Hi the first step is to evaluate weather the ligand induced response is Ca dependent or independent.

If it is completely calcium dependent or completely independent. then your life is easier.

Usually they are partially dependent, then you need to try co incubation with Ca chelator and upstream inhibitors such as Wortmanin. Subsequently you need to dissect down stream proteins.

You would get clear asnwers if you explain your experiment design.

Thank you for your help, I'm examining the downstream pathways activated by H2 relaxin on RXFP1 using this robust pCRE reporter assay. I want to be able to dissect exactly which pathways are contributing to the cAMP response produced by addition of inhibitors/activators. So far it seems as if some inhibitors have no effect on H2 stimulation of RXFP1 (even though past studies have shown that they do). How likely is it that the feedback from adjacent pathways is compensating for inhibition of another pathway? RXFP1 is a GPCR and is complex enough as it is.

targeting secondary messenger molecule would be the right approach. Life is simpler for GPCR signalling as lot of information is available. You can use specific inhibitors like LY or wortmanin, secondly you can use PLC inhibitors that work well.

Also conditioning the experiment is very essential. because lot of pathway interact, especially growt factor receptors. Its better to starve the cells with serum free media as it contain growth factors. preferable glucose free or low for syncronization. For your cell type you have to standardise the protocol.

Especially in your case, both Ca and cAMP siganlling interacts. so primarily you need to dissect amoungst these pathway.

Thank you again for your answers. I plan on directly targetting secondary messenger molecules like Ca and cAMP (which I have already done) via specific assays, however we want to test these inhibitors on the final output (pCRE) as support for information we already have. I thought that it would be a quick way of knowing which inhibitors to test/which pathways are affected, but has proven much more difficult than I first thought. I will definitely try serum free media on my cells and see if it will make a difference. Cheers!

In addition to what was already suggested, I can recommend to have vehicle and forskolin and/or cAMP (eg. Sp-cAMPS) analog controls. When vehicle does nothing (base line) and forskolin/cAMP analogs show a robust effect, I would add the pathway inhibitors like LY294002 for Pi3K, U0126 and/or PD98059 for p42/44MAPK and so forth. I am doing CRE-luciferase reporter assays in HT22 cells and this approach works okay. Also be aware that the concentrations of these inhibitors are very important and, as you said, there is crosstalk between pathways like Pi3K and MEK1/2 for example (see Benz et al., Brit. J. Pharm., 2010). Also the cAMP-pathway and the MEK1/2 pathway can be agonistic or antagonistic depending on the presence or absence of B-Raf and Raf-1 (Vossler et al., CELL, 1997).

Good luck!

Yes thank you, I am currently doing a vehicle control as well as forskolin/forskolin + inhibitor controls but have been getting variable results between each well for forskolin in presence of inhibitor. Inhibitors like LY294002 and PD98059 shouldn't effect forskolin levels right? Right now I am trying to test that the inhibitors themselves are working by trying them on other already characterised receptors but I am trying to see if there is another more direct approach. I will look into those cross-talk papers you suggested, thank you for the references!

The variable results with U0126 and LY294002 sound familar to me. Using exactly the same preincubation times of the inhibitors helped some what, but not fully. Also leaving the inibitors and/or activators in or washing after preincubation makes a difference. There are of cause many more examples for singal pathway crosstalk, you are welcome to continue the discussion!

Thanks again Erik. I am conducting all of the similar inhibitors for 30mins pre-incubation at double concentration then adding stimulants/blank after 30mins to halve the inhibitor concentration. Do you suggest washing between pre-incubation and stimulation? So that all concentrations stay constant?

Also do you have any suggestions for possible reasons why I might not be seeing an inhibitory effect when there is supposed to be one? Do you think it's worth trying multiple inhibitors on at the same time?

Sandra, I do not recommend to change between incubations, what I wanted to say is that it makes a difference if you change or not. My cells tend to wash off when I perform too many changes, so I prefer to leave everything in.

Regarding the combination of inhibitors, in our cells (HT22 and primary hippocampal neurons from the mouse) in our hands U0126 above 3 µM alone increased Akt phosphorylation (its in the Benz et al., 2010 paper, and others have meanwhile seen similar effects in other cells), so I would be careful.

BTW, if you find comments valuable I would be very pleased for an upvote!

Thanks!