AWA: Academic Writing at Auckland
A Research Methods Report helps the writer learn the experimental procedures and the ways research findings are made in that discipline (Nesi & Gardner, 2012, p. 153). The question to be investigated is often provided as part of the assignment, and there is usually less focus on existing research and much more on the methods and results of the writer's own research. An IMRD (Introduction, Methods, Results, Discussion) structure is often used. AWA Research Methods Reports include Experiment Reports, Field Reports and Lab Reports.
Title: Org27569 (allosteric modulator) effects on CP55940 (orthosteric ligand) signaling
|
Copyright: I-Fan Hsu
|
Description: How Org27569 (allosteric modulator) alters CP55940 (orthosteric ligand) signaling in cannabinoid receptor type 1(CB1)
Warning: This paper cannot be copied and used in your own assignment; this is plagiarism. Copied sections will be identified by Turnitin and penalties will apply. Please refer to the University's Academic Integrity resource and policies on Academic Integrity and Copyright.
Org27569 (allosteric modulator) effects on CP55940 (orthosteric ligand) signaling
|
Abstract Background and purpose The cannabinoid receptor type 1(CB1) have both an allosteric binding site and an orthosteric binding site. Allosteric modulators such as Org 27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} have been found to alter the response of orthosteric ligands including CP55940{(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol} in these receptors. Although previous literature have established that Org 27569 alters signaling by increasing the binding affinity of CP55940, the underlying signaling pathways are still largely debated. Therefore, this paper will examine how Org 27569 alters the signaling pathways through observing cAMP levels, internalization and ERK 1/2 phosphorylation.
Experimental approach Studies of CB1 signalling were carried out in HEK 293 cells expressing CB1 receptors. We conducted experiments using Org 27569 and CP55940 to measure binding, cAMP accumulation, receptor internalisation and ERK 1/2 phosphorylation. Key results Org 27569 increased CP55940 binding, cAMP accumulation and ERK 1/2 phosphorylation but prevented CP55940-induced receptor internalisation. Conclusions and implications In conclusion, the allosteric modulator Org 27569 seems to alter CP55940 signalling through enhanced binding to CB1 receptors, desensitisation and reduced internalisation. However, the cAMP and ERK 1/2 phosphorylation assay suggest that the effect of Org 27569 is also time-dependent which alludes tothe fact that there may be differential signalling pathways. This major finding suggests that Org 27569 may have therapeutic benefits from altering a range of complex signalling pathways. Abbreviations cAMP, Cyclic adenosine monophosphate; PTX,pertussis toxin;CB, cannabinoid; HEK-hCB1, human embryonic kidney 293 cells expressing CB1 receptor;SR141716A, N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; ERK1/2, extracellular signal-regulated kinases 1 and2;pERK, phosphorylated extracellular signal-regulated kinases;CI, confidence interval; [3H]CP55940, [3H]{(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}; Kd, dissociation constant; Ki,inhibitory constant; IC50, half maximal inhibitory concentration; Kb, dissociation constant for a competitive antagonist; EC50, half maximal effective concentration Results Radioligand competition binding assay Org 27569 addition caused a significant and concentration-dependent increase of specific [3H]bhCP55940 binding at Kd (2.5nM)(Figure 1). In contrast, CP55940 reduced the specific binding of [3H]CP55940. The parameters IC50, Ki, Kb and ternary complex constant were calculated from the curves. Org 27569 showed a pEC50 of 6.100.38, whereas CP55940 showed a pIC50 of 8.11which correlated to a pKi of 8.41. As expected, the converted Ki value (3.87210-9M, CI=7.27110-10-2.06210-8M) for CP55940 is approximately the same as the Kd value of 2.5nM. Furthermore, the pKb (5.45) and cooperativity factor (7.94) were also determined to quantify the allosteric effect of Org 27569 on orthosteric ligand binding. A
B Figure 1: Effect of Org 27569 on radioligand binding at equilibrium. The binding assay was performed on HEK-hCB1 using the CB1 agonist [3H]CP55940 (2.5nM) in the presence of either cold CP55940 (0.1nM to 1uM) or cold Org 27569 (1nM to 10uM)(A). Data was then normalized to vehicle/no treatment (B). Each data point represents mean ±SEM from a single experiment carried out in triplicate. One site - Fit logIC50 sigmoidal curves were fitted with GraphPad Prism.
