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Editor’S CornEr Cellular Logistics 2:3, 138–139; July/August/September 2012; © 2012 Landes Bioscience Kinetic and cell-based analyses of GTPase regulators nava Segev1,2 and richard A. Kahn3,* 1Landes Bioscience; Chicago, iL USA; 2department of Biological Sciences; University of illinois at Chicago; Chicago, iL USA; 3department of Biochemistry; School of Medicine; Emory University; Atlanta, GA USA Keywords: GTPases, exchange factor, ADP-ribosylation factor, GDP, GEF regulatory GtPases are often portrayed as binary molecular switches that control a wide range of cellular processes, including, but not limited to, the generation of second messengers (e.g., cAMP and inositol phosphates), intracellular traffic, cytoskeleton organization and cell proliferation. GEF stimulators and GAP inhibitors regulate the nucleotide- bound state of these proteins. Because of the relevance of GtPases and their regulators to human diseases, they comprise a major therapeutic target. Currently, most of the information about GtPase regulators comes from structure analyses. Such structural information is limited to certain conditions and does not necessarily reflect specificity or physiological activity. to address questions about specificity and mechanisms of action, kinetic and cell-based analyses of GtPase regulators is necessary. Here, we compare these two approaches in the context of regulators of Arf and heterotrimeric G-proteins. Introduction activities, they provide specificity as well than physiological concentrations. To as spatial and temporal regulation of reveal the basis of interactions between Regulatory GTPases in the heterotri- GTPase activation. GTPases and their regulators, two addi- meric G-protein and ADP-ribosylation G protein coupled receptors (GPCRs) tional approaches are currently being factor (Arf) families provide some inter- act as G-protein GEFs and the Arf GEFs employed: kinetic analyses of in vitro reac- esting parallels and contrasts in their to date all contain a conserved Sec7 tions using purified proteins, and moni- origins but also in the issues that drive domain. As described in Northup et al.,1 toring interactions in the context of cells current research and the construction of GPCRs are tremendously important for or cell lysates. While each approach has molecular models of their regulation. All clinical medicine and the pharmaceutical its advantages, it also has limitations that GTPases cycle between GTP- and GDP- industry as the number one class of target need to be considered. We think that the bound states, with consequent changes for drugs today. Though far less devel- article from Northup et al.1 argues con- in protein conformation and binding oped in chemotherapeutics, the Arf GEFs vincingly for both continued structural partners. Because the GTP/GDP ratio in have been mimicked by human pathogens work but also a clear need to couple it to cytosol is thought to be about 10, regula- (including Vibrio cholera and Legionella kinetic studies of enzymatic properties. tory GTPases would be overwhelmingly pneumophila), targeted for inhibition by In parallel, the article from Casanova2 in the active conformation except the rate fungi (Brefeldin A), or in small molecule summarizes nicely both the importance of spontaneous nucleotide exchange is screens (Golgicide A). As a result, spectac- of cell-based assays and a couple of the quite low, especially relative to the biologi- ular advances have been achieved in the approaches that are increasingly important cal process they control. Today it is gener- detailed structural analyses of each. These to research into GTPase regulation and ally assumed that activation by a guanine have allowed testable predictions of the biology, as well as cell biology in general. nucleotide exchange factor (GEF) is role of specific amino acid side chains in As always, the strongest arguments and required for the biological function of binding and hydrolysis. soundest models are those that encompass each GTPase. Indeed, nature has devised Structural analyses of GTPases and as many different approaches as possible. families of GEFs, often quite large, that their regulators were instrumental in our are specific to families or sub-families of current understanding of their mecha- Kinetic Analyses GTPases. There are typically more GEFs nisms of action. However, sole structure than their GTPase substrates and because approaches have their limitations. Both In this issue, Northup et al.1 make a the GEFs themselves are regulated and crystallography and NMR usually employ case for studying the kinetics of GEF- often contain additional domains and truncated or modified proteins in higher stimulated nucleotide exchange on Arfs *Correspondence to: Richard A. Kahn; Email: [email protected] Submitted: 10/22/12; Accepted: 10/23/12 http://dx.doi.org/10.4161/cl.22645 138 Cellular Logistics Volume 2 issue 3 Editor’S CAornEr EditoriAL and heterotrimeric G-proteins. Such stud- types of cell-based assays are discussed, being studied (Arf family members). ies can be used to test mechanistic hypoth- with variations on each type. When done without overexpression and in eses that emerge from structural studies The first type discussed is arguably not combination with compartmental mark- or obtain structure-function information a cell-based assay but a biochemical one ers, these assays are the gold