Biochemical basis for malate over production in Actinomycete spp.
MMRIResearch
1. Asparagine 284 and Tryptophan 246 mutations in human
adenosine A2a receptor affect receptor signaling function
Abstract
G protein coupled receptors (GPCRs) are the largest and most diverse set of membrane
receptors in eukaryotes. These receptors respond to multiple types of stimuli and govern many of
our physiological functions ranging from our immune function to taste and smell. A member of
the GPCR family is adenosine A2a receptor (A2aR). The receptor is triggered in response to
endogenous agonist adenosine which activates G-proteins, which in turn triggers the production
of cyclic adenosine monophosphate (cAMP) by activating the enzyme adenyl cyclase. In the
present study, we aim to determine the effects upon human adenosine A2a receptor (hA2aR)
functionality by mutating few amino acids in the receptor. cAMP levels were determined in
CHO-K1 cells expressing wild type hA2aR and two mutant of hA2aRs, namely, N284A and
W246A. The cells were stimulated with adenosine and a negative modulator of receptor –
amiloride. It was found that the created mutations completely abolished the receptor
functionality as the cAMP levels were below the detection level in CHO-K1 cells expressing
mutants of hA2aR. In wild type hA2aR, cAMP levels were increased with increasing
concentration of adenosine. Surprisingly, in the presence of amiloride, a negative modulator of
A2aR, the cAMP levels were elevated. The increase in cAMP level in the presence of amiloride
can be associated with other cellular effects of amiloride in the cell. Further experiments are
required to shed some light on A2aR efficacy in the presence of amiloride and other agonists.
2. Introduction
Modulation of signal transduction across the cellular membrane is one of the main targets for
pharmaceutical research. G-Protein Coupled Receptors (GPCRs) are the most diverse and largest
set of membrane receptors and they have been targeted for about 30% of marketed drugs
(Filmore 2004; Hopkin and Groom, 2002). Adenosine receptors, also known as rhodopsin-like,
belongs to Class A GPCRs. Four types of adenosine receptors have been identified: A1, A2a,
A2b, and A3. By binding to a specific adenosine receptor subtype, the endogenous adenosine
exerts a range of immuno modulating and cyto-protective effects. Activation of the A1 and A3
adenosine receptors lead to an inhibition of the enzyme adenylate cyclase mediated by an
inhibitory G protein (Gi), whereas A2A and A2B receptors, once activated, cause a Gs-mediated
stimulation of this enzyme (Fredholm et al., 2001).
Many representatives of Class A receptors, among them adenosine receptors, have been shown
to be allosterically regulated. An allosteric site is a ligand binding site on the protein that is
distinct from the orthosteric primary ligand binding site to which ligands such as endogenous
adenosine and synthetic derivatives thereof bind. An allosteric modulator does not have any
activity by itself, but it needs the orthosteric ligand to show its action. Amiloride and analogues
were demonstrated to be allosteric inhibitors for the A2A adenosine receptor. Amiloride
analogues increased the dissociation rate of the inverse agonist [3H]ZM 241385 from the A2a
adenosine receptor, however they did not show any effect on the dissociation rate of the agonist
[3H] CGS21680 (Gao and Ijzerman, 2000). Sodium ions (high concentrations of NaCl) rather
decreased the dissociation rate of the inverse agonist [3H]ZM241385 from the A2A adenosine
receptor in a concentration-dependent manner. The sodium ion binding site appears conserved
among class A GPCRs (Katritch et al., 2014).
These observations made us to explore the role of amino acids in the sodium ion binding pocket
in more details, specifically their role in receptor efficacy. In the present study, we mutated the
two amino acids in the sodium ion binding pocket of adenosine A2a receptor to understand the
role of these residues in receptor functionality.
3. Materials and Methods
Plasmid isolation from bacteria
pCMV6-AC-RFP-hA2aR and its mutants in same construct were maintained in bacteria E.coli
strain DH5 alpha. For plasmid isolation, bacteria was inoculated in terrific broth media
containing ampicillin and was grown overnight. Plasmid was isolated using Qiagen Maxi prep
kit as per manufacturer’s protocol and was quantified on 1% agarose gel and on EnSpire for
OD260/280 ratio.
Figure: Bacterial cultures grown overnight for plasmid isolation
4. CHO-K1 cell growth and transfection
CHO-K1 cells were grown and maintained in culture medium containing DMEM/F-12, 10%
FBS and 3 MM glutamine at 370
C and 5% CO2. Cells were passaged every two days and seeded
at 2 x 106
cells/100 mm dish for transfection. Cells were transfected with indicated plasmid (20
µg/100 mm dish) using Invitrogen’s Lipofectamine 3000 kit as per manufacturer’s protocol.
Transfected cells were micrograph using Nikon TE2000U inverted microscope. Twenty four
hours post transfection, cells were harvested and were seeded on 96 well half-area plate at
30,000 cells/well for cAMP assay.
Figure: CHO-K1 cells
5. cAMP assay and data analysis
CHO-K1 cells, transiently expressing the wild type hA2aR and its mutants, were washed with
Hanks’ balanced salt solution (HBSS) and incubated with rolipram (50 μM), adenosine (10 nM
and 100 nM), ZM 241385 (10 nM) and amiloride (10 μM and 300 μM) for 15 mins at 37°C.
