rhodopsin family

(Fredriksson et al., 2003).

Recent advances in crystallization studies have expanded our knowledge of the

structural biology of GPCRs (Jaakola et al., 2008). These findings paved the way for the

more mechanistic studies of the GPCR function and activation of G protein

(Rosenbaum et al., 2007). It was discovered that agonist binding induces a displacement

of transmembrane helix 6 of the receptor. This movement creates a space to accomodate

the C-terminus of G subunit. Insertion of the C-terminus into this cavity results in a

confomational rerrangement that opens the GDP-binding site and thus allows for

subsequent activation of the G protein by the incoming GTP. The importance of this

research was recognized by a 2012 Nobel Prize in Chemistry (Lin, 2013).

It is well established that the density of receptors at the cell surface determines the

magnitude (and duration) of the cellular response to a given signal. Two processes

directly affect the number of the receptors at the plasma membrane: (i) the delivery of

nascent proteins to their functional destination and (ii) their removal from the cell

surface. Both trafficking pathways are under strict regulation, however only the

mechanisms that control the latter are well studied and hence understood in considerable

detail (Drake et al., 2006). Therefore, this thesis will focus on the processes that occur

along the secretory pathway.

According to the current model (Dong et al., 2007) the delivery of GPCRs to their

functional destination consists of several steps, which are subject to regulation: (i)

translocation of synthesized polypeptide chains into the ER lumen, (ii) folding under the

scrutiny of ER quality control (QC), (iii) recruitment and packaging of glycosylated

proteins into COPII-coated vesicles, (iv) ER export, (v) modification and sorting of

proteins in the Golgi apparatus and (vi) export from the Golgi and trafficking to the

plasma membrane. In addition, it was reported that receptors dimerization plays a role

in the trafficking of several GPCRs, inter alia: 2-adrenergic, V2-vasopressin, D2-

dopamine receptors (Bulenger et al., 2005). On the other hand, GPCRs can be removed

from the plasma membrane by internalization, which occurs after: (i) the agonist-

induced (homologous) or (ii) the agonist-independent (heterologous) desensitization.

Once the receptor moves to the intracellular compartment, it can either recycle back to

the cell surface, where it is available to reinitiate signaling, or end its fate by lysosomal

degradation (Gainetdinov et al., 2004)

The life of a GPCR begins in the endoplasmic reticulum, precisely on a ribosome,

where it is translated from the mRNA template. The emerging polypeptide chain is co-

translationally inserted into the ER membrane via an aqueous channel, the Sec61

translocon. The transmembrane helices are released consecutively (individually or in

pairs) into the membrane bilayer through the lateral gate of the Sec61 pore (Rapoport,

2007). Upon insertion into the lipid bilayer, the helices start to assemble and re-arrange

to form an annularly packed structure. The interactions between the transmembrane

domains and the lipid environment force the protein to fold in order to attain

thermodynamical stability (Anfinsen, 1972; Bowie, 2005). Folding itself is a stochastic

process of searching for the lowest-energy protein structure, which is also the native

conformation (Dobson, 2003). The folding intermediates are characterized by exposed

hydrophobic regions, immature glycans and/or reduced thiol groups. If such unfolded

protein left the ER, these features, in particular the exposed hydrophobic segments, may

lead to unspecific interactions and aggregation with other proteins. This will be

detrimental, because aggregation interferes with the functional activity of the entrapped

proteins. Therefore, eukaryotic organisms evolved a quality control system to prevent

such errors.

The ER quality control machinery displays an array of functions: (i) it recognizes the

immature protein species and assists in their folding, (ii) it prevents the aggregation and

ER export of unfolded proteins and (iii) it either retains or directs the terminally

misfolded proteins to the ER-associated degradation (ERAD) (Anelli and Sitia, 2008).

GPCRs have been reported to interact with the quality control machinery on the luminal

and cytosolic side of the ER membrane (e.g. calnexin, calreticulin, BiP/Grp78, ERp57

and Hsp40, Hsp70, Hsp90, respectively) (Ellgaard et al., 1999; Bermak et al., 2001;

Bergmayr et al., 2013). Glycosylation plays a major role in the canonical folding

pathway of eukaryotic membrane proteins (reviewed in Pearse and Hebert, 2010): the

preformed core oligosaccharide (Glc3Man9GlcNAc2) is co-translationally transferred

from the dolichol-linked precursor to the nascent protein in the ER lumen (reviewed in

Pearse and Hebert, 2010). After translation, the glucosidases I and II remove the three

glucose residues. These glucose-trimmed glycoproteins are then inspected by the UDP-

glucose glycoprotein glucosyltransferase (UGGT), an enzyme that can discriminate

between folded and incompletely folded proteins (folding sensor) (Parodi, 1999).

UGGT adds one glucose residue to the unfolded species, thus enabling its recognition

by the ER-resident lectins calnexin or calreticulin. These lumenal chaperones form a

complex with the protein disulfide isomerase ERp57, facilitate the protein folding and

prevent the aggregation of folding intermediates. The interaction with the lectins is

abolished when glucosidase II removes the remaining distal glucose. The glycoproteins,

which are properly folded at this stage, can exit the ER. The others are reglucosylated

by UGGT and they enter again the lectin cycle until a stable fold is finally attained.

The proteins, which did not fold properly in spite of several rounds of folding cycles,

are trimmed by ER-resident mannosidases (e.g. EDEM 1, 2, 3). This modification

allows for their recognition by the components of the ER-associated degradation

machinery (ERAD). The misfolded proteins are retrotranslocated to the cytosol by

Sec61 translocon, deglycosylated, polyubiquitinated and finally degraded by the 26S

proteasome (reviewed in Stolz and Wolf, 2010). Alternatively, the misfolded protein

load can also be retained in the ER, or form intracellular aggregates. Because they can

be stained for their sugar content with the Schiff’s reagent, they are referred to as

amyloid fibrils (Dobson, 2003).

The ER quality control system ensures that only the correctly folded proteins exit the

ER, whereas the unfolded ones are retained by chaperones and the misfolded proteins

are routed to degradation. Generally, protein misfolding is caused by mutations (single

point mutations or deletions in the amino acid sequence), which destabilize the native,

three-dimensional conformation. Still, such anomalies may not necessarily affect the

function of the protein. In effect, the stringency of the chaperone and proteasome

system leads to retention and degradation of otherwise functional proteins (loss of

function) (Dobson, 2003). Several diseases originate from misfolding of GPCRs: e.g.

diabetes insipidus (V2-vasopressin receptor), hypogonadotropic hypogonadism (GnRH

receptor) or retinitis pigmentosa (opsin) (Conn et al., 2007). Other illnesses may arise

from mutations in ion channels (e.g., cystic fibrosis transmembrane conductance

regulator, CFTR/ABCC7 in cystic fibrosis) or lysosomal enzymes (e.g. alpha

galactosidase A in Fabry disease, glucocerebrosidase in Gaucher’s disease, acid

sphingomyelinase in Niemann-Pick disease) (Cohen and Kelly, 2003). The latter, so-

called lysosome-storage diseases are a group of approximately 50 rare, inherited

disorders caused by the toxic accumulation of metabolic substances in the lysosome

(Klein and Futerman, 2013). These metabolites would be normally removed, if the

pertinent enzymes were trafficked out of the ER. Apart from loss-of-function diseases,

some proteins may get misfolded beyond the ER or outside the secretory pathway. In

this case, the resulting disorder is characterized by the deposition of toxic aggregates

(gain of function). Such diseases (amyloidoses) include: e.g. Alzheimer’s disease,

Parkinson’s disease, encephalopathies such as Creutzfeldt – Jakob disease and

Huntington disease (Selkoe, 2003).

Interestingly, protein misfolding can also occur in wild type proteins. In particular,

inefficient folding of wild type proteins may be an inherent feature of many GPCRs

(Leidenheimer and Ryder, 2014). For instance, less than a half of rat luteinizing

hormone receptor is able to mature and exit the ER, whereas the rest is degraded (Pietilä

et al., 2005). The same holds true for the human opioid receptor (Petäjä-Repo et al.,

2000) and the A1-adenosine receptor (Pankevych et al., 2003).

From the studies on cystic fibrosis it has been known that glycerol can reverse the

misfolding of CFTR protein caused by the common F508 mutation (Sato et al., 1996).