standard for not available from structural studies. that is intended to capture changes in the determining the cellular location where a One nice example is using such assays to activation status of a GTPase occurring certain GTPase is stimulated by a certain address the question whether the GTPase- in the intact cells via specific pulldown GEF. Both types of cell-based analyses nucleotide-free form is a free intermediate from cell lysates. Such pulldowns require address regulator-GTPase specificity in or exists mostly when bound to the GEF. the generation of reagents that are capable a physiological context. Obviously, the Determining reaction rates and affini- of specific binding to the activated (GTP- imaging assays can also address the loca- ties is important not only for elucidating bound) GTPase with quantitative recov- tion of the interaction and, therefore, mechanisms of action, but also for phar- ery. An antibody with a conformational even highly localized interactions can be macological design and testing of poten- epitope specific to the activated species detected. tial therapeutics that target these hubs of would be a wonderful reagent but, to our cell signaling. knowledge, exists only for Ras. Instead, Perspectives While kinetic studies can be used also the most common reagents used in these for determining GEF-GTPase specific- assays consist of fusion proteins of the While both kinetic and cell-based ity, these analyses should be taken with domain from an effector that binds the approaches have their advantages, they each caution due to their reductionist nature. active GTPase, fused to a tag that can be have limitations. Therefore, getting infor- Kinetic studies employ purified proteins, used to quantitatively purify the complex mation from both approaches is essential usually expressed in bacteria, and depend from solution. GTPase-binding domains to the generation of hypotheses focused on on the availability of soluble and stable from effectors typically have the specific- mechanisms at atomic resolution and roles proteins. One important and underap- ity and affinity required of such reagents in biology/live cells as well as the ability to preciated aspect of such kinetic studies and we can expect to see many more gen- test them. Cell-based approaches can bet- is the ability (indeed the need) to charac- erated for different GTPases. The utility ter resolve issues concerning physiological terize each preparation and confirm that of such assays is not questionable. Neither regulator-GTPases specificity. Pulldown it recapitulates the native protein. For are their limitations, as pointed out by assays from cell lysates circumvent the practical reasons, these assays frequently Casanova. Caution should be used when need of other proteins or protein domains, use truncated proteins and therefore miss interpreting experiments that employ over- whereas microscopy-based assays also effects of other domains of the regulator expressed proteins as there are both theo- determine the site of interaction and are (GEF) or the substrate (GTPase), or miss retical and practical reasons to believe that superior in cases of localized stimulation effects of post-translational modifications, endogenously and exogenously expressed of a globally expressed GTPase. On the or of other molecules (proteins, lipids and GTPases or their modulators behave, other hand, kinetic analyses of purified cofactors) important for the interaction. localize and are regulated distinctly. regulators or their domains with GTPases The second type is a microscopic are instrumental for dissecting molecular Cell-Based Analyses approach that determines regulator- mechanisms suggested by structural stud- GTPase interaction inside cells and ies, which are essential for designing drug Casanova2 discusses the importance of requires fluorescent reporters. Such assays therapies. We hope that the two accompa- cell-based assays for regulation of Arfs by follow bi-molecular interactions of a nying reasoned debate papers in this issue GEFs. These assays can be used for deter- GTPase with a GEF or an effector using would encourage researchers to consider mining the physiological specificity of fluorescence microscopy. The require- including these approaches in future stud- regulator-GTPase interactions. This arti- ments of these assays for fluorescent tag- ies aimed at unraveling GTPase regula- cle also makes the point that the issues are ging and (typically) protein overexpression tion as well as drive further research into largely the same for the GEFs, which acti- are key limitations. While these are always improved methods capable of addressing vate the GTPase and downstream path- issues of concern to researchers using fluo- these important issues that are highly rel- ways, and the GAPs that may terminate rescent tags, it is particularly acute when evant to a large fraction of cell signaling or mediate the GTPase signal. Two basic the tag (GFP) is larger than the protein and regulation studies. References 1. Northup JK, Jian X, Randazzo PA. Nucleotide exchange factors: Kinetic analyses and the rationale for studying kinetics of GEFs. Cell Logist 2012; 2:140-6; http://dx.doi.org/10.4161/cl.21627 2. Casanova JE. Advantages and limitations of cell-based assays for GTPase activation and regulation. Cell Logist 2012; 2:147-50; http://dx.doi.org/10.4161/cl.22045. www.landesbioscience.com Cellular Logistics 139