Nikolai P. Skiba

Molecular determinants of selectivity in 5-hydroxytryptamine1B receptor-G protein interactions.

The recognition between G protein and cognate receptor plays a key role in specific cellular responses to environmental stimuli. Here we explore specificity in receptor-G protein coupling by taking advantage of the ability of the 5-hydroxytryptamine1B (5-HT1B) receptor to discriminate between G protein heterotrimers containing Gαi1 or Gαt. Gi1 can interact with the 5-HT1B receptor and stabilize a high affinity agonist binding state of this receptor, but Gt cannot. A series of Gαt/Gαi1 chimeric proteins have been generated in Escherichia coli, and their functional integrity has been reported previously (Skiba, N. P., Bae, H., and Hamm, H. E. (1996) J. Biol. Chem. 271, 413–424). We have tested the functional coupling abilities of the Gαt/Gαi1 chimeras to 5-HT1Breceptors using high affinity agonist binding and receptor-stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding. In the presence of βγ subunits, amino acid residues 299–318 of Gαi1 increase agonist binding to the 5-HT1Breceptor and receptor stimulation of GTPγS binding. Moreover, Gαi1 containing only Gαt amino acid sequences from this region does not show any coupling ability to 5-HT1B receptors. Our studies suggest that the α4 helix and α4-β6 loop region of Gαs are an important region for specific recognition between receptors and Gi family members.

The βγ Subunit of Heterotrimeric G Proteins Interacts with RACK1 and Two Other WD Repeat Proteins

A yeast two-hybrid approach was used to discern possible new effectors for the βγ subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gβ, each exhibiting the WD40 repeat motif. Two of these proteins, thereceptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gβγ using an anti-Gβ antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gβ1γ1 but also unexpectedly with the transducin heterotrimer Gαtβ1γ1. Gαtalone does not interact, but it must contribute to the interaction because the apparent EC50 value of RACK1 for Gαtβ1γ1 is 3-fold greater than that for Gβ1γ1 (0.1 versus0.3 μm). RACK1 is a scaffold that interacts with several proteins, among which are activated βIIPKC and dynamin-1 (1). βIIPKC and dynamin-1 compete with Gβ1γ1and Gαtβ1γ1 for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gβγ interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.

The α-Helical Domain of Gαt Determines Specific Interaction with Regulator of G Protein Signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein α subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the α subunit of transducin, Gαt. Here we report another unique property of RGS9: high specificity for Gαt. The core (RGS) domain of RGS9 (RGS9) stimulates Gαt GTPase activity by 10-fold and Gαi1 GTPase activity by only 2-fold at a concentration of 10 μm. Using chimeric Gαt/Gαi1 subunits we demonstrated that the α-helical domain of Gαt imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Gα subunits as measured by a change in fluorescence of a mutant Gαt (Chi6b) selectively labeled at Cys-210.K d values for RGS9 complexes with Gαtand Gαi1 calculated from the direct binding and competition experiments were 185 nm and 2 μm, respectively. The γ subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pγ to increase the affinity of RGS9 for Gαt. A distinct, nonoverlapping pattern of RGS and Pγ interaction with Gαt suggests a unique mechanism of effector-mediated GAP function of the RGS9.

Transducin translocation in rods is triggered by saturation of the GTPase-activating complex.

Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from transducin. Finally, we showed that the extent of transducin α subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on transducin translocation. Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase, and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.

The effector enzyme regulates the duration of G protein signaling in vertebrate photoreceptors by increasing the affinity between transducin and RGS protein

The photoreceptor-specific G protein transducin acts as a molecular switch, stimulating the activity of its downstream effector in its GTP-bound form and inactivating the effector upon GTP hydrolysis. This activity makes the rate of transducin GTPase an essential factor in determining the duration of photoresponse in vertebrate rods and cones. In photoreceptors, the slow intrinsic rate of transducin GTPase is accelerated by the complex of the ninth member of the regulators of G proteinsignaling family with the long splice variant of type 5 G protein β subunit (RGS9·Gβ5L). However, physiologically rapid GTPase is observed only when transducin forms a complex with its effector, the γ subunit of cGMP phosphodiesterase (PDEγ). In this study, we addressed the mechanism by which PDEγ regulates the rate of transducin GTPase. We found that RGS9·Gβ5L alone has a significant ability to activate transducin GTPase, but its affinity for transducin is low. PDEγ acts by enhancing the affinity between activated transducin and RGS9·Gβ5L by more than 15-fold, which is evident both from kinetic measurements of transducin GTPase rate and from protein binding assays with immobilized transducin. Furthermore, our data indicate that a single RGS9·Gβ5L molecule is capable of accelerating the GTPase activity of ∼100 transducin molecules/s. This rate is faster than the rates reported previously for any RGS protein and is sufficient for timely photoreceptor recovery in both rod and cone photoreceptors.