This activity is not restricted to glycerol. Other chemical chaperones (e.g.: DMSO or

deuterated water) have the ability to nonspecifically promote folding of proteins (Loo

and Clarke, 2007). In addition, it has been long observed that ligand binding increased

the thermal stability of their target proteins (Celej et al., 2003). Along this line, it is not

surprising that small, membrane-permeable orthosteric ligands also stabilize the native

conformation of a protein during folding in the ER. In this case, the surface expression

of folding mutants and their physiological function is restored. These molecules are

referred to as pharmacological chaperones, pharmacochaperones or pharmacoperones

(Leidenheimer and Ryder, 2014). Their main advantage is the high specificity for the

target protein, which allows for the use of low concentrations of the drug and the

reduction of the off-target effects. Additionally, pharmacochaperones promote the

correct re-folding of proteins, which are already retained in the ER. The mechanism of

action by which pharmacochaperones operate is still a matter of debate (Arakawa et al.,

2006). Arakawa et al. propose two modi operandi: The first one proposes that

pharmacochaperones bind preferably to the native-like conformations of the mutated

protein, thus stabilizing it long enough for the protein to escape the scrutiny of the ER

quality control. Accordingly, it was reported that treatment of human -opioid receptor

with opioid ligands promoted the dissociation of receptor intermediates from calnexin

(Leskelä et al., 2007). The second mechanism assumes binding of the

pharmacochaperones to early folding intermediates. This facilitates its folding by

influencing the folding kinetics.

The first study that demonstrated a GPCR rescue by the use of pharmacological

chaperones was performed on folding mutants of V2-vasopressin receptor (Morello et

al., 2000). These mutations occur in X-linked nephrogenic diabetes insipidus and cause

the retention of the V2-receptor in the endoplasmic reticulum. The effect of the mutation

was overcome by the incubation with non-peptidic antagonists. A V2-antagonist

SR49059 (Relcovaptan) was also tested in a clinical trial, where it was administered to

five patients, with a positive outcome (Bernier et al., 2006). Apart from the V2-receptor,

pharmacochaperones were also shown to rescue the folding mutants of: e.g. -opioid

receptor, 1-adrenergic receptor, D4-dopamine receptor, A1-adenosine receptor, opsin

receptor or GnRH receptor (Petajä-Repo et al., 2002; Kobayashi et al., 2009; Van

Craenenbroeck et al., 2005; Malaga-Dieguez et al., 2011; Noorwez et al., 2003;

Janovick et al., 2002, respectively). The application of pharmacochaperones to treat the

misfolding diseases is starting to receive more interest, in particular for the treatment of

lysosomal storage disorders (Parenti, 2009). A clinical trial, for instance, currently

investigates a pharmacochaperone-based therapy of Fabry disease (Giugliani et al.,

2013).

As already mentioned above in section 1.2.2, many wild type GPCRs incur a folding

problem in the ER. Therefore, it can be readily inferred that pharmacological

chaperones may facilitate correct folding, reduce the degradation and increase the

surface expression of these proteins. In fact, this phenomenon was repeatedly reported

for GPCR and non-GPCR proteins (Leidenheimer and Ryder, 2014). This has

repercussions for pharmacotherapy: It has long been known that prolonged treatment of

-adrenergic receptors with antagonists can result in exaggerated responses to

endogenous agonists, if the treatment is suddenly stopped (“propranolol withdrawal

rebound”, see Alderman et al., 1974; Miller et al., 1975), because surface receptor

levels increase (Aarons et al., 1980). Originally, the increase in receptor expression was

attributed to an antagonist-induced inhibition of endocytosis and down-regulation.

Currently, this effect is thought to reflect - at least in part - pharmacochaperoning by

cell permeable antagonists, i.e. orthosteric ligands can assist receptor folding in the ER

by binding to and stabilizing conformational intermediates on the trajectory to the stable

low energy state of the mature receptor (Morello et al., 2000; Nanoff and Freissmuth,

2012).

The correctly folded and glycosylated GPCRs have to exit ER in order to traffic to their

functional destination at the cell surface. This is achieved at the ER exit sites (ERES),

where proteins are sorted and packaged into vesicles by the cytoplasmic coat protein

complex II (COPII) (reviewed in the Venditti et al., 2014). Formation of the COPII coat

starts with activation of the small G protein SAR1 by the guanine nucleotide exchange

factor SEC12. The active, GTP-bound Sar1 associates with ER membrane via its

amphipathic N-terminal -helix, thus promoting membrane curvature. SAR1

subsequently recruits the SEC23/SEC24 heterodimer. The SEC23 moiety of this dimer

is bound to SAR1, the SEC24 moiety acts as the cargo receptor; due to it bow-tie shape

SEC23/SEC24 also contributes to stabilizing membrane curvature by forming the inner

layer of the COPII (coatomer protein II) coat on the nascent vesicle. Mutagenesis

studies indicate that GPCRs interact with the COPII complex through conserved motifs

in their C-termini, specifically in the membrane-proximal region (Dong et al., 2007).

Finally, the pre-budding complex (the cargo-loaded SEC23/SEC24-SAR1 complex) is

stabilized by recruitment of the SEC13/SEC31 heterotetramer. The SEC13/SEC31

complex forms a cage-like outer layer reminiscent of the clathrin-triskelion on the

nascent vesicle and thus induces its fission. Thereafter uncoating of the budded vesicle

is triggered by the GTPase activity of SAR1 and the vesicle is routed to the ERGIC

(ER-Golgi intermediate compartment), from where the cargo is moved to the Golgi

cisternae.

It has to be emphasized that folding and ER export processes are constitutionally

intertwined, as only correctly folded proteins can exit the ER. Moreover, they represent

a rate-limiting step in the biogenesis of GPCRs (Dong et al., 2007). This co-operation of

the folding machinery and COPII complex is elegantly explained by the

chaperone/COPII-exchange model (Keuerleber et al., 2011; Bergmayr et al., 2013).

This model was developed by investigating the fate of the A2A-adenosine receptor and

posits that chaperones of the HSP family engage the C-terminus of the A2A-adenosine

receptor on the cytosolic side of the ER membranes. Specifically, they occupy the C-

terminus, hence preventing the recruitment of COPII components (in particular cargo-

binding protein SEC24) and thus ER export. Once the receptor acquires a correct fold,

the HSP dissociate and the COPII machinery can be recruited. In this model, the

balance between retention mediated by the proteinaceous chaperones and SEC24-

mediated export determines the level of receptors at the cell surface.

Agonist-induced signaling by a given GPCR is subject to feedback control: Chuang and

Costa (1979) have shown that the ability of the 2-adrenergic receptor to re-activate

adenylyl cyclase via Gs protein was greatly reduced within seconds after actiavtion and

this effect persisted for several minutes. If the incubation with the agonist was

prolonged, the receptor disappeared from the cell surface. However, it still resided in the

intracellular compartment, as was demonstrated by radioligand binding. This example

illustrates the classical paradigm of GPCR desensitization, a process of decreased

receptor response upon continuing agonist stimulation (Hausdorff et al., 1990). Initially,

the activated receptor is recognized by one of the seven subtypes of serine/threonine-

specific GPCR kinases (GRKs), which phosphorylate the C-terminus or third

intracellular loop of the receptor (Penela et al., 2006). Two features, agonist-dependent

activation and phosphorylation, are recognized by -arrestins. These adapter proteins

require a minimum of two adjacent phosphorylated amino acids to engage the receptor

either on the cytoplasmic side of the receptor and thus prevent further G protein

interaction (Luttrell and Lefkowitz, 2002). This process occurs at the plasma membrane

and can be rapidly reversed upon the removal of the agonist. Typically, desensitization

is followed by receptor internalization, where -arrestins serve as a platform for the

recruitment of the clathrin-coat (Wolfe and Trejo, 2007). Furthermore, -arrestins also

provide a scaffold for binding of the MAPK pathway kinases, thus eliciting G protein-

independent signaling (Calebiro et al., 2010). During internalization, the arrestin-

receptor complex is directed into clathrin-coated pits, where it undergoes the dynamin-

dependent endocytosis (Hanyaloglu and von Zastrow, 2008). Subsequently, the

receptors that are sequestered from the cell surface are either (i) recycled back to the

plasma membrane (resensitization) or (ii) targeted for degradation in lysosomes

(downregulation) (Drake et al., 2006). The prerequisite for receptor recycling is a

dissociation of -arrestins and dephosphorylation of the receptor; this is accomplished

by the low pH within the endosome, which allows for dissociation of the agonist. The

resulting release of -arrestin renders the phosphorylated residues accessible to

phosphatases. Lysosomal targeting is contingent on receptor ubiquitination.

