Annie Otto-Bruc

Dissociation of GDP Dissociation Inhibitor and Membrane Translocation Are Required for Efficient Activation of Rac by the Dbl Homology-Pleckstrin Homology Region of Tiam

Small G proteins of the Rho/Rac/Cdc42 family are associated with lipid membranes through their prenylated C termini. Alternatively, these proteins form soluble complexes with GDI proteins. To assess how this membrane partitioning influences the activation of Rac by guanine nucleotide exchange factors, GDP-to-GTP exchange reactions were performed in the presence of liposomes using different forms of Rac-GDP. We show that both non-prenylated Rac-GDP and the soluble complex between prenylated Rac-GDP and GDI are poorly activated by the Dbl homology-pleckstrin homology (DH-PH) domain of the exchange factor Tiam1, whereas prenylated Rac-GDP bound to liposomes is activated about 10 times more rapidly. Sedimentation experiments with liposomes reveal that the DH-PH region of Tiam1 forms, with nucleotide-free prenylated Rac, a membrane-bound complex from which GDI is excluded. Taken together, these experiments demonstrate that the dissociation of Rac-GDP from GDI and its translocation to membrane lipids favor DH-PH-catalyzed nucleotide exchange because the steric hindrance caused by GDI is relieved and because the membrane environment favors functional interaction between the DH-PH domain and the small G protein.

GTP-dependent binding of Gi, Go and Gs to the γ-subunit of the effector of Gt

The γ‐subunit of the cGMP‐phosphodiesterase (PDEγ) of retinal rods forms a tight complex with the activated α‐subunit of transducin (GtαGTPγS). We observe that while PDEγ is not the physiological effector of other Gα subtypes, it can still detectably interact with them. This interaction is strong with Gilα and Gi3α (K d ≈ 10 nM) and weaker with Goα and Ggα (K d ≈ 1 μM). For all these Gα subtypes, similar intrinsic fluorescence changes are observed upon PDEγ binding. Moreover, similar relative decreases in affinity are obtained when the GDP forms of Gilα, Gi3α or Gtα are used in lieu of the GTP forms. This points to a conserved GTP‐dependent effector‐interaction domain.

[16] Use of α-toxin-permeabilized photoreceptors in in vitro phototransduction studies

Publisher Summary In vitro assays utilizing purified retinal proteins have been a cornerstone of biochemical studies of phototransduction. However, many studies have relied on purification and assay techniques that destroy photoreceptor integrity, and consequently, require the evaluation of dynamic enzymatic interactions under nonphysiological conditions. In vitro study of phototransduction, using intact photoreceptors, is an appealing alternative to conventional biochemical techniques. This strategy preserves conditions for enzymatic interactions that more closely resemble those found in vivo . A variety of permeabilization strategies have been developed to overcome the physical barrier imposed by the plasma membrane. One of these techniques, toxin-mediated poration, has been used in a growing number of in vitro studies in both whole cells and membranebound organelles. This technique was valuable in our own studies of rhodopsin phosphorylation in intact photoreceptors. The utility of the α-toxin as a permeabilizing agent has been substantially enhanced through the engineering of gateable pores composed of mutant α-toxin proteins.

Interaction of the Retinal Cyclic Gmp-Phosphodiesterase Inhibitor with Transducin (Gt) and with Other G-Proteins (Gi, Go and Gs)

Heterotrimeric G-proteins act as signal transducers at the cytoplasmic face of the cell’s plasma membrane by interacting first with an activated receptor and then with an effector such as an enzyme or a channel (Gilman, 1987; Ross, 1989). The first interaction leads to the activation of the G protein, i.e., the switching of its α-subunit from an inactive, GDP-bound state to an active, GTP-bound state. The G-protein a-subunit (Gα), now bearing GTP, binds to and turn on an effector. G-protein effector coupling is commonly studied by monitoring changes in the levels of a second messenger, which reflects effector activity. However, such measurements are generally too indirect to allow accurate determinations of the kinetic parameters that describe the G-protein effector interaction. Here, we summarize recent studies where intrinsic tryptophan fluorescence was used as a biophysical means to monitor the interaction between transducin (Gt), the G-protein of the visual system and its effector target, the retinal cGMP-phosphodiesterase (PDE).