Budded baculovirus particles as a source of membrane proteins for radioligand binding assay: The case of dopamine D1 receptor.
INTRODUCTION
G-protein-coupled receptors have become very important drug targets and therefore ligand binding assays for these receptors are an essential part of drug discovery. Among a variety of experimental systems, the radioligand binding assay has remained as one of the main methods in this field for decades. Usually cell membranes or tissues are used in these experiments, however in this article we demonstrate that baculoviruses produced in Sf9 cells display recombinant receptors on their surface and therefore can be used in radioligand binding assay.


METHODS
We have used baculoviruses with dopamine D1 receptors as a model system to validate the usage of this receptor preparation in radioligand binding experiments. In order to collect membrane receptors and separate free radioligand, we have applied FilterMate Harvester with Filtermat B.


RESULTS
Using baculoviruses with dopamine D1 receptors as a model system, we have shown that this is a suitable preparation for conventional radioligand binding assay. Here, all the experiments were performed using the dopamine D1-like receptor specific radioligand [3H]SCH23390. There were no significant differences between binding parameters determined in membranes of Sf9 cells and baculovirus particles. Similar pIC50 and Ki values were also determined in competition binding assays with HEK293 cell membranes.


DISCUSSION
All the results obtained with baculovirus preparation were in good agreement with the data obtained in parallel experiments with membrane preparations from Sf9 and HEK293 cells expressing dopamine D1 receptors. Shape uniformity, homogeneous distribution and slow sedimentation of the membranes are some of the advantages of baculovirus preparations, which prove them as promising source of membrane proteins for routine and high throughput analysis.
['Allikalt|Anni|A|', 'Rinken|Ago|A|']
D000818:Animals D016367:Baculoviridae D001552:Benzazepines D001667:Binding, Competitive D002460:Cell Line D018491:Dopamine Agonists D018492:Dopamine Antagonists D057809:HEK293 Cells D006207:Half-Life D006801:Humans D008565:Membrane Proteins D011869:Radioligand Assay D019275:Radiopharmaceuticals D017447:Receptors, Dopamine D1 D018411:Spodoptera D014771:Virion
Budded baculovirus Radioligand binding assay G-protein-coupled receptor Methods Dopamine D(1) receptor