Adenosine is a purine nucleoside, comprising an adenine base attached to a ribose

moiety. Hence, it serves as a building block of nucleic acids and energy transfer

molecules, e.g. ATP. Already in 1929 it was appreciated that adenosine is also a

signaling molecule: injection of adenosine caused a drop in heart rate (Drury and Szent-

Györgyi, 1929). Today, we know that by signaling through its four receptors (A1, A2A,

A2B and A3) adenosine plays a regulatory role in all cells and tissues of the body,

mediating e.g. vasodilation, cellular hyperpolarization and inhibition of neurotransmitter

release, inhibition of platelets aggregation etc. (Fredholm et al., 2001). In the nervous

system, adenosine is considered a neuromodulator, because it is neither stored nor

released from synaptic vesicles as a classical neurotransmitter (Ribeiro et al., 2003).

However, very recently, adenosine was shown to be present in synaptic vesicles

prepared from rat brain (Corti et al., 2013). Because adenosine affects essentially all

tissues and is formed during hypoxia, Newby introduced the concept of the ‘retaliatory

metabolite’ as a conceptual framework to rationalize the diverse actions of adenosine

(Newby, 1984): this model posits that the actions of adenosine are aimed to protect the

cell against metabolic stress/hypoxia and to restore homeostasis: hypoxia and metabolic

stress result in high tissue levels of adenosine; adenosine is released into the

extracellular space via equilibrative transporters or formed in the extracellular space by

sequential dephosphorylation of ATP and/or ADP (see below). Stimulation of adenosine

receptors results in a concerted response, which dampens energy demand e.g., by

hyperpolarizing cells) and increases energy supply (by vasodilation). In addition,

adenosine also orchestrates long term protective responses by promoting angiogenesis

and by dampening the inflammatory response.

As adenosine exerts its physiological effects via its receptors, it is therefore crucial to

regulate its extracellular level. Adenosine is produced both, within cells and in the

extracellular space (Figure 1). The intracellular pool is generated by (i) a cytosolic 5’

nucleotidase which hydrolyses adenine nucleotides (AMP, ADP and ATP) or to smaller

extent (ii) by S-adenosylhomocysteine hydrolase which is responsible for the

breakdown of S-adenosylhomocysteine (Deussen, 2000). Extracellularly, adenosine

stems from the concerted action of ecto-ATPase (ATP to ADP), ecto-ATP-

diphosphohydrolase (ATP and ADP to AMP) (CD39) and ecto-5’ nucleotidase (AMP to

adenosine) (CD73) which degrade the adenine nucleotides (Zimmerman, 2000).

Nevertheless, adenosine has a very short half-life, as it is rapidly taken up into the cell

through nucleoside transporters and metabolized by adenosine deaminase to inosine or

by adenosine kinase to AMP. Therefore, injection of adenosine only causes a very

short-lived drop in heart rate. This is exploited in the treatment of paroxysmal

supraventricular tachycardia: intravenous administration of adenosine or of its precursor

ATP results in a transient atrioventricular block, which lasts less than 10 seconds but

suffices to terminate the paroxysm and restore sinus rhythm (Eltzschig, 2009; Moser et

al., 1989). Because of its short half-life, adenosine is only active locally as an autacoid

and therefore it does not produce long range actions like a classical circulating hormone

(Newby, 1984).

Any rise in the energy expenditure leads to degradation of ATP and consecutive

generation of adenosine, which in turn can be transported across the cell membrane

through nucleoside transporters. Movement by gradient-dependent passive diffusion is

conducted by the ubiquitous bidirectional equilibrative nucleoside transporters (ENTs,

which belong to the SLC29/solute carrier-29 family). These are further divided by their

sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Boleti et al., 1997). The

second form of adenosine transport is translocation by a Na+-dependent, concentrative

nucleoside transporters (CNTs, which belong to the SLC28 family) (Gray et al., 2004).

Under normoxia, the concentration gradient favors the flow of adenosine from the

extracellular compartment into the cell, where it is metabolized. Accordingly, inhibition

of ENTs, e.g. by dipyridamole, increases extracellular adenosine levels. Overall, the

combined action of metabolizing enzymes and transporters ensures the very low

adenosine levels at physiological conditions, which oscillate in the nanomolar range (40

– 460 nM) (Ballarin et al., 1991).

Adenosine is generated from the degradation of ATP. It diffuses accordingly to its gradient through bidirectional equilibrative nucleoside transporters, which are inhibited by dipyridamole. Adenosine kinase and adenosine deaminase are the major metabolising enzymes of adenosine. The former is inhibited by 5-iodotubercidin, whereas the latter by EHNA. During hypoxia equilibrative nucleoside transporters and adenosine kinase are inhibited. ADO indicates adenosine; INO, inosine; AMP, adenosine monophosphate; ATP, adenosine triphosphate; AK, adenosine kinae; ADA, adenosine deaminase; ATPC, ATP-permeable channel; ENT1, ENT2, equilibrative nucleoside transporter 1, 2. Adapted from Görlach et al. (2005). The situation changes when energy demand exceeds its production and the cellular pool

of ATP (up to ~10 mM) is rapidly depleted, a condition that occurs during metabolic

stress or decrease in oxygen supply (ischaemia/hypoxia) (Linden, 2001). As a

consequence, hydrolysis of ATP translates into the increase of intracellular adenosine

level and its subsequent outflow through the nucleoside transporters. In addition, ATP is

released by damaged cells (Fredholm et al., 2011), erythrocytes (Bergfeld and Forrester,

1992) and upon neurotransmitter release. Released ATP is rapidly hydrolyzed to

adenosine by CD39 and CD73 (see above). Apart from the direct effect on adenosine

generation, hypoxia induces also upregulation of CD39 and CD73 (Eltzschig et al.,

2003), inhibition of adenosine kinase (Decking et al., 1997) and down-regulation of

equilibrative nucleoside transporters ENT1 (Eltzschig et al., 2005). Generally, the

metabolism of hypoxic tissue shifts towards increased production of adenosine, which

rises up to the micromolar range in the extracellular space and thus engages its four

receptors. Literature reports differ on the level of adenosine increase, ranging from 2 to

100-fold (reviewed in Latini and Pedata, 2001). Such discrepancies arise from the level

of oxygen used in the studies, the duration of hypoxia, the type of tissue and – last but

not least - the analytical method employed. As pointed out above, extracellular

adenosine is not exclusively of hypoxic/ischaemic origin: adenosine is formed from

ATP released by neuronal activity; accordingly, it can also be stimulated by

glutamatergic transmission or by KCl-induced depolarization (Latini and Pedata, 2001).

Extracellular adenosine mediates its effects by four receptors: A1, A2A, A2B and A3,

which belong to the GPCR (class A/rhodopsin-like) family. They can be distinguished

by their cognate G proteins and by the effects, which they exert on adenylyl cyclase and

cAMP formation. A1- and A3-receptors inhibit the formation of cAMP by coupling to Gi

and/or Go, whereas A2A-and A2B-receptors stimulate adenylyl cyclase by coupling to Gs;

the A2B-receptor also couples to Gq and thus stimulates IP3-formation. The receptors

vary also in their affinity for endogenous ligand; the A1, A2A and A3 are already

activated by basal levels of adenosine; in contrast, A2B are low-affinity receptors. The

adenosine receptors are antagonized by methylxanthine derivatives, of which the most

conspicuous example is caffeine with the notable exception of the A3-receptor, which

binds methylxanthines with low affinity (Fredholm et al., 1999). Receptor subtypes are

highly conserved among species; only the sequence of the A3-receptor varies

substantially throughout evolution (Fredholm et al., 2001). All receptors but the A2A-

receptor have a palmitoylation site at the C-terminus. Since the determination of crystal

structure of the A2A-adenosine receptor (Jaakola et al., 2008; Dore et al., 2011; Xu et

al., 2011) it has been possible to model the 3D-structures of the other subtypes and

possibly, to design new drugs (Jacobson, 2013).