Cyclic AMP assay The effect of Org 27569 on CP55940-mediated inhibition of forskolin-stimulated cAMP production was measured. The cAMP standard curve was performed to calculate the dynamic range (between 10-8Mand 10-10M) of the assay and to estimate the quantity of cAMP produced by the cells. CP55940 alone showed a slight decrease in cAMP levels at 10-10Mand 10-9M but started to increase gradually at concentrations above 10-8M (Figure 2). CP 55940 co-treated with Org 27569 showed that Org 27569 prevented the inhibition of cAMP accumulation by CP55940. This was seen as a slight increase in cAMP levels; at CP55940 concentrations higher than 10-8M, Org 27569 increased cAMP levels above those produced by forskolin alone. However, the discrepancy in cAMP levels between CP55940 alone and CP55940 co-treated with Org 27659 was not obvious. This was because CP55940 alone (10-6M-10-11M) did not show much difference in cAMP levels (difference was too small to be fitted with a sigmoidal curve) from having only 20 minute equilibration and 20 minute drug stimulation times. The minimal discrepancy could also be from having no data at 10-9M CP55940 with 10 uM Org 27659 since the detection reagent was absent. In contrast to previous studies, CP55940 has been shown to inhibit cAMP levels in a concentration-dependent manner. However, at high concentrations of Org 27659 in the presence of CP55940, the maximum cAMP levels were significantly larger than those produced by forskolin alone. A
B
Figure 2: Cyclic AMP production in hCB1-expressing cells. (A) Effect of CP55940 (10-6-10-11M) alone and CP55940 (10-6-10-11M) combined with Org 27569 (10uM) on forskolin-stimulated cAMP production. The standard curve was used to interpolate the unknown cAMP concentrations (B). The cAMP levels were detected by the cAMP LANCE assay. Data represent the mean values ±SEM from a single experiment carried out in triplicate (A) and duplicate (B).ORG27569+CP55940 (10-9M) fell outside the dynamic range (lost sensitivity) and was therefore excluded. Log (inhibitor) vs. response (three parameters) sigmoidal curves were fitted with GraphPad Prism.
Trafficking assay to detect receptor internalization The receptor trafficking assay was conducted to determine whether Org 27569 altered CB1 receptor internalization in combination with the orthosteric ligand CP55940. Results showed that the surface fluorescence on HEK-hCB1 cells increased with increasing concentrations of Org 27569 when CP55940 was kept at a single concentration of 100nM (Figure 3). In comparison, cells treated with 100nM CP 55940 only showed less fluorescence on the surface. Collectively, these findings suggest that CP55940 caused internalization of the CB1 receptor; whereas the allosteric modulator Org 27569 produced concentration-dependent inhibition of CP55940-induced receptor internalization. These findings are consistent with what was previously reported by Cawston et al. The data was also normalized with no primary antibody as 0% and vehicle as 100% (Figure 3 B). CP55940 alone and CP55940 co-treated with 10-5M Org 27569 showed approximately 50% and 100% cell surface receptor, respectively. Normalizing fluorescence levels to the number of cells present did not improve the data and was therefore excluded. A
B
Figure 3: Org 27569 inhibits CP55940-induced internalization of CB1 receptors. HEK-hCB1 cells were stimulated with CP55940 (100nM) alone or in combination with Org 27569 (10-5-10-9M) and treated with primary (anti-HA) and secondary antibody. Cells were then stained by HOERST nuclear stain. (A) Level of fluorescence on the cell membrane. (B) Fluorescently tagged HEK-hCB1 surface normalized to vehicle/no treatment. Each point represents mean ±SEM from a single experiment carried out in triplicate. Log (agonist) vs. response (three parameters) sigmoidal curves were fitted with GraphPad Prism.
