GPCR Signaling - Science topic

There are several alternative methods available to measure G protein-coupled receptor (GPCR) signaling without relying on radio-labeled GTP binding. These methods provide valuable insights into GPCR activation and downstream signaling events. Here are some commonly used techniques:

1. Fluorescent GTP Analogs: Fluorescently labeled GTP analogs, such as mant-GTP or Bodipy-GTP, can be used to monitor GTP binding to G proteins. These analogs emit fluorescence upon binding to G proteins, allowing for real-time detection of GPCR activation. Fluorescence resonance energy transfer (FRET) techniques can also be employed to measure GTP binding indirectly.

2. Bioluminescence Resonance Energy Transfer (BRET): BRET is a proximity-based assay that measures protein-protein interactions. In the context of GPCR signaling, BRET can be used to detect interactions between GPCRs and downstream signaling molecules, such as G proteins or β-arrestins. This technique provides insights into GPCR activation and downstream signaling events.

3. Calcium Imaging: GPCR activation often leads to intracellular calcium mobilization. Calcium imaging techniques, such as fluorescence microscopy using calcium-sensitive dyes (e.g., Fluo-4), can be employed to measure changes in intracellular calcium levels upon GPCR activation. These measurements provide information about GPCR signaling and downstream calcium-dependent processes.

4. Phosphorylation Assays: GPCR activation often triggers phosphorylation events mediated by protein kinases. Phosphorylation assays, such as western blotting or immunofluorescence using phospho-specific antibodies, can be utilized to detect and quantify GPCR-induced phosphorylation of downstream signaling proteins. This approach provides insights into GPCR signaling pathway activation.

5. cAMP Assays: GPCRs can activate intracellular signaling pathways, such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP assays, such as enzyme-linked immunosorbent assays (ELISAs) or fluorescence-based assays using cAMP-sensitive dyes, can be employed to measure changes in cAMP levels upon GPCR activation. These measurements provide information about GPCR signaling and downstream cAMP-dependent processes.

The C-Myc-tagged protein contains 11 amino acids, which can still recognize their corresponding antibodies when expressed in different protein frameworks as antigenic epitopes. The c-Myc tag has been successfully used in Western-blot hybridization, immunoprecipitation and flow cytometry, and can be used to detect the expression of recombinant proteins in target cells.

In theory, the larger the molecular weight of the protein tag, the greater the impact on the function of the protein itself, so macromolecular tags are generally only used for detection or protein purification. If the molecular weight of the C-Myc tag is relatively small, the immunogenicity is weak, and generally speaking, it will not affect subsequent research and applications.

Hi guys,

the big problem in the field is the semantical imprecision. What do people really mean when referring to beta-arrestin-mediated signaling? What exactly is this? Is it arrestin-modulation of G protein signaling or arrestin signaling in the absence of G protein activity, ie truly G protein-indepedent signaling? For the latter there is no proof in the literature, however arrestin contribution to G protein signaling has been seen by many. If we use arrestin signaling for both scenarios, no surprise there is confusion. Inititially I liked the idea of arrestins signaling in their own right but I am afraid it is no more than a preconceived concept that does no longer hold since we have the tools to really challenge the concept. The signaling bias literature is full of papers with unsupported claims and I predict it will take years for our field to recover because some proponents of arrestin signaling are very protective of the arrestin transducer model. You cannot imagine that publishing my paper was close to impossible, I was caught in a mafia of people who tried everything to kill my paper. A nightmare for the first author, he left academic science, he was disgusted...

Anyway, when you read literature, be critical, don't believe every word of a high impact paper, look at the data and dare to come up with your own interpretation! We all have the same data in the bias field, what differs is our interpretation!

hi

you can used from PC3 & MDA-MB-231( good expression in mRNA and protein level )

good luck

If you're inhibiting G12/13 in a cell line, you can also use the pGL4.34[luc2P/SRF-RE/Hygro] vector in a reporter gene assay to look at RhoA activation. It might be a bit easier than showing RhoA activation in pulldown assays.

For what it's worth... I decided to keep my stock solution at RT and my experiments were successful and I never had a problem keeping the CNO in solution.

It worked thanks !

I used an ELISA based kit from Amersham. It was useful although a little expensive. It is not difficult but it would require a whole day and it has a very detailed protocol. I recommend it. Good luck!!!