In my thesis, I focused on the A1-adenosine receptor, as it is responsible for the majority

of the short-term effects that follow a hypoxic insult. The A1-receptor is expressed at

high levels (i.e. 1 pmol/mg) in the brain (cortex, hippocampus, cerebellum) and testis;

intermediate levels (20 to 100 fmol/mg) can be found in skeletal muscles, kidneys,

adipose tissue and liver (Fredholm et al., 2001). Among the adenosine receptor

subtypes, it has the highest affinity for adenosine (Dunwiddie and Masino, 2001). Upon

activation by its agonist, A1-receptor couples to pertussis toxin sensitive G proteins of

the Gi and Go subfamily. The -subunits of these G proteins further translate the signal

by inhibition of adenylyl cyclase, which leads to decreased accumulation of intracellular

cAMP (Freissmuth et al., 1991). Apart from that, Gi coupling may also (i) activate

ATP-sensitive potassium channels in cardiac myocytes (Kirsch et al., 1990) or (ii)

inhibit Q, P, N-type Ca2+ channels, as shown in rat cultured neurons (Scholz and Miller,

1991). The signal generated by the A1-receptor is also transduced by the -complex of

its cognate G proteins. Accordingly, the -subunits might (i) activate phospholipase

C 2 and 3, which leads to release of Ca++ from intracellular stores (Akbar et al., 1994)

or (ii) activate the G protein-gated inward rectifier K+ channels (Kofuji et al., 1995).

The A1-adenosine receptor is comprised of 326-amino-acids. A short C-terminus (36-

amino acids) with a palmitoylation site at Cys309 is responsible for stabilizing helix-8

(proximal region of the C-terminus) in the plasma membrane (Libert et al., 1992).

Mutations in the conserved NPxxY(x)5,6F sequence of helix-8 or its truncation result in

the retention of the receptor in the endoplasmic reticulum, thus reducing the number of

functional receptor at the cell surface (Malaga-Dieguez et al., 2010; Pankevych et al.,

2003).

Adenosine signaling which increases substantially during hypoxia, ischaemia or

inflammation is aimed at restoring the metabolic homeostasis and enhancing tissue

repair. Generally, the receptor-mediated short-term mechanisms (via high-affinity A1-

and A2A-receptors) increase oxygen supply and/or decrease oxygen demand (e.g.

vasodilation, reduction of neurotransmitter release and neuronal firing rate) and in the

long-term (predominantly via the A2A- receptor and low-affinity A2B-receptor) they

regenerate the injured tissue (e.g. reduction of inflammation, stimulation of

angiogenesis and cell proliferation) (Fredholm, 2007). The examples given below

describe the main axes of adenosine-induced protection that correspond with the

retaliatory metabolite concept.

Lack of oxygen in the brain tissue, usually caused by an ischaemic insult (e.g. stroke),

starts a cascade of events, which lead to neuronal cell death (Bickler and Donohoe,

2002). Inhibition of oxidative phosphorylation results in a decrease of ATP levels,

which further translate into stopping of Na+/K+ pump and in the end, depolarization of

the cell membrane. These changes initiate influx of Na+ and Ca2+ and release of

glutamate into the extracellular space. Subsequent activation of NMDA receptors

triggers further Ca2+ influx and thus aggravates the damage via the glutamate cascade

(excitotoxicity). In effect, neurons die from swelling, necrosis or apoptosis (Lipton,

1999). In order to survive, mechanisms evolved which limit neuronal energy demand.

The neuroprotective action of adenosine is elicited via the A1-receptor and encompasses

two major mechanisms. (i) Presynaptically, adenosine signaling inhibits release of

excitatory neurotransmitters (mainly glutamate) through mechanism that limits Ca2+

entry (Fredholm et al., 2005a). (ii) At postsynaptic sites, increased K+ conductance

causes hyperpolarization and thus inhibits activation of NMDA receptors (Dunwiddie

and Masino, 2001). Along this line, adenosine has also beneficial anticonvulsant effect

in epilepsy experimental models and protects against seizure-dependent cell death

(Boison, 2006). In contrast, it is not activation but blockade of the A2A-receptor

signaling by antagonists, which elicits a neuroprotective effect (Phillis, 1995; Behan and

Stone, 2002). Interestingly, studies performed in newborn rats show that the effect of

A1-receptor signaling is detrimental on neuronal survival (Turner, 2004). This may

reflect a differential mechanism of neuronal cell death in adult and newborn animals

(Fredholm et al., 2005).

Adenosine administered intravenously is used in the clinic as an antiarrhythmic drug for

the treatment of supraventricular tachycardias (Mitchell and Lazarenko, 2008). Its

protective effect in the heart is elicited predominantly by the A1-receptor signaling in

the sinus node and the atrioventricular node (Bellardinelli et al., 1989). Upon activation,

A1-receptor induces negative chrono-, dromo- and inotropic effects. In addition,

signaling by A2A and A2B-receptor induces vasodilation in most of the vascular beds,

except for renal arteries, where adenosine acts as a vasoconstrictor (Osswald, 1975).

Other vascular-related effects include inhibition of platelet aggregation and inhibition of

P-selectin expression via A2A-receptor (Sandoli et al., 1994; Minamino et al., 1998).

Another cardioprotective (occurs also in neuronal tissue) response triggered by

adenosine is the phenomenon of ischaemic pre-conditioning (Lankford et al., 2006;

Gidday, 2006). Briefly, short periods of sublethal ischaemia induce a tolerance to

subsequent episodes of decreased oxygen content. In the original study of Murry et al.,

a 75% reduction in histological infarct size was observed after 40 minutes of ischaemic

episode (Murry et al., 1986). The mechanism is still not fully understood, however A1-

and A3-receptor signaling is involved (Thornton et al., 1992; Liu et al., 1994).

Interestingly, the pre-conditioning effect can also occur in a remote organ, which was

not subjected previously to ischaemic insult (remote pre-conditioning) (Schulte et al.,

2004).

In the inflamed and/or hypoxic tissue, injured cells together with activated immune cells

release ATP into the extracellular space. Its degradation by CD39/CD73 pathway and

the resulting adenosine accumulation promotes in turn anti-inflammatory and

immunosuppressive effects (Ohta and Sitkovsky, 2001). These effects are mediated

mainly via A2A-receptors expressed on various immune cells (Sitkovsky et al., 2004).

For instance, neutrophils exhibit reduced phagocytosis, degranulation, oxidative burst

and leukotriene formation upon activation of the A2A-receptor (Cronstein, 1994).

Similarly, in the presence of A2A-receptor agonists, monocytes release less TNF- and

lymphocyte activation is reduced (Link, 2000; Lappas et al., 2005). Activation of A2A-

receptors is beneficial in e.g. experimental rheumatoid arthritis (Floegel et al., 2012) or

lung inflammation (Reutershan et al., 2007). Accordingly, A2B-receptor activation also

dampens immune response in e.g. inflammatory bowel disease (Frick et al., 2009) or

sepsis (Csoka et al., 2010). However, A2B-agonists were found pro-inflammatory in

asthma models (Hasko et al., 2009). Therefore, the final outcome of the A2B-receptor

signaling depends on the cell type (Ham and Rees, 2008). In general, adenosine acting

via the Gs-coupled adenosine receptors stops inflammation and protects the remaining

healthy tissues (Sitkovsky and Ohta, 2005).

Apart from immediate effects that restore tissue oxygenation, adenosine can also ensure

long-term oxygen supply by stimulation of angiogenesis (Adair, 2005). Angiogenesis is

contingent on the proliferation of endothelial cells and their subsequent sprouting.

Endothelial cells express A2A-receptors; their stimulation promotes endothelial cell

proliferation (Sexl et al., 1995) via a Gs-independent signaling pathway, which leads to

activation of MAP kinase via RAS (Sexl et al., 1997; Seidel et al., 1999). This effect

also operates in vivo as shown in a wound healing studies (Montesinos et al., 2004).

Possibly, suppression of thrombospondin 1 also plays a role (Desai et al., 2005). In

addition, A2B-receptor also contributes to angiogenesis, because it stimulates the

secretion of VEGF and interleukin-8 in endothelial and mast cells (Feoktistov et al.,

2002 and 2003) and endothelial cell proliferation (Dubey et al., 2002). Stimulation of

A1-receptor on monocytes may also be relevant to angiogenesis because this enhances

the release of VEGF (Clark et al., 2007). These long term effects of endogenous

adenosine are beneficial in hypoxia and inflammation; however, they can be exploited

by tumor cells: solid tumors are hypoxic; hence the accumulation of extracellular

adenosine may contribute to angiogenesis and thus promote tumor growth. Similarly,

tumor cells can capitalize on the increased adenosine levels, because they dampen the

immunological response within the tumor (Spychala, 2000).

In the retaliatory metabolite concept, adenosine is a two-faceted molecule (Newby et

al., 1984): it is a signal of metabolic distress and its concentration rises in proportion to

energy consumption. Secondly, adenosine has a beneficial aspect: when released into

the extracellular space, adenosine dampens cellular metabolism by acting on inhibitory

A1-adenosine receptors and thus counteracts the impact of hypoxia. My working

hypothesis is based on this retaliatory metabolite concept. In addition, it relies on the

well established fact that pharmacochaperoning of GPCRs by their orthosteric and

allosteric ligands enhances the rate at which these proteins fold and thus enhances their

cell surface expression. The higher density of receptors at the plasma membrane

augments the magnitude of the cellular response by increasing Bmax or by shifting the

concentration-response curve of agonists to the left.

