Essay: Engage Readers with this Unraveling Intricacies of GPCRs: How Chinese Hamster Ovary Cells Respond to Forskolin, Calcitonin, and Serotonin



Abstract

Here we report the characterisation of endogenously expressed receptors in Chinese hamster ovary (CHO) cells, which couple in a negative or positive manner to adenylyl cyclase, as measured by cAMP linked luciferase reporter gene assay. Luciferase expression in reporter cells was found responsive to changes in forskolin concentration. Calcitonin caused a stimulatory effect on the AC pathway, measured in an increase of luciferase expression. Serotonin was found to cause an inhibition of forskolin: stimulated luciferase expression.

Introduction

G-protein coupled receptors (GPCR) are a part of a family of membrane bound receptors (1) that function by responding to extracellular stimuli (i.e. binding of ligands such as neurotransmitters and hormones), subsequently initiating a variety of intercellular signalling events. (10) Upon agonist binding to GPCR conformational change occurs, which results in activation of a heterodimeric G protein that consists of 3 subunits, ,  and . (10) Activated subunits  and  interact with effector proteins such as enzymes or ion channels (8), leading to a change in intercellular secondary messenger, such as cAMP. (8)

Many physiological processes such as vision, neuronal transmission and intercellular communication is mediated via GPCR signalling. (12) Dysregulation of GPCR signalling can result in a range of diseases, (12) making GPCR of interest as pharmaceutical targets, with 36% of all drugs targeting them. (9)  Functionally coupling GPCRs to reporter genes, such as luciferase, allows for pharmacological characterisation of these receptors. (7)

Forskolin:

The dose response curve seen in figure 1a. was used to estimate the EC50 for the drug forskolin. The estimated EC50 was found to be =10-6.14 = 0.72M.

It is observed a linear increase of luciferase expression in response to forskolin concentrations between 0.1M and 3.01M. However, a decrease in luciferase expression can be observed at forskolin concentrations higher than 30M.

There is also observed a decrease in luciferase expression at a forskolin concentration of 10M, the error bar at this point is large.

Calcitonin:

The dose response curve seen in figure 1b. Was used to estimate the EC50 for the drug calcitonin. The EC50 was estimated to be = 10-10.79 = 0.000016M.

It´s also observed that “steady” increase of the luciferase expression starts at a calcitonin concentration of 0.0000025M and the curve starts to platter at calcitonin concentrations higher than 0.0001M. In addition, the highest luciferase expression was observed at a calcitonin concentration of 10M.

Serotonin:

The ideal dose response curve seen in figure 1c. Was used to estimate the IC50 for the drug serotonin. The IC50 was estimated to be = 10-7.23 = 0.059M

The lowest expression of luciferase is observed at a serotonin concentration of 10M. In addition, as seen in figure 3, the luciferase expression (%) decreased in response to an increase serotonin concentration.

Forskolin VS. Calcitonin

The estimated EC50 value (0.000016M) of calcitonin is lower than that of forskolin (0.72M). Meaning that a lower concentration of the drug calcitonin is needed to give half maximal response, compared to the concentration needed for forskolin. In addition, the curve steadily rises at a calcitonin concentration of 0.0000025M. However, for the drug forskolin, the curve starts to steadily rise at a concentration of 0.1M.

A significant decrease of luciferase expression (%) is not observed at high calcitonin concentration, however can be observed at high forskolin concentrations.

Forskolin VS. serotonin

1M of the drugs forskolin was used when making the drug dilutions for serotonin. The concentration of forskolin added is close to the EC50 value for forskolin. In addition, as seen in figure 1 and 3, an increase in the concentration of serotonin results in a decrease in luciferase expression. However, an increase on concentration of forskolin results in an increase of luciferase expression (%).

Discussion

Upregulation of luciferase expression is responsive to an increase of intercellular cAMP levels. This is because cAMP activates protein kinase A (PKA) by binding to its regulatory subunits. (3) The activated PKA in turn phosphorylates the transcription factor, cAMP response element (CRE) binding protein, resulting in its binding to CRE that is located in promoter region of the luciferase gene. (3)  Intercellular cAMP synthesis is catalysed by an activated AC (4), and thus by stimulating or inhibiting the AC, luciferase expression is regulated. By measuring the dose-dependent response of luciferase expression when treating CHO cells with different drugs the receptor endogenously expressed in these cells can be characterised.

+control was removed to remove basal level of luciferase expression.

CHO cells treated with forskolin.

Forskolin is known to bind directly to AC in a catalytic split of C1 and C2 domain (5), and activates most forms of AC, independent of receptors. (5)

As seen in figure 1a, luciferase expression increases in a linear fashion between forskolin concentration of 0.1M and 3.01M. This suggest that in that range of forskolin concentration the drug has a positive effect on luciferase expression.  

