Cell signalling – Discover G-LISA to study Small GTPases

As is already known, multiple families of small GTPases (Ras, Arf and Rho families) make up the Ras cell signalling - GTPase molecular cycleGTPase superfamily. Due to their role as molecular switches in cell signalling (active GTP-bound state vs inactive GDP-bound states) and in many other cellular responses (cytoskeletal reorganization, regulation of transcription, apoptosis…), these small GTPases are the subject of intense investigation to try to understand the mechanisms that regulate activation and inactivation of this proteins.

In this post, I invite you to discover a revolutionary way to study these cell signalling events by quantifying the level of active small GTPases in your experimental model.

As said above, the Ras, Arf and Rho families belong to the Ras GTPase superfamily; known to be crucial in cell signalling. Among them, the Ras and Arf families consist of many small G-proteins, including Ras and Ral isoforms in the former while Arf isoforms populate the latter.  The Rho family of small GTPases consists of at least 20 members, the most extensively characterized of which are the RhoA, Rac1 and Cdc42 proteins.

As mentioned in a previous post, Cytoskeleton have developed two ways to measure the level of activation of a Ras, Arf, and Rho protein. The pull down assay and the new G-LISA assay. Even if each assay has its advantages, the G-LISA has been developed to address some shortcomings of the Pull Down assay such as: Long experimental time, large amount of sample required , not very consistent and not suitable for plate reader technology.

Cell signalling - Simple procedure of the G-Lisa assay

Fig 1. Simple procedure of the G-Lisa assay

What are the advantages of the G-LISA technology for your cell signalling studies?

Based on the ELISA principal, the G-LISA assay uses a 96-well plate coated with a binding domain of a Ras, Arf, or Rho-family effector protein.  The active GTP-bound form of the Ras, Arf, or Rho-family protein, but not the inactive GDP-bound form, from a biological sample will bind to the plate, respectively.  The bound active Ras, Arf, or Rho-family protein is then detected by incubation with a specific primary antibody followed by a secondary antibody conjugated to HRP. The signal is then developed with OPD or chemiluminescence reagents.

As shown in Fig.1 these assays are based on a simple and fast (<3 hours) protocol, they require only small amounts of sample (5-50 µg cell protein) and they yield quantitative and accurate data.

G-LISA assays not only improve current experimental design, but enable technology to facilitate cell signalling experiments that were not possible with traditional pull-downs. For instance, cell cultures in 3D matrices grown for 10 or more days can now be analysed for GTPase activation (1). The G-LISA assays also provide detection accuracy and sensitivity that allows analyses of GTPase activity in preparations previously off-limits with pull-down assays (2,3).

Recent studies directly compared the G-LISA activation assays with pull-downs and the advantages for studying cell signalling are clear:

  • G-LISA assays are superior due to the ability to use small amounts of protein (1,4,5), their greater sensitivity (1,3,5) and quantitative measurements (2).
  • The G-LISA kits are available in either luminometric or colorimetric detection versions.  The assays are identical except for the final detection step.  In general: the luminometric assays are more sensitive, while the colorimetric assays have a slightly lower level of variance.

Cell Signalling studies with G-LISAs – Practical experimental results

The G-LISA kits have been tested on several mammalian cell lines and with different well-known Rho activating stimuli (download the list of tested cell types).  All kits give results that are similar to those seen with conventional cell signallingpull-down assays.  Luminometric and colorimetric assays give comparable results.

To illustrate all this point, here are typical G-Lisa results:

Cell signalling -  practical results obtained with Cytoskeleton's G-LISAs

Fig. 2 : Cell signalling studies – Measurement
of RhoA activation by LPA in different cell type.
Blue = Serum Starved, Yellow = Activated.

In Figure 2, you can see the measurement of RhoA activation after treatment by Lysophospatidic acid (LDA) in different cell types with the RhoA G-LISA Activation Assay (#BK121). Here, Swiss 3T3 (mouse), A431 (human) and HeLa (human) cells were first serum starved and then stimulated by LPA. For this test 25 µg of lysates were subjected to the G-LISA assay, and the data shown relative luminescence units (RLU) over background signal (wells incubated with lysis buffer alone instead of cell lysates).

 

Cell signalling -  practical results obtained with Cytoskeleton's G-LISAs

Fig. 3 : Cell signalling studies – Rho activity measured in Swiss 3T3 cells treated with the Cell Permeable Rho Inhibitor (CT04)

Now in Swiss 3T3 cells the activation of Rhoa has been measured after treatment of these cells with different concentrations of a Cell permeable Rho Inhibitor (CT04). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 0.20, 0.50 and 2.0 µg/ml of CT04 for 4 h in serum free medium at 37°C, then activated with 100 µg/ml calpeptin for 10 min.  Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (#BK124).  Note: At 2.0 µg/ml CT04 for 4 h results in almost complete (90%) inhibition of RhoA activity. Figure 3 we can clearly see a decrease of RhoA activation after treatment by increase concentration of the CT04.

Cell signalling -  practical results obtained with Cytoskeleton's G-LISAs

Fig 4. Cell signalling studies – Time course of RhoA activation by LPA, calpeptin and CNF (Cat.# CN03) in Swiss 3T3 cells

And finally to compare the effect of different compound, a time course of RhoA activation was performed on Swiss 3T3 cells (fig. 4). The 3T3 cells were treated by 10 µg/ml LPA, 100 µg/ml calpeptin (Cat.# CN01) and 1 µg/ml CNF (Cat.# CN03) in Swiss 3T3 cells. Serum-starved cells were stimulated with activator for the times shown in the graph and RhoA activation was analysed with the G-LISA kit (#BK121). LPA stimulation leads to a rapid and transient activation of RhoA, which peaks at 2-3 min and quickly declines to basal levels. Activation by calpeptin leads to a signal increase between 10-30 min which declines to basal after 60 min. Activation by CNF leads to signal increase after 60 min which is stable for up to 12 h.

For more convenience and ease of use in your cell signalling experiments, each G-LISA kits contain all the reagents needed for complete cell signalling studies.  All you need are your cell or tissue samples, a platform shaker and a microtiter plate compatible luminometer or spectrophotometer.  You’ll have enough material in the kit for 96 activation assays.  The affinity wells can be separated from each other, so you can run anywhere from 2-96 assays per experiment and each kit can be used for numerous separate experiments.

Which cell signalling pathway and small G proteins can be measured with these kits?

G-LISA assays are available for:

Arf1 Arf6 Cdc42 Rac1 Rac1/2/3RalA Ras RhoA RhoA/Rac1/Cdc42 combo

Don’t hesitate to browse this cell signalling Small GTPase product guide to find others products suited to your applications.

Do you need some tips on choosing the right G-LISA Small GTPases Activation Assays for your cell signalling experiments? Don’t hesitate to contact our specialist through the comments form below with your questions, or to directly get in touch with your local tebu-bio office – we’ll be pleased to help!

Frédéric Samazan
Written by Frédéric Samazan
Frédéric Samazan is a Product Manager at tebu-bio, and an avid football fan in his spare time.