Thus, the working hypothesis of my thesis posits that, within the cell, endogenously

produced adenosine acts as a pharmacological chaperone on its cognate A1-receptor and

promotes its folding. This hypothesis predicts that pharmacochaperoning by

endogenous adenosine should occur in the physiologically relevant context, namely

during hypoxia. Accordingly, I verified my hypothesis in a step-wise fashion by several

independent approaches:

1. I created a condition that results in intracellular accumulation of adenosine and

tested the pharmacochaperoning activity of adenosine by using a folding-

deficient A1-receptor mutant as a folding sensor.

2. I examined if the effect was specific for the A1-adenosine receptor.

3. I determined if the effect was also observed for the wild type A1-receptor.

4. I confirmed that hypoxia led to an increase in surface levels of A1-receptors and

linked this to adenosine accumulation.

Results of this chapter are presented in the form of my first-author manuscript published

in the journal ‘Molecular Pharmacology’.





I, Justyna Kusek hereby declare that the work presented in the following publication is

largely my own. Contributions of each author of the manuscript are specified as

follows: Cloning of FLAG-A1-R, A1R-YFP and A1R-Y288A-YFP constructs was carried

out by Laura Malaga-Dieguez. Mutant V2R constructs were a generous gift of Ralf

Schülein (Leibniz Institute for Molecular Pharmacology, Berlin). Subcloning of these

constructs was performed by Edin Ibrisimovic and Qiong Yang.

The stable cell line expressing Flag-A1-R was generated by Erjola Musaraj. Qiong Yang

created cell lines stably expressing Flag-V2-I318S and V2-T273R-GFP receptors.

Martin Witek performed the HPLC analysis.

I carried out all remaining experiments presented in the publication. I prepared all

figures presented in this publication, except for Figure 5B and supplementary Figure 4,

which was done by Dr. Michael Freissmuth.

Dr. Michael Freissmuth, Dr. Christian Nanoff and Dr. Christian Gruber contributed to

research design, data analysis and the writing of the publication mentioned above.

β

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Fig. 1

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SR 121463 inhibitors c.

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Suppl. Fig. 1

Kusek J., Yang Q., Witek M., Gruber C.W., Nanoff C. and Freissmuth M. Chaperoning of the A1-adenosine receptor by endogenous adenosine – an extension of the retaliatory metabolite concept. Molecular Pharmacology

Supplementary Figure 1. Detection of intracellular adenosine accumulation in HEK293 cells subjected to a combined inhibition of adenosine kinase, adenosine deaminase and adenosine transport or to hypoxia. HEK293 cells were grown in medium (DMEM) containing 10% FCS until they were about 80% confluent; subsequently they were incubated in the absence of FCS for 4 h (B) or 16 h (C) with a combination of 2 μM EHNA, 0.5 μM 5-iodotubercidin and 10 μM dipyridamole (inhibitors c.) or under hypoxic conditions (5% O2) for 1 h (D). After the incubation, the cells were washed with phosphate-buffered saline (PBS); the cells were lysed by addition of 2.5% perchloric acid (1 ml/ 10 cm dish). The cells were scraped off the plate and transferred to an Eppendorf tube containing 30 μl EHNA (3.3. mM) and idotubercidin (0.67 mM) to prevent the enzymatic degradation of adenosine. The samples were sonicated for 3 min on ice to extract the intracellular content and centrifuged at 4°C for 5 min at 7,000 g. The supernatants were snap-frozen in liquid nitrogen and neutralized after thawing with 4.2 M KOH, followed by centrifugation at 4°C for 15 min at 16,000 g. The supernatants were freeze-dried. The lyophilized material was taken up in 0.2 ml H2O and immediately applied to the HPLC (Hitachi La Chrom system equipped with a L7100 pump, L7200 autosampler and L7455 UV detector). Separation was done on a Zorbax SB-Aq column (Agilent, 250 mm × 4.6 mm ID, 5 μm) and eluted in 0.2 M ammonium acetate (solvent A) and acetonitrile (solvent B). The following gradient was applied using a flow rate of 1.5 ml/min: 0 min, 0% B, 0-6.5 min, 20% B; 6.5–8 min, 80% B; 8–8.1 min, 95% B; 8.1-9.9 min, 95%; 9.9-10 min, 0% B. A) The whole analytical RP-HPLC chromatogram of a sample from untreated cells spiked with adenosine as a standard (the other panels represent only a part of the full chromatogram, for the sake of clarity). B, C, D) Overlayed chromatograms of untreated (red line) and treated samples (black line). E) Overlayed chromatograms of untreated (red line), untreated spiked with adenosine standard (200 ng/ml) (black line) and adenosine standard only (200 ng/ml) sample (grey line). The chromatograms are representative of at least three independent experiments. Of note, there was an increase in cAMP upon treatment of cells with the combination of inhibitors. This reflects the activation of A2B-receptors, which are endogenously expressed in HEK293 cells, by the accumulation of adenosine.


Suppl. Fig. 2

A1R

Gβ

55

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A B


Supplementary Figure 2. Effect of the adenosine transport inhibitor NBMPR on the accumulation of mature A1-adenosine receptor-Y288A. HEK293 cells stably expressing A1-receptor-Y288A fused to YFP were incubated for 24 h with vehicle (lane labeled untreated), 0.5 μM iodotubercidin and 2 μM (EHNA/IODO) and a combination of 0.5 μM iodotubercidin, 2 μM EHNA and 10 μM NBMPR (EHNA/IODO/NBMPR). A) Membranes (20 µg/lane) prepared from these cells were separated electrophoretically and the A1-receptor level was detected by immunoblotting for YFP. Gβ-subunits were visualized as a loading control. B) Receptor levels were determined by incubating membranes (10 µg) prepared from these cells with a single concentration of [3H]DPCPX (5 nM). Shown is the specific binding. Non-specific binding was determined in the presence of 10 µM XAC (<20% of total binding) and subtracted from total binding to give specific binding.


A

B

C

Suppl. Fig. 3

Supplementary Figure 3. Confocal images of HEK 293 cells stably expressing A1-receptor-Y288A. Cells were incubated for 24 h in the presence of (A) vehicle, (B) with 1 μM DPCPX or (C) 2 μM EHNA, 0.5 μM iodotubercidin and 10 μM dipyridamole and the receptor visualized via the fused YFP moiety. Images were captured with identical pinhole settings and processed in parallel (with the same settings for brightness and contrast); the scale bar indicates 5 or 10 μm. The right hand panels represent a quantification of the pixel distribution along a line bisecting individual cells (n=10/condition).


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total bindingnon-specific binding (XAC 10 µM) dipyridamoleiodotubercidinEHNA comb. inhibitors

Suppl. Fig. 4

Supplementary Figure 4. Direct effect of 5-iodotubercidine, EHNA and dipyridamole on radioligand binding to A1-adenosine receptor. Membranes from HEK293 cells stably expressing A1-receptor fused to YFP (10 µg per assay) were pre-incubated for 30 min with adenosine deaminase (ADA, 1 unit/ml) to deplete residual adenosine. Subsequently, the membranes were incubated with [3H]DPCPX (11 nM) and the inhibitors at the indicated concentration (dipyridamole: 1, 10 and 100 µM; 5-iodotubercidin: 0.05, 0.5 and 5 µM; EHNA: 0.2, 2 and 20 µM) or their combination (2 μM EHNA, 0.5 μM iodotubercidin and 10 μM dipyridamole) for 1 h at 20°C. Non-specific binding was defined by the antagonist XAC (10 µM). Data are means ± S.E.M from three separate experiments, performed in duplicates. None of the inhibitors caused appreciable inhibition of binding up to the concentration employed for the pharmacochaperoning experiments, i.e., 10 µM dipyridamole, 0.5 µM iodotubercidin and 2 µM EHNA. The same is true for the combination of inhibitors. Modest inhibition (by about 10% was only observed at 10-fold higher concentrations, i.e., 20 µM EHNA and 100 µM dipyridamole.