Proteomic Profiling of a Layered Tissue Reveals Unique Glycolytic Specializations of Photoreceptor Cells

The retina is a highly ordered tissue whose outermost layers are formed by subcellular compartments of photoreceptors generating light-evoked electrical responses. We studied protein distributions among individual photoreceptor compartments by separating the entire photoreceptor layer of a flat-mounted frozen retina into a series of thin tangential cryosections and analyzing protein compositions of each section by label-free quantitative mass spectrometry. Based on 5038 confidently identified peptides assigned to 896 protein database entries, we generated a quantitative proteomic database (a “map”) correlating the distribution profiles of identified proteins with the profiles of marker proteins representing individual compartments of photoreceptors and adjacent cells. We evaluated the applicability of several common peptide-to-protein quantification algorithms in the context of our database and found that the highest reliability was obtained by summing the intensities of all peptides representing a given protein, using at least the 5–6 most intense peptides when applicable. We used this proteome map to investigate the distribution of glycolytic enzymes, critical in fulfilling the extremely high metabolic demands of photoreceptor cells, and obtained two major findings. First, unlike the majority of neurons rich in hexokinase I, but similar to other highly metabolically active cells, photoreceptors express hexokinase II. Hexokinase II has a very high catalytic activity when associated with mitochondria, and indeed we found it colocalized with mitochondria in photoreceptors. Second, photoreceptors contain very little triosephosphate isomerase, an enzyme converting dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. This may serve as a functional adaptation because dihydroxyacetone phosphate is a major precursor in phospholipid biosynthesis, a process particularly active in photoreceptors because of the constant renewal of their light-sensitive membrane disc stacks. Overall, our approach for proteomic profiling of very small tissue amounts at a resolution of a few microns, combining cryosectioning and liquid chromatography-tandem MS, can be applied for quantitative investigation of proteomes where spatial resolution is paramount.

Functional roles of the two domains of phosducin and phosducin-like protein.

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the βγ subunit complex (Gβγ) and blocking Gβγ association with Gα subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each constituting approximately half of the molecule. We investigated the functional roles of the two domains of phosducin and phosducin-like protein in binding retinal Gtβγ. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound Gtβγ with a 2.5-fold greater affinity than phosducin-like protein; 2) phosphorylation of phosducin decreased its affinity by 3-fold, principally as a result of a decrease in k 1; and 3) most of the free energy of binding comes from the N-terminal domain with a lesser contribution from the C-terminal domain. In assays measuring the association of Gtβγ with Gtα and light-activated rhodopsin, both N-terminal domains inhibited binding while neither of the C-terminal domains had any effect. In assays measuring membrane binding of Gtβγ, both the N- and C-terminal domains inhibited membrane association, but much less effectively than the full-length proteins. This inhibition could only be described by models that included a change in Gtβγ to a conformation that did not bind the membrane. These models yielded a free energy change of +1.5 ± 0.25 kcal/mol for the transition from the Gtα-binding to the Pd-binding conformation of Gtβγ.

Proteasome overload is a common stress factor in multiple forms of inherited retinal degeneration

Inherited retinal degenerations, caused by mutations in over 100 individual genes, affect approximately 2 million people worldwide. Many of the underlying mutations cause protein misfolding or mistargeting in affected photoreceptors. This places an increased burden on the protein folding and degradation machinery, which may trigger cell death. We analyzed how these cellular functions are affected in degenerating rods of the transducin γ-subunit (Gγ1) knockout mouse. These rods produce large amounts of transducin β-subunit (Gβ1), which cannot fold without Gγ1 and undergoes intracellular proteolysis instead of forming a transducin βγ-subunit complex. Our data revealed that the most critical pathobiological factor leading to photoreceptor cell death in these animals is insufficient capacity of proteasomes to process abnormally large amounts of misfolded protein. A decrease in the Gβ1 production in Gγ1 knockout rods resulted in a significant reduction in proteasomal overload and caused a striking reversal of photoreceptor degeneration. We further demonstrated that a similar proteasomal overload takes place in photoreceptors of other mutant mice where retinal degeneration has been ascribed to protein mistargeting or misfolding, but not in mice whose photoreceptor degenerate as a result of abnormal phototransduction. These results establish the prominence of proteasomal insufficiency across multiple degenerative diseases of the retina, thereby positioning proteasomes as a promising therapeutic target for treating these debilitating conditions.

Periaxin is required for hexagonal geometry and membrane organization of mature lens fibers

Transparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell.

Mechanistic Basis for the Failure of Cone Transducin to Translocate: Why Cones Are Never Blinded by Light

The remarkable ability of our vision to function under ever-changing conditions of ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity of rods and cones. One such mechanism involves massive translocation of signaling proteins, including the G-protein transducin, into and out of the light-sensitive photoreceptor outer segment compartment. Transducin translocation extends the operating range of rods, but in cones transducin never translocates, which is puzzling because cones typically function in much brighter light than rods. Using genetically manipulated mice in which the rates of transducin activation and inactivation were altered, we demonstrate that, like in rods, transducin translocation in cones can be triggered when transducin activation exceeds a critical level, essentially saturating the photoresponse. However, this level is never achieved in wild-type cones: their superior ability to tightly control the rates of transducin activation and inactivation, responsible for avoiding saturation by light, also accounts for the prevention of transducin translocation at any light intensity.