ERK 1/2 phosphorylation assay The AlphaScreenSureFire p-ERK 1/2 assay was conducted to investigate whether Org 27569 activates MAPK members such as ERK1/2 after showing that Org 27659 caused cellular internalization. Both CP55940 alone and CP55940 co-treated with Org 27569 showed time-dependent stimulation of ERK1/2 during the 10-minute time course. CP55940 combined with Org 27569 gave a slightly higher maximum pERK response in comparison to CP55940 at 5 minutes (Figure 4). However, CP55940 alone showed smaller responses than CP55940 combined with Org 27659 later towards the end of the 10 minute period. Although previous literature have also demonstrated that CP alone or in combination with Org 27569 had peak responses at 5 minutes, their results however showed more significant increases in pERK levels with CP55940 and Org 27569 co-treatment.
Figure 4: Time course of phospho-ERK signaling in hCB1-expressing cells. HEK cells expressing hCB1 receptors were treated with either vehicle, CP55940 (100nM) or CP55940 (100nM) combined with the allosteric modulator Org 27569 (10uM) for a period of 10 minutes. The pERK activity was measured through the AlphaScreenSureFirep-ERK1/2 assay. Data represent mean values ±SEM from a single experiment carried out in duplicate. Sigmoidal curves were fitted with GraphPad Prism.
Discussion Allosteric modulators such as Org 27569 have been shown to display contradictory effects on the cannabinoid CB1 receptor. Therefore, experiments were conducted to investigate how Org 27569 modulates CP55940-mediated effects on HEK-hCB1 cells. These experiments involved assessing the binding affinity, cAMP production, receptor internalisation and pERK activity of Org 27569 alone or in combination with CP55940. The equilibrium competition binding assay was conducted to investigate whether Org 27569 altered agonist binding to CB1 receptors. The competition between the unlabelled and radioligand form of CP55940 was characterized by a pIC50 of 8.11. In contrast,Org 27569 significantly increased the specific binding of [3H]CP55940in a concentration-dependent manner with a pEC50 of 6.100.38 (Figure 1A). Org 27569 also showed an α value of 7.94 which is greater than 1, indicative of its positive allosteric modulatory effects on [3H]CP55940 binding. These findings are consistent with previous studies whichalso conducted dissociation kinetic experiments to demonstratethat the allosteric binding to CB1 receptors causes conformational changes to increase agonist affinity for the orthosteric binding site(1). In brief, Price et al (2012) suggested that [3H]CP55940 displays both rapid and slow dissociation kinetics, which represents its binding to low-affinity uncoupled receptors (R) and high-affinity activated (R*) receptors, respectively. The addition of Org 27569 significantly decreased the binding in both states which correlates to the positive cooperativity observed in the equilibrium binding assay (1). Ahn et al (2012) further investigated the binding of [3H]CP55940 with Org 27569 in a highly constitutively active T210I receptor and a constitutively inactive T210A receptor. They demonstrated that Org 27569 significantly increased [3H]CP55940 binding for the inactive T210A receptor but had no effect on the active T210I receptor (2). Therefore indicating that Org 27569-induced agonist binding is dependent on receptor state (2). Keeping this in mind, the next question raised is how there is divergence in Org 27569-induced binding affinity and efficacy. CP55940 is commonly known as an agonist that binds CB1 receptors to stimulate Gαisignalling (3). This protein in turn inhibits adenylate cyclase to prevent cAMP production. The cAMP assay conducted showed that CP55940 combined with Org 27569 increased cAMP levels that were more than those produced by forskolin alone (Figure 2).Our elevatedcAMP response was however not significantly higher than the CP55940-induced response compared to other studies (3).Much of the debate lies within how Org 27569 alters the CP55940-mediated cAMP response. Org 27569 is suggested to inhibit Gαi-mediated CP55940-induced inhibition of cAMP production through Gαs signalling instead of Gαi inverse agonism (3). This is because SR141716A—a known inverse agonist has its effects blocked by PTX treatment, and the effects of Org 27569 appears PTX-insensitive (3). However, the