The paper models the reaction by solving a system of 3 ODEs

R+An <-> RAn <->R'An

where R is the receptor, An an antagonist, RAn the receptor-antagonist complex and R' the isomerised receptor-antagonist complex.

I would like to model the reaction

R+L <-> RL <-> R'L

R+I <-> RI <-> R'I

where R is a receptor, L radioligand, RL the receptor-ligand complex, R'L the receptor-ligand complex with conformational changes, I an in inhibitor, RI the receptor-inhibitor complex and R'I the receptor-inhibitor complex with conformational changes. This would be a system of 4 ODEs.

Thank you very much for the suggestions, I think I shall try the ELISA which is more affordable to me

Rcsb.org has links to Molecular Graphics Software (http://www.rcsb.org/pdb/static.do?p=software/software_links/molecular_graphics.html).

you can quantify the expressed receptors using quantitative GFP fluorescence similar to that described for soluble protein on https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315830/.

It might depend on the activation pathway you want to measure. Receptors can often continue to signal after internalisation (e.g. MAPKs, and even sometimes G proteins). 

If internalisation is stopping the signal, then you could try inhibiting internalisation (e.g. the dynamin inhibitor dynasore or  use < 18 C temperature).

It is all very receptor subtype dependent - some don't seem to desensitise and/or internalise at all. 

What specifically are you trying to achieve?

Dear Rahnama,

I think you can find it from this:

To produce a GST fusion protein containing a PH domain (and then perform "pull-down" assays) may serve almost equally than yeast two hybrid l to isolate PH domain-binding proteins

We did the 2 approaches in this paper that hopefully may be useful to you.

Thank you both for your replies.

Dear Alice, 

Freezing is an important distress for the cells and it can change their metabolism and could also affect gene expression patterns. In my experience, cells respond very different when frozen compared to fresh. Membranes are one of the most frequent and important damage, hence it compromise the cell compartimentalization and the overall response to any external compound they are exposed to. It cound also make intracellular proteins to release to the medium, even if your protein of interest is a secreted one, you might find higher amounts because of this phenomenon.

Also cell viability is strongly compromised but, depending on the cell type, you may have a non-negligible amunt of "diying" cells which mask the effect of create artifacts.

Hope my thoughts could be helpful. Good luck and best regards.

Dear Kolla,

I suggest first you establish a dose-response relationship with a known agonist for reference. Then you measure the dose-response with your compounds alone as well as together with the agonist. If a compound is an antagonist, you will see no change of basal activity if measured alone, while observe a parallel right-shift of the agonist dose-response curve when measured together. In case of an inverse agonist you also observe  a shift when measured together, but must observe a decline in the dose-response curve with increasing concentration below the basal level. This can be observed only if a measurable basal activity is present. If a potential inverse agonist compound is identified this way, then you might design binding assays to see where the compound binds and how it affects the binding of agonist (to differentiate the functional effect from an allosteric negative co-operativity effect), but this is a more complicated issue and you need purified protein for that.

Regards,

Karoly

Good evening.

My name is Maria maniou. I would like to take part in the writing of the article. How many words Ι will need to write ? Until when Ι should hand it over; And finally I would like to tell me what exactly it should contain.

Have you tried Arginine  Pyroglutamate?

Dear Ashgar

You would need an estimate of the variance of your data. This implies that you should determine your EC50 values in at least three independent experiments. This will give you 3 EC50 values from which you can calculate a mean and a standard deviation. You can then compare the mean of your standard agonist with the mean of another ligand, using a simple test like the t-test. You can also compare the mean of your standard agonist with the means of a number of other ligands, using ANOVA type tests, followed by post tests. Finally you can use the full data set for the EC50 determinations and test all data against each other. A good textbook on biostatistics would be 

Best wishes for your research

Birger

I would try a fusion protein but in the C-terminus. Constructs for instance for BRET are fusion proteins of a GPCR with a BRET donor (Renilla luciferase) and a BRET acceptor (eg. yellow fluorescent protein). These fusion proteins go to the membrane and GPCRs are functional. A variation of this approach would be to attach half of your protein to the C terminus of the GPCR and half to another protein that goes to the membrane (assuming that the two halves will "meet", i.e. that two halves would produce the entire protein). 

BHK-21 is not in this list:

---

Since BHK-21 cell line has been used for recombinant GPCR expression

. . . I find it hard to believe that nobody has ever looked at (and reported on) the expression of endogenous GPCRs of these cells. But unbelievable things do happen.

