GPCRs Signalling and expression

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Mag
Mag's picture
GPCRs Signalling and expression

I have GPCRs from transfected CHO for binding assays. The problem is that it no longer couples to G-protein or inhibits c-AMP, but the antibody says that the receptor is highly expressed on the cell surface.
What could be a problem with the GPCRs that explain it?
I would appreciate any help.

saswati1
saswati1's picture
did you check whether you had

did you check whether you had any problems checking the cAMP levels? I mean , it may be possible that something is wrong with cAMP assay

Jama
Jama's picture
How did u determin the

How did u determin the expression is on the cell surface? did you fix the cells if so you need to make them more permeable. Try 100% cold (-20) mathanol

Jama

Bluejay
Bluejay's picture
Expression level is also

Expression level is also important.  From review of the literature, generally 500 to 100,000 receptors per cell is needed with many cells in the 2000 to 50,000 level.  The presence of G proteins should not be a problem since cells tend to more Gi and Ga and Gs G-proteins.  Another issue with cAMP assays is the lysis of cells to release the cAMP.  Some kits include 0.1M HCl and others suggest small amounts of detergent.  You should check that the cells are lysed.  Sometimes additional sonication or centrifugation would be useful.