Oleg G. Kisselev

The G-Protein βγ Complex

Abstract The vast majority of signalling pathways in mammalian cells are mediated by heterotrimeric (αβγ) G proteins. Reviewed here is regulation of signal transduction by the βγ complex at different protein interfaces: subunit–subunit, receptor–G protein and G protein–effector. The role of diverse β and γ subunit types in achieving specificity in signalling and potentially unidentified functions for these subunits also are discussed.

Efficient interaction with a receptor requires a specific type of prenyl group on the G protein gamma subunit.

Post-translational prenylation of the carboxyl-terminal cysteine is a characteristic feature of the guanine nucleotide-binding protein (G protein) γ subunits. Recent findings show that the farnesylated COOH-terminal tail of the γ1 subunit is a specific determinant of rhodopsin-transducin coupling. We show here that when synthetic peptides specific to the COOH-terminal tail of γ1 are chemically modified with geranyl, farnesyl, or geranylgeranyl groups and tested for their ability to interact with light activated rhodopsin, the farnesylated peptide is significantly more effective. These results show that an appropriate isoprenoid on the G protein γ subunit serves not only a membrane anchoring function but in combination with the COOH-terminal domain specifies receptor-G protein coupling.

Rhodopsin Controls a Conformational Switch on the Transducin γ Subunit

Abstract Rhodopsin, a prototypical G protein-coupled receptor, catalyzes the activation of a heterotrimeric G protein, transducin, to initiate a visual signaling cascade in photoreceptor cells. The βγ subunit complex, especially the C-terminal domain of the transducin γ subunit, Gtγ(60–71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin α subunit by light-activated rhodopsin. We report that this domain is unstructured in the presence of an inactive receptor but forms an amphipathic helix upon rhodopsin activation. A K65E/E66K charge reversal mutant of the γ subunit has diminished interactions with the receptor and fails to adopt the helical conformation. The identification of this conformational switch provides a mechanism for active GPCR utilization of the βγ complex in signal transfer to G proteins.

G-protein betagamma-complex is crucial for efficient signal amplification in vision.

A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. Although much is now known about the role of the α-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary βγ-complex in this process remains a mystery. Here, we show that elimination of the transducin γ-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin βγ-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions.

Rhodopsin-Transducin Interface: Studies with Conformationally Constrained Peptides

To probe the interaction between transducin (G(t)) and photoactivated rhodopsin (R*), 14 analog peptides were designed and synthesized restricting the backbone of the R*-bound structure of the C-terminal 11 residues of G(t)alpha derived by transferred nuclear Overhauser effect (TrNOE) NMR. Most of the analogs were able to bind R*, supporting the TrNOE structure. Improved affinities of constrained peptides indicated that preorganization of the bound conformation is beneficial. Cys347 was found to be a recognition site; particularly, the free sulfhydryl of the side chain seems to be critical for R* binding. Leu349 was another invariable residue. Both Ile and tert-leucine (Tle) mutations for Leu349 significantly reduced the activity, indicating that the Leu side chain is in intimate contact with R*. The structure of R* was computer generated by moving helix 6 from its position in the crystal structure of ground-state rhodopsin (R) based on various experimental data. Seven feasible complexes were found when docking the TrNOE structure with R* and none with R. The analog peptides were modeled into the complexes, and their binding affinities were calculated. The predicted affinities were compared with the measured affinities to evaluate the modeled structures. Three models of the R*/G(t)alpha complex showed strong correlation to the experimental data.

Electron paramagnetic resonance studies of functionally active, nitroxide spin-labeled peptide analogues of the C-terminus of a G-protein alpha subunit.

The C-terminal tail of the transducin alpha subunit, Gtalpha(340-350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogues of Gtalpha(340-350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin-label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin-label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC(343)-Gtalpha(340-350) revealed greater immobilization of the nitroxide when compared to that of the N-terminally modified Proxyl-Gtalpha(340-350) analogue. A doubly labeled Proxyl/TOAC(348)-Gtalpha(340-350) was examined by DEER spectrocopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin-labels in this GPCR-G-protein alpha-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface.

Conformational Changes in the Phosphorylated C-terminal Domain of Rhodopsin during Rhodopsin Arrestin Interactions

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.

Modulating G-Protein Coupled Receptor/G-Protein Signal Transduction by Small Molecules Suggested by Virtual Screening

Modulation of interactions between activated GPCRs (G-protein coupled receptors) and the intracellular (IC) signal transducers, heterotrimeric G-proteins, is an attractive, yet essentially unexplored, paradigm for treatment of certain diseases. Regulating downstream signaling for treatment of congenital diseases due to constitutively active GPCRs, as well as tumors where GPCRs are often overexpressed, requires the development of new methodologies. Modeling, experimental data, docking, scoring, and experimental testing (MEDSET) was developed to discover inhibitors that target the IC loops of activated GPCRs. As proof-of-concept, MEDSET developed and utilized a model of the interface between photoactivated rhodopsin (R*) and transducin (Gt), its G-protein. A National Cancer Institute (NCI) compound library was screened to identify compounds that bound at the interface between R* and its G-protein. High-scoring compounds from this virtual screen were obtained and tested experimentally for their ability to stabilize R* and prevent Gt from binding to R*. Several compounds that modulate signal transduction have been identified.

The arrestin‐bound conformation and dynamics of the phosphorylated carboxy‐terminal region of rhodopsin

Visual arrestin binds to the phosphorylated carboxy‐terminal region of rhodopsin to block interactions with transducin and terminate signaling in the rod photoreceptor cells. A synthetic seven‐phospho‐peptide from the C‐terminal region of rhodopsin, Rh(330–348), has been shown to bind arrestin and mimic inhibition of signal transduction. In this study, we examine conformational changes in this synthetic peptide upon binding to arrestin by high‐resolution proton nuclear magnetic resonance (NMR). We show that the peptide is completely disordered in solution, but becomes structured upon binding to arrestin. A control, unphosphorylated peptide that fails to bind to arrestin remains highly disordered. Specific NMR distance constraints are used to model the arrestin‐bound conformation. The models suggest that the phosphorylated carboxy‐terminal region of rhodopsin, Rh(330–348), undergoes significant conformational changes and becomes structured upon binding to arrestin.

G-protein alpha and beta-gamma subunits interact with conformationally distinct signaling states of rhodopsin.

Light activated rhodopsin interacts with domains on all three subunits of transducin. Two of these domains, the C-terminal regions of the alpha and gamma subunits mimic the ability of transducin to stabilize the active conformation of rhodopsin, metarhodopsin II, but display different roles in transducin activation process. Whether the interactions are with the same or different complimentary sites on Meta II is unknown. We have used chemo-selective thioalkylation of rhodopsin and UV/visible spectroscopy to show that interactions with transducin C-terminal domains can be selectively disrupted. These data provide evidence that formal structural determinants on Meta II for these domains of transducin are different. In a set of complimentary experiments we examined the reactivity of Meta II species produced in the presence of the Gtalpha and Gtgamma subunit peptides to hydroxylamine. Analysis of the rates of Meta II decay confirms that the conformational states of Meta II when bound to Gtalpha and Gtbetagamma represent distinct signaling states of rhodopsin.