In your 'results' sheet you get bottom and top values (lines 3 and 4) representing the upper and lower asymptotes of your DRC.  Set the difference to 100 for your reference curve/compound and relate values from other curves to it.

Hi Qian,

I am not sure why (perhaps a founder effect), but the most traditional way to report Ca2+ indicator signal is dF/F0. 

However, a change in that measure tells you very little in terms of biological activity (it's just a relative approximation of the concentration of calcium). You'll likely benefit from further processing to infer the underlying activity.

For example, nonnegative matrix deconvolution is often used to estimate the spike trains that cause the calcium signal increase. 

Edit: just saw you are interested in GPCRs so likely not using GCaMP strictly to infer spike activity but rather to see the GPCR activity. For that there is no need to deconvolve. See for example this publication from a member of my lab.

I am not sure of throwing any light but what is your aim?  SEAP assay or screening delta opioids?  

If the latter, measure cAMP by RIA, ELISA or the Cisbio kit in the presence of blockers of PDE. This will enhance your throughput and let you forget SEAP.  

On the agonist side you could try for example forskolin, which would mimic Gs activation.

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. 

There is more to consider than just the maths here. I would studiously avoid any situation in which you have around 10% DMSO as an intermediate dilution. It's all too easy to exceed aqueous solubility via this route and precipitate compound. If you start with 10mM DMSO stock then I would make the first into aqueous 1/100 at least. I would combine this idea with the comment above about keeping the same final conc of DMSO in all wells, (a VERY good idea). Therefore Step 1 would be to make an initial series of dilutions in neat DMSO. If you do this in micronic tubes / racks then this and all subsequent steps can be performed with a multichannel pipette. Step 2: transfer 10ul of each neat DMSO dilution to empty 1ml micronics. Step 3  Add 990ul of your culture medium to all tubes. This should ensure fast even dilution which avoids compounds exceeding aqueous solubility. Top compound conc is now 100uM. Now add 20ul to 180ul of cells. Top conc of compound is now 10uM and final conc of DMSO is 0.1%. This should be fine for most cells, especially CHO.K1 which are very robust, even if exposure is for 3-4 days. I would nevertheless include a zero DMSO control just for good practice, and to eliminate potential synergy with any other non-specific parameters.

Hmm, perhaps you can look at the papers that are cited in these two methods. One of them uses mastoparan as a positive control for mitochondrial swelling and the other uses it for lipid disruption. 

I suppose that in order to affect GPCR signalling you need to lower your concentration beyond what is used for disruption. At least these methods will give you an upper limit. 

My tutorial walks through this entire process:

Hello,

When PKA is activated, the regulatory domain (C subunit) split from the main enzyme.  You can simply measure the mass of the C subunit in your system.There are antibodies against this subunit.  You can do either histochemistry, ELISA, Western blottong,

Cheers

See a published report on this

It's unlikely you could detect GPCR interaction with G proteins via co-IP. When GPCRs are activated, they promote the exchange of GDP with GTP in the Galpha subunit. The Galpha-GTP then dissociates from receptors and beta-gamma complex. 

I  perform PSI and protein to protein blast, against PDB and found the following  match:

1. PBD ID: 1PV8 (42% match), 3OLP (30%), 1E51 (42%) and 3NA5 (27%).

All were chain A. Check whether these hits are GPCR or not and to which class they belong.

In determining the classes, I can perform statistical analysis for you, but I need more information. on the frequency of GPCR hits classifications, you got and those that did not see it as a GPCR , still analysis based on decision to classify it under the classes. whether class A or B. Also i need results on the hits within classes as well to give you a decision based on classes.

If materials involves any drug, L-DOPA increases striatal CB1 mRNA in rats (according with Zeng et al., 1999).

In addition, it seems that alcohol reduces CB1 expression in some brain structures while increases it in some others (but I dont remember the ref.)

Donot boil or heat the sample; this is what my colleagues who work with GPCRs suggest and do. They are not sure why this works. It likely prevents aggregation. Try it out to see if this improves your western blots.

Hi Anastasia, 

How many cysteines do you have in your protein? How many of them are in disulphide bonds?

How quickly are you heating your sample?

