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Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
Published on: January 27, 2014
A2a/D2 receptor interactions are not observed in COS-7 cells transiently transfected with dopamine D2 and adenosine
P Snaprud1, P Gerwins, M G Caron
1Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
Abstract:
The rat D2 receptor and the dog A2a receptor subcloned into the pXM vector were transiently transfected into COS-7 cells using the DEAE-dextran method. The transfected cells expressed approx. 200 fmol D2 receptors/mg protein and approx. 5 pmol/mg protein of the A2a receptor as judged by binding experiments with [3H]raclopride [or[3H]-N-propyl-apomorphine (NPA)] and [3H]-CGS 21680, respectively. The high affinity KD values were 0.43 and 19 nM for D2 and A2a receptors, respectively, in agreement with results obtained from other cells and tissues. The non-selective adenosine receptor agonist NECA stimulated cAMP accumulation both in non-transfected and transfected COS-7 cells with only a slight difference in potency, suggesting that most of the stimulation is due to activation of A2b receptors known to be present on virtually every cell. The two A2a selective agonists CGS 21680 and CV-1808 were essentially inactive in transfected COS-7 cells, but were very active in PC-12 cells known to possess functional A2a receptors. Dopamine did not decrease cAMP accumulation in the transfected COS-7 cells. CGS 21680 (30 nM) did not affect the binding characteristics of D2 receptors in the co-transfected COS-7 cells in contrast to the increased KH, KL and RH values found previously in rat striatal membranes after CGS 21680 treatment. The present findings indicate that transiently transfected A2a and D2 receptors in COS-7 cells have normal binding properties, but couple poorly to adenylyl cyclase, despite the presence of Gs protein and adenylyl cyclase in these cells. Our results also demonstrate that the previously reported interactions between A2a receptors and D2 receptors do not occur when only the receptor proteins are expressed in COS-7 cells, suggesting that the two receptor molecules do not interact directly to influence binding characteristics.
Insights
Transiently transfected dopamine D2 and adenosine A2a receptors in COS-7 cells exhibit normal binding but poor signaling. These receptors do not directly interact, suggesting other factors mediate previously observed A2a-D2 receptor interactions.
Area of Science:
- Pharmacology
- Molecular Biology
- Cell Biology
Background:
- Dopamine D2 receptors and adenosine A2a receptors are G protein-coupled receptors involved in various physiological processes.
- Previous studies suggested potential interactions between D2 and A2a receptors in certain cellular contexts.
- COS-7 cells are a common cell line for transient transfection and receptor expression studies.
Purpose of the Study:
- To investigate the expression, binding properties, and signaling capacity of rat D2 and dog A2a receptors transiently transfected into COS-7 cells.
- To determine if co-expression of D2 and A2a receptors in COS-7 cells leads to altered binding characteristics or functional interactions.
- To elucidate the coupling of these receptors to adenylyl cyclase in a heterologous expression system.
Main Methods:
- Transient transfection of COS-7 cells with rat D2 and dog A2a receptor constructs using the DEAE-dextran method.
- Radioligand binding assays using [3H]raclopride or [3H]-N-propyl-apomorphine for D2 receptors and [3H]-CGS 21680 for A2a receptors.
- Measurement of cyclic AMP (cAMP) accumulation in response to receptor agonists to assess adenylyl cyclase activity.
Main Results:
- Transfected COS-7 cells expressed functional D2 and A2a receptors with high affinity binding properties.
- Adenosine receptor agonists stimulated cAMP accumulation, primarily via endogenous A2b receptors, not the transfected A2a receptors.
- Dopamine did not affect cAMP levels, and A2a selective agonists did not alter D2 receptor binding characteristics, even in co-transfected cells.
- Despite expressing functional receptors, adenylyl cyclase coupling was poor for both D2 and A2a receptors in COS-7 cells.
Conclusions:
- Transiently expressed D2 and A2a receptors in COS-7 cells possess normal binding but exhibit impaired coupling to adenylyl cyclase.
- The absence of observed interactions between D2 and A2a receptors in this system suggests that direct physical interaction between the receptor proteins may not be the primary mechanism for their previously reported interplay.
- These findings highlight the importance of cellular context and potential accessory factors in mediating receptor-receptor interactions and signaling.
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