[3 H] D

PCPX

bou

nd (c

pm)

untreate

d

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EHNA DIP

0

1000

2000

3000

4000

5000

6000


Suppl. Fig. 5

Supplementary Figure 5. Effect of 5-iodotubercidin, EHNA or dipyridamole on the accumulation of A1-adenosine receptor-Y288A detected by radioligand binding. A) HEK293 cells stably expressing A1-receptor-Y288A fused to YFP were incubated for 24 h with vehicle (untreated), 1 μM DPCPX, 0.5 μM 5-iodotubercidin, 2 μM EHNA or 10 μM dipyridamole. Receptor levels were determined by incubating membranes (10 µ g) prepared from these cells with a single concentration of [3H]DPCPX (5 nM). Shown is the specific binding. Non-specific binding was determined in the presence of 10 µM XAC. Data are means ± S.E.M from 2 independent experiments, performed in duplicates.

1 2 4 8 24 1 2 4 8 24 incubation

time

hypoxia normoxia

A1R

Gβ

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55

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tio M

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Suppl. Fig. 6

Supplementary Figure 6. Time-dependent effect on the accumulation of mutant A1-receptor-Y288A upon hypoxic incubation. A) HEK293 cells stably expressing A1-receptor-Y288A fused to YFP were incubated for 1, 2, 4, 8, or 24 hours under hypoxia (5% O2, left) or normoxia (right). The membranes (20 µg/lane) prepared form these cells were electrophoretically resolved and the receptor detected by blotting for the YFP-moiety (upper blot). Immunodetection of G protein β-subunits served as loading control (lower blot). B) The bar diagram shows the densitometric quantification of the receptor levels, analyzed by ImageJ software. The relative optical density of mature (M, upper) and core (C, lower) glycosylated species was determined and presented as the M:C ratio. Data are means from two independent experiments, error bars represent S.E.M.

SR 121463 5% O2

fold

ove

r con

trol

untreated hypoxia 1 h hypoxia 2 h0.0

0.5

1.0

1.5

55

[kDa] ctrl 1h 2h

5% O2

V2R

Gβ

A B

C


Suppl. Fig. 7

Supplementary Figure 7. Effect of hypoxia on the accumulation of the folding-deficient V2-vasopressin receptor mutants. A) HEK293 cells stably expressing V2-receptor-T273R-GFP (A, B) were incubated under normoxic or hypoxic (5% O2) conditions for 1 or 2 h. The membranes (10 µg/lane) prepared form these cells were electrophoretically resolved and the receptor detected by blotting for the YFP-moiety (upper blot), whereas G protein β-subunits detection served as loading control (lower blot). B) The bar diagram shows the densitometric quantification of the receptor levels, analyzed by ImageJ. The pixel density of the receptor band (~ 55 kDa) was determined and normalized by setting the mean density observed in untreated control cells as 1. Data are means from two independent experiments, error bars represent S.E.M. C) HEK293 cells stably expressing N-terminally Flag-tagged V2-receptor-I318S were incubated under normoxia, with 5 μM SR121463A or under hypoxia (5% O2) for 24 hours. The receptors surface expression was determined by quantifying fluorescence intensity (emitted by Alexa Fluor® 488-labelled secondary antibody against the primary anti-FLAG antibody), by flow cytometry. Shaded histograms represent normoxic samples and open histograms indicate the treatment with a pharmacochaperone (SR121463A, left) or hypoxia (right). The experiment was performed in duplicates.

The concept of pharmacological chaperones - i.e. low molecular weight ligands that

assist folding of their respective proteins - is grounded on solid experimental evidence

(Morello et al., 2000; Loo and Clarke, 2007). These exogenous compounds may bind

ortho- or allosterically and in most cases are of the synthetic origin. Recently,

Leidenheimer and Ryder pointed out a post-translational regulatory mechanism, where

endogenous ligands could act as “cognate ligand chaperones”, thus facilitating folding

of their cognate proteins (Leidenheimer and Ryder, 2014). In our work, I developed this

idea by showing that endogenous adenosine can chaperone its cognate A1-adenosine

receptor in the physiological context.

The working hypothesis was based on the findings demonstrating that hypoxia caused

intracellular accumulation of adenosine (Latini and Pedata, 2001), decreased the activity

of nucleoside transporters ENT1 (Chaudary et al., 2004) and inhibited adenosine kinase

(Decking et al., 1997). Moreover, blocking of the enzymes that metabolize adenosine

led to the increase of its content. For instance, perfusion of isolated guinea-pig hearts

with EHNA or iodotubercidin resulted in the elevated adenosine level in epicardial and

venous fluid (Ely et al., 1992). Accordingly, in the present work, the increased

intracellular level of adenosine was achieved by concerted inhibition of adenosine

deaminase, adenosine kinase and equilibrative nucleoside transporters or by

withholding oxygen from the cell culture atmosphere (i.e, by hypoxia). In both

instances, the intracellular accumulation of adenosine was documented by HPLC. I

determined that this accumulation of endogenous ligand caused upregulation of the A1-

adenosine receptor at the cell surface, similarly to the action of the A1-antagonist

DPCPX. For this purpose, I employed (i) an ER-retained receptor with a mutation in the

conserved NPxxY(x)5,6F motif as a folding sensor and (ii) the wild type receptor.

Importantly, it was crucial to block the equilibrative nucleoside transporters with

dipyridamole or NBMPR, as otherwise the mere blocking of adenosine deaminase and

adenosine kinase, did not translate into the accumulation of the A1-receptor. The

accumulated receptor had mature glycosylation pattern and was binding-competent,

with a radioligand affinity comparable to that of the wild type receptor. Flow cytometry

and confocal microscopy confirmed that the up-regulated receptor resided at the cell

surface. A possible non-specific (off-target) effect of the inhibitors was ruled out,

because they did not cause any upregulation of the mutated V2-vasopressin receptor,

which, however, was subject to pharmacochaperoning by a cognate ligand, the V2-

antagonist SR 121463. Hypoxia recapitulated the effect, which the combination of

inhibitors elicited on the surface levels of both, mutated and wild type A1-receptor.

Intracellular adenosine generated in this manner up-regulated the mature A1-receptor,

whereas it did not affect the expression level of the V2-vasopressin receptor.

I conclude that the observed accumulation of the A1-adenosine receptor at the cell

surface is caused by the pharmacochaperoning effect of the endogenous adenosine.

Several lines of evidence support this conclusion: (i) Accumulation of endogenous

adenosine enhanced the surface expression of the A1-adenosine receptor, but not of the

other GPCR representative that is responsive to pharmacochaperoning (V2-vasopressin

receptor). (ii) The up-regulated receptors were located on the cell surface, had mature

glycosylation pattern and were binding-competent. (iii) The cocktail of inhibitors did

not inhibit the ER associated degradation (ERAD); in fact, blockage of ERAD with

kifunensine resulted in the accumulation of the immature, binding-incompetent receptor

species, but did not suffice to enhance surface levels of the receptor. (iv) The cocktail of

inhibitors did not affect the rate of the transcription: the experiments were performed in

the heterologous system, where the expression of both, the A1-receptor and the V2-

receptor was driven from a CMV promoter. In addition and more importantly, the

cocktail of inhibitors also up-regulated the A1-receptor, even when protein synthesis

was blocked by cycloheximide.

Upon inhibition of ER associated degradation by kifunensine (cf. Figure 2B), a core-

glycosylated and binding-incompetent species of the A1-Y288A-receptor accumulated.

This observation is consistent with earlier findings, which documented that - even in the

case of the wild type receptor – a large fraction was trapped in the ER in an apparently

misfolded state, which failed to bind a radioligand antagonist (Pankevych et al., 2003)

In fact, protein misfolding and degradation has been documented in several wild type

GPCRs (see section 1.2.2). Presumably, it is an inherent feature of the folding process

and its stochastic nature that trap a part of the forming proteins in incorrect

conformation. As cycloheximide treatment largely reduced the number of core-

glycosylated, ER-bound receptors, we propose that pharmacochaperones such as

DPCPX or endogenous adenosine act on the newly synthesized receptor.