49 C sounds like a very reasonable melting temp. for a quite stable GPCR. And like Brian said you might have a two-step denaturation so part of it is (on average) melted at 49 C and another part at 56.5 C. 

I think it looks like an artifact though. What wavelength are you exciting at? And what machine?

Glucose isomerase is a very nice control for CPM, much better than betalactoglobulin in my experience. Look at my paper on TSA with SYPRO Orange (you can download from my profile) and try the experiment where you remove Mg2+ from the glucose isomerase (helps solubilise the microcrystals it is sold in) and then compare with and without MgSO4.

Okay, I just checked and you can't buy glucose isomerase from Hampton Research any more. If there are some protein crystallographers at your institution then go and ask them if they have some in the fridge that you can borrow a smidge of. It's a small, matte plastic bottle with a white label. There's some blue printed on it and it says Hampton Research and glucose isomerase 33 mg/ml in microcrystalline solution.

Other control experiments to do: If you add urea or guanidinium then the melting temperature should go down. This is also in my paper. 

I hope some of this helps and that you have time to give the extra details I asked for.

Best of luck!

While serum is a common positive control, some cells do not migrate toward it. For example, we find that pancreatic carcinomas migrate less toward serum or its major component, LPA, when compared to BSA. 10% serum can have very large amounts of LPA. At high levels, LPA and serum downregulate the main migratory LPA receptor, LPA1. At these levels of LPA, LPA2 receptor remains, but it does not always stimulate migration. I would recommend 100nM LPA as a good chemoattractant. But always remember to coat the transwells with matrix (e.g. fibronectin, collagen, laminin). Serum is handy because it supplies matrix (fibronectin and vitronectin) along with the chemoattractant. Personally, I didn't find the fluoroblock wells to work very well. My lab continues to count them manually.

Since your tissue is so small and precious, you may try to measure cAMP indirectly via its effects on PKA activation. One such way would be to do your treatment, fix the tissue with PFA, slice it and do IHC for phospho-PKA substrate (antibody from Cell Signaling). This is not specific for PKA, but gives you a hint.

There are also examples in the literature for IHC of cAMP and cGMP using antibodies against these  (http://www.jbc.org/content/272/50/31489.full) and the antibodies are commercially available (http://www.antibodyresource.com/search/Antibodies/2319c8e0-28e2-529b-9030-bdb39bb25769/camp/application/IHC-P).

Good luck!

Erik

It is difficult to understand whether the siRNA experiment is poorly explained in this thread or poorly conceived. In general, GEFs and GAPs are upstream of small GTPases. Therefore, knockdown of the GEF using siRNA and measuring Rac1 or cdc42 activation makes sense.

But the converse experiment in which you knockdown the GTPases is problematic. If you think about it, cells deficient in Rac1 will not make lamellipodia, and therefore you won't observe anything localizing to this type of structure, because it no longer exists. So you don't really learn anything meaningful about a direct protein-protein interaction between the GEF and the GTPase from this type of experiment. I think this is the reason that both David and Yan Cai were critical.

I agree with Filipe.  Also, try spiking your lysates with exogenous cAMP as a technical control. Protease activity could digest the ELISA Ab.

Cells should normally incorporate [3H]-myo-inositol during overnight culture. I have done that with COS-7, HEK-293 and CHOk1 cells - always worked without any problems. In my case optimal dose was 5ul [3H]-myo-inositol per ml of cell culture media. I assume you want to do the inositol phosphate accumulation assay. You can look up some of my papers, for example one of these: http://www.ncbi.nlm.nih.gov/pubmed/21036226 or http://www.ncbi.nlm.nih.gov/pubmed/22846991 for some details.

Thank you Daniel. I will make a pilot using DTT in presence or absence of ATP and compare that with non-activated PTX.

The PAQR family is a very ancient family, just like the GPCRs, but its numbers in human (11) is much smaller in GPCR numbers in human (300+).

Are there other family of membrane-bound proteins in human, with 7 transmembrane motifs, has this reverse topology? I don't think so. If there is, we would already have found them as all human genes are known now. You can easily validate my claim here by running  a TM model against all human protein sequences, and identify those with 4+ TM domains (so you don't miss any due to TM model's inaccuracy).  If you find any other members, that are not GPCR, and not PAQR, I think you get a paper. If that new family has the reverse-orientation to GPCR, then it is another PAQR-like family. Good luck with your hunting.