cAMP production with Org 27569 alone was unaffected by PTX treatment suggesting it could be a biased agonist that actives the Gαs pathway itself (3). This result is in contrast to Cawston et al (2013), where the Org 27569-induced increase in cAMP levels is PTX sensitive, indicating it was inverse agonism at Gαiand not Gαs-mediated. Furthermore, the real-time cAMP BRET assay showed that Org 27569 did not alter the initial inhibition of cAMP accumulation by CP55940, but it significantly increased cAMP levels after 5 minutes (4). Since this delay in cAMP response did not seem to be from switching between Gαito the Gαs pathway, the next most reasonable explanation is that Org 27569 rapidly desensitizes the receptor to adopt a constitutively inactive form (4). Interestingly, studies did in fact show that Org 27569 reduced the time to repolarise without changing the extent of hyperpolarisation (4). This major finding suggests that it enhances desensitisation of the receptor (4). Furthermore, experiments were conducted to investigate whether Org 27569 causes receptor desensitisation through CB1-mediated GIRK activation and subsequent channel closing (4). Results showed that AT-20 cells challenged with somatostatin after CP55940 treatment with or without Org 27569 produced similar hyperpolarisation as somatostatin alone (4). Therefore, indicating that the Org 27569-induced desensitisation was mediated at the level of CB1 and not the ion channel (4). Normally, receptors are internalised following desensitisation via phosphorylation (4). Surprisingly, figure 3 showed thatOrg27569 inhibited CP55940-induced receptor internalisation in a concentration-dependent manner—a finding also shown by Cawston et al.(2013). Studies were also done to see whether Org 27569-induced internalisation is B-arrestin dependent. Bailleet al, (2012) investigated if Org 27569 blocks B-arrestin recruitment since CP55940 induces internalisation through B-arrestins. Although Org 27569 alone did not affect B-arrestin recruitmentit seemed to inhibit CP55940-induced B-arrestin recruitment, suggesting that desensitisation may not be B-arrestin mediated (4). Therefore, indicating the existence of other pathways contributing to Org-27560-mediated desensitisation (4). One possibleexplanation is that the desensitized conformation of CB1 induced by Org 27569 is different to CP55940 alone, and is less favourable for B-arrestin recruitment and hence internalisation (4). The ERK 1/2 phosphorylation assay showed that CP55940 co-treated with Org 27569 had slightly higher maximum levels of pERK at 5 minutes in comparison to CP55940 alone. Although the maximum phosphorylation response was achieved in previous studies, our Org 27569 and CP55940 combined pERK response was rather insignificant (2,5). Thiswas most likely from using a low concentration of 100 nM CP55940 in our experiment.Nevertheless, Ahn et al. (2012) demonstrated that Org 27569 alone caused significantly higher increase in transient ERK 1/2 phosphorylation compared to stimulation by CP55940 alone, and the peak at 5 minutes was decreased by co-treatment with Org 27569 and CP55940 compared to Org 27569 alone. This alludes to competition between G protein-mediated and G-protein independent pathways (2). However, it is important to note that Org 27569-mediated pERK responses are CB1-mediated since cells without CB1 receptors did not increasepERK levels (2). Based on this premise, Ahn et al.(2012) have also summarised the effects of Org 27569: Firstly, it is not Gs-dependent. Secondly, it may induce differential signalling pathways by blocking interaction sites with G proteins, altering the intracellular structure of the receptor or promoting different oligomerisation states of receptors (2). In conclusion, the allosteric modulator Org 27569 seems to alter CP55940 signalling through enhanced binding to CB1 receptors, desensitisation and reduced internalisation. However, the cAMP and ERK 1/2 phosphorylation assay suggest that the effect of Org 27569 is also time-dependent which alludes to the fact that there may be differential signalling pathways. Therefore, to gain better insights of how Org 27569 modulates CB1 receptor signalling, future experiments may include other forms of allosteric modulators to observe if they also alter signalling in a time-dependent manner.
References
|
