ABSTRACT
Low-molecular-weight guanosine triphosphatase proteins (small GTPases, a form of G-protein distinct from heterotrimeric G-proteins) were among the first
5.1 Small GTPase Biosensor Design 137 5.2 Rho-Family GTPases 145
5.2.1 Rac1 145 5.2.2 RhoA 147 5.2.3 Cdc42 148
5.2.3.1 Dye-Based Cdc42 Biosensors 149 5.2.3.2 Cdc42 Translocation Biosensors 150 5.2.3.3 FRET-Based Cdc42 Biosensors 151
5.3 Ras-Family GTPases 153 5.3.1 Ras 153
5.3.1.1 Ras Translocation Biosensors 154 5.3.1.2 FRET-Based Ras Biosensors 154
5.3.2 Rap 156 5.4 Other GTPase Families 157
5.4.1 Ran 157 5.4.2 Arf6 158
5.5 Conclusion 159 References 160 Supplementary Readings 166
molecules targeted for study with biosensors. Their long and interesting history illustrates the evolution of biosensor design, principles, and approaches. Although there are some exceptions, GTPase proteins almost always exist in one of two conformations-an inactive conformation bound to GDP and an active conformation bound to GTP. It is only in this active conformation that GTPases can interact productively with their downstream effector proteins (Figure 5.1). In general, the activation of GTPases is regulated by three classes of upstream proteins: guanine nucleotide exchange factors (GEFs), which mediate the binding of GTP; GTPase activating proteins (GAPs), which accelerate the hydrolysis of GTP to GDP; and guanine nucleotide dissociation inhibitors (GDI), which bind the GDP-bound form of the proteins (Bar-Sagi and Hall 2000; Reuther and Der 2000; Takai et al. 2001; Jaffe and Hall 2005). The goal of most biosensors is to track the transient localization and formation of the GTPbound, “activated” conformation of the GTPase in living cells and animals. For example, the cycling between active and inactive nucleotide states is tightly coupled to changes in membrane localization for many GTPases. In other cases, regulatory mechanisms such as phosphorylation and degradation can influence the activity and localization of the protein. In designing a biosensor, one strives for maximum sensitivity by producing the brightest possible sensor, and for some designs, the greatest possible difference between the fluorescence
Figure 5.1 The Rho GTPase regulatory cycle. Rho GTPases are molecular switches that cycle between GDP-bound (inactive) and GTP-bound (active) states. Guanine dissociation inhibitors (GDIs) sequester GDP-bound GTPases in the cytoplasm. At the membrane (potentially still associated with GDIs), GTPases are activated by guanine nucleotide exchange factors (GEFs), which promote the exchange of GDP for GTP. Active GTPases interact with multiple effector proteins that govern a wide range of cell responses and behaviors. GEF activity is opposed by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis, thereby returning GTPases to their GDP-bound state.