A decrease in luciferase expression is seen at a forskolin concentration of 10M, however, the error bar is large at this point. (figure 1a). Which could suggest that the decrease seen could be due to human error.

At forskolin concentrations higher than  30M there is seen a significant decrease in luciferase expression (figure 1a), which is also seen in other studies (2). Previous studies found that the transcription factor ICER (inducible cAMP early repressor) negatively regulate luciferase gene expression (3). This could explain why it´s observed a decreased luciferase expression at high forskolin concentration. Furthermore, other studies found that ICER is responsive to cAMP concentrations, (2) and is only expressed at elevated intercellular cAMP levels. (2) In addition, it was seen a direct correlation between the inhibition of the luciferase expression and forskolin induced ICER expression. (2). Interestingly, it was found that Cos-7 cells, which lacs ICER, did not show a decrease in the expression of luciferase at high concentrations of forskolin. (2) This additional level of gene transcription regulation highlights the importance of investigating crosstalk between transcription factors responsive to intercellular cAMP levels in cAMP-linked reporter gene assay.

CHO cells treated with salmon calcitonin

Luciferase expression was found to increase in a linear fashion between calcitonin concentrations 0.0000025M and 0.0001M. (Figure 1b). However, low levels of calcitonin do seem to have an effect on upregulation of luciferase expression.  The curve starts to platter at calcitonin concentration higher than 0.0001M. (Figure 1b) Suggesting increasing the calcitonin concentration above this will not significantly further increase luciferase expression.

In agreement with other studies, (1) sensitivity of luciferase expression in response to change of calcitonin concentration suggests the presence of an endogenously expressed calcitonin receptor in CHO cells. The response of increased luciferase expression when increased calcitonin concentration indicates that the calcitonin receptor is positively coupled to AC via G-protein, thus acts to activate the effector protein leading to increased intercellular cAMP levels and subsequently upregulate gene transcription of luciferase reporter gene. (3) All forms of AC are found to be stimulated by G protein class, Gs, through its binding to the C2 domain of AC. (5)

In contrast to forskolin stimulated luciferase expression, decrease in luciferase expression was not observed at a high concentration of calcitonin. However, interestingly it has been reported that CHO cells that overexpress calcitonin receptors a strong inhibition of luciferase expression occurs at high calcitonin concentration. (2)

CHO cells treated with serotonin.

When the drug dilutions for serotonin was made, 1M of forskolin was added. As previously seen (figure 1a), forskolin at this concentration have on an effect on upregulation of luciferase transcription.

 In agreement with other studies (1) a decrease of forskolin stimulated luciferase expression (figure 1c) occurs in response to increased serotonin concentration. Sensitivity of forskolin: stimulated luciferase expression in response to change in serotonin concentrations suggests the presence of an endogenously expressed serotonin receptor in CHO cells. Serotonin receptors that are known to be negatively coupled to AC via G-proteins is the 5HT receptor (1). The 5HT GPCR signal via G protein class Gi which are known to block the ability of forskolin to stimulate AC. (5) Thus, the inhibition of AC results in intercellular cAMP levels to decrease and subsequently CREB is not activated, therefore does not bind to the CRE for the upregulation of luciferase transcription.

In addition, it was investigated if serotonin effects the basal levels of luciferase expression. (1) As mentioned previously, serotonin shows an inhibition of forskolin stimulated luciferase expression. However, previous study stated that the basal levels of luciferase expression was not altered by serotonin. (1)

Investigating drug potency.

The EC50 of calcitonin (0.000016M) was found to be much lower than EC50 of forskolin (0.72M) and IC50 of serotonin (0.059M). This indicates that calcitonin is a more potent drug than forskolin and serotonin.

As mentioned, forskolin is found to bind directly to AC, (5) and thus by-passing the GPCR signalling. Subsequently, when by-passing GPCR loses some signal amplification that occurs through GPCR binding.

Alternative approaches

In addition to a cAMP-linked reporter gene assay, other additional approaches may be used to investigate GPCR signalling and presence of the receptor.

• Pertussis or cholera toxin. G-protein class Gi and Gs is known to be pertussis and cholera toxin sensitive, respectively. (1) Therefore, by treating cells with either pertussis or cholera toxin one can study the effect on the GPCR. A study found that when treating cell with pertussis no significant effect was seen on forskolin stimulated luciferase expression. (1) However, abolished the inhibition of forskolin stimulated luciferase expression of 5HT receptor signalling. (1) Suggesting the 5HT receptor mediated response is most likely via a pertussis toxin sensitive g-protein, Gi (1). In addition, GPCR that signal via cholera toxin sensitive G proteins, Gs, can be identified by treating cells with cholera toxin. (1)

• cAMP assays are used to investigate GPCR signalling by measuring cellular cAMP levels that are dependent on the activity of AC. (11) Which in turn is regulated by GPCR coupled to G-proteins subunits Gs or Gi/o. Gs