Alexey E. Granovsky

Regulation of transducin GTPase activity by human retinal RGS.

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory γ-subunit of PDE (Pγ), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin α-subunit, Gtα GDPAlF 4 − , while its binding to GtαGTPγS or GtαGDP was weak. hRGSr and Pγ did not compete for the interaction with Gtα GDPAlF 4 − . Affinity of the Pγ-Gtα GDPAlF 4 −interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of Gtα GDPAlF 4 −binding to Pγ labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PγBC). Binding of hRGSr to Gtα GDPAlF 4 −complexed with PγBC resulted in a maximal ∼40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for Gtα GDPAlF 4 −(K d 35 nm). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s−1. Addition of Pγ to the reconstituted system reduced the GTPase level accelerated by hRGSr (k cat 0.085 s−1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 μm rhodopsin these rates exceeded 0.7 s−1. Our data suggest that effects of hRGSr on transducin’s GTPase activity are attenuated by Pγ but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.

Identification of the γ Subunit-interacting Residues on Photoreceptor cGMP Phosphodiesterase, PDE6α′

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory γ subunit (Pγ). Chimeric proteins between cone PDE6α′ and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pγ. Substitution of the segment PDE5-(773–820) by the corresponding PDE6α′-(737–784) sequence in the wild-type PDE5 or in a PDE5/PDE6α′ chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pγ. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pγ-binding region, PDE6α′-(750–760), revealed PDE6α′ residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pγ. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met758 and Gln752directly bind Pγ. A model of the PDE6 catalytic site shows that PDE6α′-(750–760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pγ to Met758 would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.

Roles of the Transducin α-Subunit α4-Helix/α4-β6 Loop in the Receptor and Effector Interactions

The visual GTP-binding protein, transducin, couples light-activated rhodopsin (R*) with the effector enzyme, cGMP phosphodiesterase in vertebrate photoreceptor cells. The region corresponding to the α4-helix and α4-β6 loop of the transducin α-subunit (Gtα) has been implicated in interactions with the receptor and the effector. Ala-scanning mutagenesis of the α4-β6 region has been carried out to elucidate residues critical for the functions of transducin. The mutational analysis supports the role of the α4-β6 loop in the R*-Gtα interface and suggests that the Gtα residues Arg310 and Asp311 are involved in the interaction with R*. These residues are likely to contribute to the specificity of the R* recognition. Contrary to the evidence previously obtained with synthetic peptides of Gtα, our data indicate that none of the α4-β6 residues directly or significantly participate in the interaction with and activation of phosphodiesterase. However, Ile299, Phe303, and Leu306 form a network of interactions with the α3-helix of Gtα, which is critical for the ability of Gtα to undergo an activational conformational change. Thereby, Ile299, Phe303, and Leu306play only an indirect role in the effector function of Gtα.

Identification of Effector Residues on Photoreceptor G Protein, Transducin

Transducin is a photoreceptor-specific heterotrimeric GTP-binding protein that plays a key role in the vertebrate visual transduction cascade. Here, using scanning site-directed mutagenesis of the chimeric Gαt/Gαi1 α-subunit (Gαt/i), we identified Gαt residues critical for interaction with the effector enzyme, rod cGMP phosphodiesterase (PDE). Our evidence suggests that residue Ile208 in the switch II region directly interacts with the effector in the active GTP-bound conformation of Gαt. Residues Arg201, Arg204, and Trp207are essential for the conformation-dependent Gαt/effector interaction either via direct contacts with the inhibitory PDE γ-subunit or by forming an effector-competent conformation through the communication network between switch II and the switch III/α3-helix domain of Gαt. Residues His244 and Asn247 in the α3 helix of Gαt are responsible for the conformation-independent effector-specific interaction. Insertion of these residues rendered the Gαt/i chimera with the ability to bind PDE γ-subunit and stimulate PDE activity approaching that of native Gαt. Comparative analysis of the interactions of Gαt/i mutants with PDE and RGS16 revealed two adjacent but distinct interfaces on transducin. This indicates a possibility for a functional trimeric complex, RGS/Gα/effector, that may play a central role in turn-off mechanisms of G protein signaling systems, particularly in phototransduction.

Probing functional interfaces of rod PDE γ-subunit using scanning fluorescent labeling

In the dark, the activity of the rod cGMP phosphodiesterase (PDE) catalytic α- and β-subunits (Pαβ) is inhibited by two γ-subunits (Pγ). On light stimulation of the photoreceptor cells, the GTP-bound α-subunit of visual G-protein transducin (GtaGTP) displaces the Pγ-subunits from their inhibitory sites on Pαβ, leading to the effector enzyme activation. We designed a number of Pγ mutants, each with a single cysteine residue evenly distributed at a different position along the Pγ polypeptide chain. These cysteine residues served as sites for the introduction of the environmentally sensitive fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (BC). Analysis of the interactions of Pαβ and Gta with the fluorescently labeled Pγ mutants suggests two distinct functional interfaces of Pγ. The Pαβ/Pγ interface is formed essentially by the C-terminus of Pγ and by the N-terminal portion of the Pγ polycationic region, Pγ-24-45, whereas the Pγ/Gta interface includes the C-terminal portion of Pγ-24-45 and the region surrounding Pγ Cys68. Such functional organization of Pγ may represent an important element for the PDE activation mechanism during transduction of visual signals.

Assays of G protein/cGMP-phosphodiesterase interactions

Activation of cGMP phosphodiesterase (PDE) by the photoreceptor G protein transducin (G 1 ), is a key event in the vertebrate visual transduction cascade. The assays of cGMP hydrolysis by activated PDE have been a major tool for monitoring the interaction between transducin and PDE, and initial studies mainly relied on such assays. The presence of rod outer segment (ROS) membranes or lipid vesicles significantly enhances the effectiveness of PDE stimulation because of the formation of an active membrane-bound complex between G 1 α and PDE and allows an increase in assay sensitivity. However, membrane-supported activation of PDE by transducin is a complex process that depends on a number of factors such as type and concentration of membranes {vesicles), binding of PDE, binding of G 1 α to membranes, and the intact stale of lipid modifications on Pαβ and G 1 α. Furthermore, PDE activation is not a direct monitor of transducin binding to the enzyme. This is evident from studies of G 1 α mutants that bind but fail to activate PDE, or interact with the effector weakly without causing enzyme activation.