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’.
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
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
EndoH - + - - + - + - - + - PNGaseF - - + - - - - + - - +
wild type A1-receptor untreated DPCPX IODO/EHNA/DIP
M
C D 55
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Fig. 3
inhibitors c. SR 121463
Flag-V2-receptor-I318S
SR 121463 inhibitors c.
V2-vasopressin-T273R-GFP
A
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Fig. 4
DPCPX inhibitors c.
A1-receptor-Y288A-YFP
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Fig. 5
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Fig. 8
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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.
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
Suppl. Fig. 2
A1R
Gβ
55
35
[kDa]
A B
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 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.
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
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).
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
Compound (µM)0,0 0,1 1,0 10,0 100,0
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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.
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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
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.
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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
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.
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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
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:
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
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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