Dear Akhilesh, go through following papers. These papers might be helpful for you.

Cancer Res. 2006 Dec 15;66(24):11831-9.

Oncogene. 2013 May 16;32(20):2534-42.

Clin Cancer Res. 2011 Aug 1;17(15):4996-5004.

You can perform real time PCR to check expression at mRNA level or western blot/IHC to see protein expression.

All the best.

Vikram 

GTPγS or PTX are good options.

One extra point to consider is that mutations very often affect expression level and this alone can affect signalling. Sometimes these effects will be quite drastic in either a positive or negative direction. Although some changes are quite predictable many are not.

I did Westerns a few years ago, but had the same problem.  The way I worked around it was to determine which were the beta-arrestin2 and 1 bands (there were two bands for beta-arrestin2 in the wild-type mice I got from Dr. Lefkowitz), by seeing which band(s) were absent in the beta-arrestin1 and 2 KO brain tissue.  I always got labeling of beta-arrestin1 with the beta-arrestin2 Ab, but the beta-arrestin1 Ab was pretty clean and didn't show beta-arrestin2.

If you're labeling cells and not Western blots, I don't know what to tell you, if there's not a newer, more specific Ab available for Barr2.

It is just a matter of setting up your machine. The one we have is a CFX96 from bio-rad. http://www.bio-rad.com/pt-br/category/real-time-pcr-detection-systems. 

Mannan, intriguing!

G-Protein-coupled receptors: the release of second messengers is instrumental for the initiation of a series of intracellular events, including phosphorylation and the activation of enzymes; these enzymic changes, in turn, can activate transcription factors, intricately linked to cell identity. DNA transcription factors thus can influence the reacquisition of cellular memory post transcription (depending on the “intactness” (and robustness) of factors associated with transcription during this process, which would be acquired by the cell consequent to transcription.

Thank you!!

You could employ MicroScale Thermophoresis! This method would allow you to measure ligand-receptor interactions as well as receptor dimerization. Furthermore, you could investigate interactions in a buffer system including detergents using the purified receptors, or you could alternatively measure the interactions in liposomes or nanodiscs. Expressing fluorescent fusion proteins you even do not need to purify the receptors at all. Please find more information for MST measurements to investigate membrane proteins and dimerization in the attached publications!

use Tris glycine gels and run with 1mM DDT and LDS loading buffer and Do NOT heat the samples

PAR-1 is found on nearly every cell tested, so it is likely on THP-1 cells. And we find CCR1 is expressed by them as well.

An odd number allows N-terminal extra cellular and C-terminal intracellular (usually) and 7 helices permit the possibility to form a pore of ideal size for ions and small molecules.

Calcium is the best way to go. Simple, reproducible, highly sensitive, large signal-to-noise ratio. All you need is a fluorescence microscope, a suitable fluorophore (Fura-2 if you have the optics/light source to use this ratiometric assay; Fluo-4 if the only thing you have is a fluoresceine cube), ionomycin to transform fluorescence data into actual calcium concentrations. I personally use Fluo-4AM for all our work. It is less sensitive than the ratiometric Fura-2 assay, but since we do simultaneous imaging of other variables, Fluo-4 works best for us.

Hello,

there are two nice papers published in Endocrinology that can probably give you a good list of genes. See Datson et al 2011 and Polman et al 2013.

Good luck!

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?

Thanks for the reply but we are looking to cleave in in vitro or in vivo sytem......to make it more clear we want to cleave it in cells transfected with receptor and then to check its translocation...............and also in mouse tissues...can we do that

As was written above, all of the GPCRs that I have tagged with fluorescent proteins tolerated this addition very well. Yet many aspects of intracellular function could have variable kinetics, efficacy, and binding. Concerning the pathway of activation that you are interested in, it would be good to monitor those efficacy. Depending on the G protein, the arrestin binding, GRKs, internalisation, expression rates, dimerisation, etc, that you might be looking into, there could be some changes. But in my personal experience the twelve different GPCRs that I coupled to either GFP, EGFP, CFP or mCherry did not have any functional outcome that I noticed (yet there certainly would be variations to some degree, perhaps within the experimental variability that couldn't be easily monitored; no statistically different changes that I could observe), nor was I doing a full range of pleiotropic activation of all known interacting proteins. So the suggestion I have is simply to fully investigate the pathways that interest you in comparison to an untagged variant.