Many studies showed that membrane-permeable ligands (agonists or antagonists)

rescued the cell surface expression of the retained GPCR mutant and wild type

receptors, e.g. -opioid, V2-vasopressin, dopamine D4 or bradykinin B1 receptor

(Leskela et al., 2007; Wüller et al., 2004; Van Craenenbroeck et al., 2005; Fortin and

Marceau, 2006; respectively). The present findings are consistent with these earlier

studies; they also conform and extend the work of Pankevych et al. (2003) and Malaga-

Dieguez et al. (2010). From a mechanistic perspective, I propose that adenosine

(similarly to the antagonist DPCPX) stabilized folding intermediates of the A1-receptor

and thus prevented their recognition by ER quality control and ERAD proteins. It is also

attractive to speculate that binding of adenosine to the newly folded A1-receptor

promotes its dissociation from proteinaceous chaperones. In fact, several studies have

shown that administration of pharmacological chaperones interrupts the interaction of

calnexin with rhodopsin (Noorwez et al., 2009), the V3-vasopressin (Robert et al., 2005)

or the -opioid receptor (Leskela et al., 2007) in the ER. The maturating GPCR proteins

may also interact with cytosolic chaperones that bind to their C-terminus and probe the

folding state (section 1.2.3). Interestingly, the overexpressed Hsp40 protein DRiP78

was shown to retain the mutated D1-dopamine, M2-muscarinic (Bermak et al., 2001)

and A1-adenosine receptors (Malaga-Dieguez et al., 2010) in the endoplasmic

reticulum.

Chaperoning action of endogenous ligands on the folding of their cognate proteins was

already reported: e.g., intracellular GABA treatment enhanced the expression the wild

type GABAA-receptors and rescued a mutated version of the receptor that was retained

within the secretory pathway (Eshaq et al., 2010). Moreover, the retinol-binding protein

was up-regulated upon attachment of retinol in the ER (Melhus et al., 1992) and choline

treatment facilitated the maturation of recombinant 4 2 nicotinic acetylcholine

receptor (Sallette et al., 2005). There is one previous example, which suggests that

endogenous agonists (or fragments thereof) may also operate as pharmacochaperones on

GPCRs. Dopamine can exert a pharmacochaperoning effect on dopamine D4 receptors

(DRD4) in cells, where its intracellular accumulation is supported by means of the

dopamine transporter/SLC6A3 (Van Craenenbroeck et al., 2005). Cells co-expressing

the wild type and the M345T folding mutant of DRD4 together with the dopamine

transporter showed the increased receptor levels at the cell surface, upon pre-treatment

with dopamine. However, in vivo, the dopamine transporter in the plasma membrane

operates in a relay with the vesicular monoamine transporter, which sequesters the

dopamine in the vesicles. This arrangement keeps the level of free intracellular

dopamine low. It is therefore questionable that there is a physiological situation, where

dopamine accumulates to levels capable of pharmacochaperoning one of its cognate

receptors during their synthesis in the ER. In contrast, intracellular adenosine increases

rapidly upon hypoxic insult. This creates a condition where ligand and its cognate

protein share a spatial proximity. Therefore, to the best of my knowledge, this is the

first demonstration of GPCR pharmacochaperoning by endogenous ligand in a

physiologically relevant context.

As already outlined in section 1.3 of this thesis, adenosine functions as a retaliatory

metabolite. I propose a ramification of this concept: adenosine not only activates its four

receptors and thus induces the protective responses; it also promotes the folding and

surface expression of A1-adenosine receptor (Figure 2). My data showed that

intracellular adenosine, which had been generated by inhibition of its metabolising

enzymes and transport or by hypoxia, pharmacochaperoned the A1-adenosine receptors.

I surmise that this action may include other adenosine receptors. In fact, this conjecture

is supported by earier observations: anoxia of 3-6 hours increased the cell surface

expression of A2A-adenosine receptors in undifferentiated PC-12 cells (Arslan et al.,

2002). The authors concluded that this change was not attributable to increased

transcription or translation but resulted from receptor re-distribution, because adenosine

receptors were translocated from intracellular compartments to the cell membrane. In

hindsight, I interpret the data as to indicate that anoxia elicited accumulation of

intracellular adenosine that facilitated folding and ER exit of the A2A-receptors.

Intracellular accumulation of adenosine (ADO) during hypoxia facilitates folding and stabilizes the correct conformation of the A1-adenosine receptor (A1R) in the endoplasmic reticulum. In effect, a higher number of mature receptors exit the ER and reach the cell surface. As a consequence, increased density of A1-receptors at the plasma membrane translates into enhanced A1-signaling, thus affording the protection against hypoxia.

Adenosine-induced pharmacochaperoning is an extension of the retaliatory metabolite

concept: it introduces an intrinsic positive feedback loop mechanism, which magnifies

the action of accumulating adenosine. Possibly this mechanism operates most

effectively in the tissues most susceptible to hypoxic insults. In this scenario, sudden

oxygen deprivation induces depletion of the cellular ATP pool (millimolar

concentration) and subsequent accumulation of intracellular adenosine. Adenosine

promotes the ER exit and surface expression of those A1-receptors, which reside in the

ER. Protein folding is a very rapid process; the conformational space is explored by

folding trajectory over a timescale of seconds to minutes; thus adenosine may act as a

chaperone before diffusing out of the cell along its gradient, while nucleoside

transporters are still available. Accordingly, in my experiments upregulation of the A1-

receptor was also detected by immunoblotting and by radioligand binding after 1 hour

of hypoxia. Longer periods of hypoxia did not increase the effect, in contrast to the

treatment with EHNA, iodotubercidine and dipyridamole: here, the A1-receptor

accumulated over time due to constant inhibition of adenosine deaminase, adenosine

kinase and nucleoside transporters, respectively. It was shown that longer oxygen

deprivation (8 – 20 h) decreased the activity of ENT1, possibly via PKC mechanism

(Chaudary et al., 2004) and caused down-regulation of adenosine kinase (Decking et

al., 1997). These changes would contribute to accumulation of intracellular adenosine.

Therefore, I propose that adenosine pharmacochaperoning occurs predominantly at the

beginning of the hypoxic phase and again, when the nucleoside transporters and

adenosine kinase are inhibited and down-regulated. The second phase of chaperoning

could possibly account for the the loss of the internalized receptors from the plasma

membrane and sustain the A1-signalling.

In my work, I observed an upregulation of the wild type A1-receptor by about 20% after

24 hours incubation under hypoxia. It is intuitively expected that such effect is

beneficial and contributes to tissue protection: for instance, cardioprotection afforded by

A1-receptor signaling depends on the number of these receptors (Dougherty et al.,

1998). In fact, overexpression of the A1-receptor leads to enhanced protective response

against hypoxia, as shown for transgenic mouse hearts (Matherne et al., 1997).

Accordingly, upregulation of A1-receptor by endogenous adenosine or other orthosteric

ligands is expected to have a beneficial effect in case of hypoxia/ischaemia. Evidence in

support of this conjecture is available: chronic administration of the antagonist DPCPX

protected hippocampal cells from global forebrain ischaemia (von Lubitz et al., 1994).

Similarly, other studies reported the protective effect of chronic administration of

caffeine (or other adenosine receptor antagonists), in contrast to acute treatment

(reviewed in Jacobson et al., 1996). This effect was linked to the upregulation of A1-

receptors; I propose that this upregulation resulted from the pharmacochaperoning

action of the antagonists.

In this thesis, I focused on the anterograde trafficking of the A1-receptor through the

secretory pathway; I did not study the extent to which the A1-receptor was subject to

internalization. Desensitization and internalization of the A1-adenosine receptor has

been the subject of much debate (reviewed in Mundell and Kelly, 2011 and Klaasse et

al., 2008). Hypoxic treatment produced variable results on receptor expression (Rebola

et al., 2003; Coelho et al., 2006; Castillo et al., 2008; Hammond et al., 2004). In

general, these studies concentrated on the long-term changes of A1-receptor density

(>12 hours), which can be also influenced by neuronal degeneration and not only

internalization rate. The current view is that desensitization of the A1-receptor is slow

when compared to other adenosine receptors. This can be rationalized by taking into

account that the intracellular portions of the A1-receptor are devoid of the multiple

serine and threonine residues, which are required for phosphorylation by GRKs. In the

absence of multiple phosphorylation sites, recruitment of arrestins and arrestin-

dependent desensitization cannot occur.

According to the current view on pharmacological chaperoning of membrane proteins,

the ligands have to permeate the ER membranes in order to exert their function. This

notion comes from the fact that the binding pocket of the maturating proteins is located

in vicinity to the luminal side of the ER. Therefore, it is not clear how dopamine or

choline (both are hydrophilic molecules) can chaperone the DRD4 and 4 2 nicotinic

acetylcholine receptor, respectively. One of the explanations is the presence of

transporters in the ER membranes, as has been shown for a mutated, ER-resident GAT-

1 transporter. This protein transfers GABA as efficient as the wild type protein (Scholze

et al., 2002). In order to exert this function, the transporter would have to work in a

reverse manner, which is known for the glutamate, dopamine or equilibrative nucleoside

transporters (Kanai et al., 2013; Mortensen and Amara, 2003; Gu et al., 1995;

respectively). Along this line, adenosine could enter the ER lumen by means of active

transport. In fact, the equilibrative nucleoside transporter-3 (ENT3/SCL29A3) is located

to intracellular membranes (Baldwin et al., 2005); as a consequence it could possibly

work in the ER under our experimental conditions.

