Hi Chiara Mazzotti,

I think that the endogenous arrestin could not be a problem (it's expression is probably much smaller than the arrestin-GFP). The translocation of arrestin to plasmamembrane can be observed both in HeLa and HEK293, so i think it should not be a problem. One problem could be that if the expression of arrestin GFP is too high, and the expression of your receptor is low, you can not see its translocation due the high cytoplasmatic background. I would try to find cells with relative low arrstin-GFP expression, there could be the translocation more pronounced. Are you sure the receptors are in the same cell than the arrestin-GFP? Using an (other color) fluorescent labeled receptor could help to find cells with high receptor/lower arrestin expressing cells. BTW, which 7TM receptor are you using? Bests,

Bence Szalai

To improve translocation of arrestin you could add the c-tail of the V2R to your receptor. It helps in seeing robust translocation.

 Bence Szalai, I am sure that my cells produce also my receptor because I previously stain it with a fluorescent label.

che agonista usi? prova dosi crescenti di agonista...

Chiara,

From our experience, most of the overexpressed arrestin-GFP in cells does not function properly probably due to misfolding. Only very few cells will have a functioning arrestin that will translocate in response to your ligand. I would suggest making stable cell lines.

Hesham

Your HeLa cells may not have high enough expression of GRKs, or the wrong subtypes of GRK, or the wrong subcellular localisation of GRKs. So although the agonist is activating the receptor, it is not being phosphorylated in a way that allows arrestin recruitment. Or the phosporylation pattern only causes transient arrestin interaction which you cannot see in your assay. Try transfecting in GRKs, or using a different cell-line. Its not unusual to see cell-specific patterns of receptor signalling based on differential expression of signalling partners. 

Hi Chiara,

Did changing your level of gfp-arrestin do the trick? maybe you need to reconstruct your plasmid. is there a linker between gfp and arrestin and where is the tag? also, r u doing live imaging or fixed staining? Hoping this gets resolved.

-ED

Datan, the plasmid was a gift from another lab and it was supposed to work. I am doing live imaging! I will soon retry with HEK293T

Hi Mazzotti,

Maybe you are not facing any problem. Do you know when or where your receptor recruits beta-arrestins...Is it before or after being internalized? In the cell membrane or in the cytoplasm after formation of recycling endossomes?

You should be aware that, for some GPCRs, recruitment of beta-arrestins is not necessairly made in the cell membrane. In fact, there are some receptors that internalize independently of beta-arrestins but need them for signalling and/or recycling. In such cases, the scaffold of the receptor is made in the cytoplasm after internalization by clatrin-coated pits. 

Good luck

Adriana Rodrigues

it might be also possible that your ligand is degradet immediately. you may change to serum-free medium with several wash steps immediately before adding the ligand. ....as it is a peptide ligand you may consider that also...

all the best !

Steffen Schuessler

hi Steffen, I tried using the same protocol as before using HEK293T and I didn't see arrestin delocalization.

Now I will try a starvation step with serum free-medium as you suggested! indeed, I read many articles that use this method. 

Thank you!

hi Chiara, you may also add some inhibitors, bacitracin is cheap and unspecific. guess you may need something for matrix-metalo-proteinases which sit at the cell membran.

all the best !