In my previous post Small GTPases: Measuring small G protein activation I looked at state-of the-art methods for measuring the activation of small G proteins, such as RhoA, Rac1, Cdc42, and the proto-oncogen Ras. Today, I invite you to explore some methods for measuring the activity of Guanine nucleotide Exchange Factors (GEFs).

Small G Protein Activation

Fig. 1: Small G protein activation cycle.

GEFs stimulate the release of GDP to allow the binding of GTP (which activates the small G protein) and of GTPase-activating proteins (GAPs) which stimulate the GTPase activity of small G proteins to inactive them

The Ras superfamily of small G proteins consists of more than 150 members which fall into five main families: Ras, Rho, Ran, Rab and Arf.

The Rho subfamily consists of proteins like RhoA, Rac1, and Cdd42. These proteins have been shown to be involved in the regulation of actin dynamics, thus playing a crucial role in processes like cell movement, intracellular transport, and organelle development. While RhoA affects actin stress fibers, Rac1 exhibits effects on lamellipodia and Cdc42 on filopodia.

Small G proteins  cycle between the inactive, GDP-bound form and the active, GTP-bound form (Fig. 1). The balance of the GTP to GDP bound state underlies the switch mechanism as they turn from an activated (GTP form) to inactive state (GDP form). The balance of GTP to GDP bound states is controlled by catalytic proteins that either increase the rate of exchange of GDP for GTP (GEFs), increase the GTPase activity (GAPs), or prevent the exchange of GDP (Guanosine Nucleotide Dissociation Inhibitor or GDI).

p50 RhoGAP activity measured by GTP hydrolysis by RhoA and Rac1 protein.

Fig. 2: p50 RhoGAP activity measured by GTP hydrolysis by RhoA and Rac1 protein. Sample 1 = Rac1 only, sample 2 = Rac1 and p50 RhoGAP, sample 3 = RhoA only, sample 4 – RhoA and p50 RhoGAP, and sample 5 – p50 RhoGAP alone. Reactions were incubated at 37ºC for 20 min followed by the addition of CytoPhos reagent for 10 min to determine the phosphate generated by the hydrolysis of GTP.

It has been shown that the GAP family of proteins is large (70 members) and potentially important for changing a cell from a normal to disease status. Likewise a number of GEF proteins have been identified as oncogenes and are involved in human disease such as cancer. Interestingly, the expression of GEF proten is tissue or cell type specific, providing a therapeutic potential for cancer treatment.

In order to facilitate the exploration of this molecular switch, ready-to-use GAP and GEF activity assays have been designed for research applications.

Reveal the GAP activity

The majority of assumed GAP proteins have only been implicated by homology to contain GAP activity. Thus the real function of putative GAPs can be elucidated with dedicated GAP Assay Kits (Fig. 2. Source: Cytoskeleton, Inc.) which include small GTPase proteins (Ras, RhoA, CDC42 and Rac1). Interestingly, these assays enable the screenings of ligands and inhibitors for pharmaceutical studies.

Detect putative GEF activity

The classical approach to measure GEF activity is based on radioactive methods. To eliminate the need for these labeling methods, fluorescent GEF assay have been released by using fluorescent guanine nucleotide analog mant (N-methylanthraniloyl – excitation at 360nm (+/-10nm) – emission  at 440nm (+/-20nm)).

Once bound to GTPases, the fluorophore emission intensity increases dramatically approximately 2 fold. Therefore, the enhancement of fluorescent intensity in the presence of small GTPases and GEFs will reflect the respective GEF activities of known or unknown proteins (Fig. 3).

Cdc42 exchange assay.

Fig. 3: Cdc42 exchange assay. The small GTPases Cdc42 and the human Dbs protein were tested. The reactions were conducted in a 96 well black flat bottom half area plate format. Each reaction contains 1 μM GTPases, 50 μg/ml BSA, 20 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl2, and 0.8 μM mant-GTP with or without the presence of 0.5 μM human Dbs(DH/PH) protein.

Like the GAP assay kit, the Rho GEF Exchange Assay Kit comes with human Cdc42, Rac1 and RhoA proteins as potential targets for the putative GEF to be measured. This assay is ideal for measure the activity of putative GEFs but also to screen for GEF inhibitors in a high throughput format.

Interested in measuring GAP and GEF activity?

Feel free to leave your questions or comments below!

 

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Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.