Secondly, the ER membranes are considered to be more permeable than other cellular

membranes (Le Gall et al., 2004): isolated ER vesicles and ER membranes allow for the

permeations of small (< 5kDa), charged molecules. In addition, ATP is present in the

ER, but it is not clear how it is transported from the cytosol. Recently, it has been

shown that ATP transport to ER is Ca2+-controlled (Vishnu et al., 2014). One could

speculate that adenosine uses similar way of entry as ATP, is formed by hydrolytic

cleavage from ATP in the ER or it diffuses passively through the ER membranes.

The third explanation assumes that endogenous adenosine binds to the nascent A1-

receptor in the ER from the cytosolic site. Molecular dynamics simulations of of

rhodopsin (Grossfield et al., 2008) and of the 2-adrenergic receptor (Romo et al., 2010)

demonstrate that the core and binding pocket of these GPCRs is significantly hydrated.

For instance, in rhodopsin, the bulk water rather than the structural water plays a role in

hydrolytic cleavage of the chromophore (Jastrzebska et al., 2011). Similarly, internal

water has been also found in the structure of adenosine A2A receptor (Piirainen et al.,

2011; Xu et al., 2011). This structural feature is supposedly shared by other rhodopsin-

like GPCRs as water molecules enter the core of the protein through the highly

conserved NPxxY and D(E)RY motifs on the cytoplasmic site (Grossfield et al., 2008).

Therefore, it is conceivable that adenosine penetrates the A1-adenosine receptor through

the hydrated core, induces corrective re-arrangement of the side-chains and finally

occupies the binding pocket.

In summary, this work provided a proof that endogenous adenosine can chaperone the

A1-adenosine receptor and enhance its cell surface expression. Moreover, this

mechanism extends the concept of adenosine as the retaliatory metabolite and

contributes to its protective effect. Last but not least, it opens new directions in studying

the role of endoplasmic reticulum and intracellular ligand-receptor interactions.

The endoplasmic reticulum has been suggested to serve as a storage reservoir of

folding.competent intermediates (Leidenheimer et al., 2014). Such a pool of proteins

could be easily released and traffic to the cell surface upon the receiving a metabolic

signal in form of its ligand. This would serve as a positive feedback loop mechanism for

the ligand-receptor systems which evolved to react to sudden changes in the

environment in order to restore homeostasis in the afected tissue. For example,

prostaglandin E2 receptor, EP2 is up-regulated after the inflammation in the tissue (Kras

et al., 2013), which may be also account for - in addition to changes in transcription - by

the pharmacochaperoning action of the prostaglandin E2 in the endoplasmic reticulum.

A similar action may also be conceivable for the G protein-coupled estrogen receptor

GPR30 (Filardo and Thomas, 2012) or endogenous metabolites which reach high

concentration levels (e.g. fatty acids, lactate, ketone bodies, succinate, bile acids) and

act on the group of recently deorphanized GPCRs (GPR40 and GPR120, HCA

receptors, GPR109a and GPR109b, GPR91 and TGR5, respectively). These receptors

have evolved to sense the levels of substrates or intermediates of energy metabolism

and to regulate metabolic functions, accordingly to their concentration changes (Tonack

et al., 2013). These are the new directions to study in order to explore new roles of the

ER. Considering that, the endoplasmic reticulum should not only be viewed as a an

important stage in protein biogenesis, but also as a hub, which also receives

environmental cues and reacts accordingly, balancing export and degradation of the

newly synthesized proteins.

4.

Materials and methods of this chapter were performed as described in the publication below:





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Justyna Kusek19 May 1982 in Szczecin, Poland

Polish

PhD program ‘Cell Communication in Health and Disease (CCHD) in Neurobiology at the Institute of Pharmacology, Medical University of Vienna, Austria. Supervisor: Prof. Michael Freissmuth; Thesis title: Chaperoning of A1-adenosine receptor by endogeous adenosine - an extension of the retaliatory metabolite concept.

Adam Mickiewicz University, Poznan, Poland. Graduated in July, 2007; Supervisor: Przemyslaw M. Mrozikiewicz, MD; Thesis title: The influence of interleukin-6 and tumor necrosis factor gene polymorphisms on bone mineral density in postmenopausal women.

, Paris 7 Denis Diderot University, Paris, France. Supervisor: Sarah Boudaly, PhD; Laboratory project: Polymorphonuclear Neutrophil - Dendritic Cell Cross-talk: Study of the Molecular Mechanisms that Controls the Interaction, February-July 2006.

Faculty of Pharmacy, Medical University of Poznan, Poland, October 2004 – June 2005.

PhD Student at the Institute of Pharmacology, Medical University of Vienna, Austria.

internship at the Institute of Pharmacology, Medical University of Vienna, Austria.

Project Assistant, Early Stage Researcher in Marie-Curie Project ‘Healthy hay’; Technical University of Vienna, Vienna, Austria

Scientific Assistant at the Research Institute of Medicinal Plants, Poznan, Poland.

Laboratory Assistant at the Research Institute of Medicinal Plants, Poznan, Poland.

Cambridge Certificate of Advancement in EnglishMittelstufe Deutsch B2 certificate

B2 DELF certificatenative

2012: Award for the best oral presentation at 6th Meeting of the Study Group ‘Receptors and Signal Transduction’ of the German Biochemical Society ‘Young Science meets experience’, Reisenburg bei Ulm, Germany

, Yang Q, Witek M, Gruber CW, Nanoff C, Freissmuth M (2014) Chaperoning of the A1-adenosine Receptor by Endogenous Adenosine - An Extension of the Retaliatory Metabolite Concept Mol Pharmacol mol.114.094045; published ahead of print October 29, 2014, doi:10.1124/mol.114.094045Thill J, Regos I, Farag MA, Ahmad AF, , Castro A, Schlangen K, Carbonero CH, Gadjev IZ, Smith LM, Halbwirth H, Treutter D, Stich K (2012) Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin). Phytochemistry 82:67-80.

, Seremak-Mrozikiewicz A, Drews K, Mikolajczak P, Czerny B, Maciejewska M, Bogacz A, Derebecka-Holysz N, Barlik M, Mrozikiewicz PM (2008) The influence of interleukin-6 and tumor necrosis factor alpha gene polymorphisms on bone mineral density in postmenopausal women. Ginekol Pol 79(6):426-31. (Article in Polish).

Christian W. Gruber, Florian Puhm, Christian Nanoff and Michael Freissmuth Pharmacochaperoning of the ER-retained A1-adenosine receptor. Poster presentation at FEBS-EMBO lecture course “Biomembranes: Molecular Arxhitecture, Dynamics and Function”; June 10th – 20th, 2013; Cargese, Corsica, France

Christian W. Gruber, Florian Puhm, Christian Nanoff and Michael Freissmuth Pharmacochaperoning of the ER-retained A1-adenosine receptor. Poster presentation at Gordon Research Conference on Molecular Pharmacology; April 28th – Mai 3rd, 2013; Barga, Italy

Christian W. Gruber, Christian Nanoff and Michael Freissmuth Pharmacochaperoning of the ER-retained A1-adenosine receptor. Oral presentation at 1st Polish-German Biochemical Societies Joint Meeting; September 11-14th, 2012; Poznan, Poland

Christian W. Gruber, Christian Nanoff and Michael Freissmuth Pharmacochaperoning of the ER-retained A1-adenosine receptor. Oral presentation at 6th Meeting of Study Group ‘Receptors and Signal Transduction’ of the German Biochemical Society ‘Young Science meets experience’ June 15 – 16th, 2012; Reisensburg bei Ulm, Germany.

Christian W. Gruber, Christian Nanoff and Michael Freissmuth Pharmacochaperoning of the ER-retained A1-adenosine receptor. Poster presentation at 8th MUW PhD Symposium May 13 – 14th, 2012; Vienna, Austria.

, Christian W. Gruber, Christian Nanoff and Michael Freissmuth; The interactome of the ER-retained A1-adenosine receptor. Poster presentation at 36th European Symposium on Hormones and Cell Regulation, October 13 – 16th, Mont Sainte Odile, France

Documents

doctoral thesis corrected - Medizinischen Universität Wien · LL '(&/$5$7,21 The work presented in this thesis was performed at the Center for Physiology and Pharmacology, Medical