Intracellular cAMP levels were determined using Cisbio HTRF cAMP dynamic kit as per
manufacturer’s protocol. The data was analyzed using GraphPad Prism Software.
Figure: Principle of CisBio HTRF cAMP dynamic kit
6. Results
Plasmid isolation, quantification
Plasmids, pCMV6-AC-RFP-hA2aR and its mutants, were isolated in high concentration (2.0
µg/µl each) and were quantified on gel and on EnSpire. Plasmids were of high purity as OD260/280
ratio was 1.8 in all three samples and there was no RNA/protein bands on the gel.
7. CHO-K1 cells transfection
Cells were 80-90% confluent at time of transfection. Transfection efficiency was about 70-80%
in all three samples.
8. N284A and W246A completely abolished the receptor signalling
Using a standard curve with known values of cAMP, the concentrations of intracellular cAMP
in CHO-K1 cells expressing hA2aR and its mutants, N284A and W246A, were determined.
Standard Curve Plotted graph of samples with known values of cAMP production (nm).
9. Figure: cAMP production in CHO-K1 expressing wild type A2aR. Treatment: 10 nM
Adenosine, 100 nM adenosine, 10 nM ZM241385and 10 µM amiloride.
When compared to DMSO control, there was approx. two fold increase in intracellular cAMP
level in CHO-K1 cells stimulated with 100 nM of adenosine. In the presence of inverse agonist,
ZM241385, there was not any detectable amount of cAMP. When cells are stimulated with 100
nM adenosine and 10 µM of amiloride, cAMP levels were further elevated by 2.5 fold when
compared to its DMSO control.
10. Figure: cAMP production in CHO-K1 expressing wild type A2aR: Treatment: 10 nM
Adenosine, 100 nM adenosine, 10 nM ZM241385and 300 µM amiloride.
Again, similar trend was observed. cAMP production increased two fold when adenosine was
added at 100 nM concentration. Addition of inverse agonist, ZM241385, completely halted the
cAMP production. When cells are stimulated with 100 nM adenosine and 10 µM of amiloride,
cAMP levels were further elevated by 1.5 fold when compared to 100 nM adenosine without
amiloride.
11. Figure: cAMP production in CHO-K1 expressing wild type A2aR: Treatment: 10 nM
Adenosine, 100 nM adenosine, 10 nM ZM241385 and 10 µM amiloride.
The concentration of cAMP produced in both, CHO-K1 cells expressing mutants of hA2aR -
N284A and W246A, was below the detection limit. It indicates that receptor functioning was
completely abolished if these two amino acids were mutated. Even, in the presence of 10 µM
amiloride, cAMP levels remains below the detection limit.
12. Figure: cAMP production in CHO-K1 expressing wild type A2aR: Treatment: 10 nM
Adenosine, 100 nM adenosine, 10 nM ZM241385 and 300 µM amiloride.
Again, negligible amount of cAMP was observed in CHO-K1 cells expressing mutants of
hA2aR. Treatment with increased concentration of amiloride (300 µM), does not make any
difference to the cAMP levels when compared to non-amiloride treated cells.
13. Discussion and Conclusion
The endogenous agonist adenosine binding to the receptor triggered an increase in the production
of cAMP. As shown in the results, the study suggests that the asparagine 284 and tryptophan 246
are crucial amino acids for the adenosine A2a receptor signaling as these mutations indicated a
total loss in the receptor signaling. According to Massink et al., all mutations in the sodium ion
binding pocket impact the A2a receptor signaling significantly (Massink et al., 2014). It was
found that the N284A mutation negated the effect of sodium ion binding. The absence of
tryptophan in W246A mutation has a positive effect on binding of both agonist and inverse
agonist in the presence of sodium ion (Massink et al., 2014). ZM241385 is a known inverse
agonist of A2aR (Lopes et. al 1999). The studied mutations have no effect on the inverse agonist
binding to the A2aR. In the presence of amiloride, in addition to adenosine, cAMP levels were
elevated indicating that amiloride acts as a positive modulator of adenosine A2aR. However,
other studies supported the theory that amiloride acts as a negative modulator. These studies are
based on membrane binding assays and not on functional assays such as measuring the
concentration of secondary messenger cAMP. In bindings assays, the receptor is studied at
membrane level only whereas in functional assays, like cAMP, whole cell physiologically active.
Also, amiloride is an allosteric modulator of several GPCRs, including diverse sub-families of
adenosine, aminergic, and gonadotropin-releasing hormone receptors (Gao and Ijzerman, 2000;
Gao et al.,2003; Heitman et al., 2008; Hoare et al., 2000; Pauwels, 1997). So, increased cAMP
levels on amiloride treatment can be because of the other cellular effects of amiloride.
In summary, site directed mutagenesis may elucidate the role of amino acids within the receptor.
Amino acid residues asparagine 284 and tryptophan 246 in adenosine A2aR have been shown to
be an integral part for receptor signalling. Mutations in these amino acids have completely
destroyed receptor functionality. These observations shed some light on importance of studied
amino acids in receptor signaling in the presence of agonist, inverse agonist and modulator.
14. Acknowledgement
I would like to thank Dr. Edward Amento and other program directors, Manmeet Kaur (program
coordinator), Harpreet Kaur (senior mentor), Saalini Sastry (junior mentor), my AP Biology
teacher Dara Levy-Homestead High School, and MMRI staff for guidance and assistance in
completing this project.
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