Hello Zhiyuan,

I worked with the D3 subtype in function for few time... I was working with the HTRF kit to study the cAMP production (quite similar than yours) in HEK 293 and COS cells.

What I can tell you is dopamine is not a good ligand at all... I had a small production of cAMP (D1-like response) due to a better response of endogenous expression of some receptors which bind dopamine. But this was not detect in radio-ligand assay with untransfected cells.

You have to find some selective agonists of the D3 receptor and test with mock cells if you get a cAMP response as control... I'm sorry... At this level, I stop my experiments... I cannot suggest you more...

Best

Guillaume

As Guillaume says, CHO and HeLa clones can express small endogenous levels of GPCR, hen in your case D1R is among them you cannot see the D3R inhibitory effect. Selective D3R agonist may help out and if you are worried about adenylate cyclase, test inhibitors of adenylate cyclase as a control.

hi Guillaume，

Thanks four suggestions.

I am preparing to purchase the 7-OH-DPAT to do the cAMP Glo. I am wondering whether you have combine the forskolin and dopamine to detect the response of dopamine receptor D3.

Best

zhiyuan

Thanks Erik, I will share my experiment progress with everyone ,hoping get the  problem solved

Hi Zhiyuan,

Yes. I was using forskolin for activation of AC and dopamine to activate the D3. As I told you, I was only able to see a slight cAMP production with dopamine (EC50 around 5 uM). This was observed with transfected and mock cells and was absent by adding spiperone (dopamine R antagonist).

I know that D3 was really a problem in functional study... I used bromocriptine but not really successful... One other problem I heard was that FSK is too powerful (by activating all the AC in the cells...)

I want to suggest you to contact some companies sailing such functional kit and ask if they are able to have a good signal with D3. We were in contact with Cisbio (http://www.htrf.com/homepage) and they were developing new strategies for these receptors...

Good luck with your project!

Best

Guillaume