RGS9, a GTPase Accelerator for Phototransduction

AbstractThe rod outer segment phototransduction GAP (GTPase-accelerating protein) has been identified as RGS9, a member of the RGS family of Gα GAPs. RGS9 mRNA expression is specific for photoreceptor cells, and RGS9 protein colocalizes with other phototransduction components to photoreceptor outer segment membranes. The RGS domain of RGS9 accelerates GTP hydrolysis by the visual G protein transducin (Gαt), and this acceleration is enhanced by the γ subunit of the phototransduction effector cGMP phosphodiesterase (PDEγ). These unique properties of RGS9 match those of the rod outer segment GAP and implicate it as a key element in the recovery phase of visual transduction

Coevolutionary Signals in Metabotropic Glutamate Receptors Capture Residue Contacts and Long-Range Functional Interactions

Upon ligand binding to a G protein-coupled receptor, extracellular signals are transmitted into a cell through sets of residue interactions that translate ligand binding into structural rearrangements. These interactions needed for functions impose evolutionary constraints so that, on occasion, mutations in one position may be compensated by other mutations at functionally coupled positions. To quantify the impact of amino acid substitutions in the context of major evolutionary divergence in the G protein-coupled receptor subfamily of metabotropic glutamate receptors (mGluRs), we combined two phylogenetic-based algorithms, Evolutionary Trace and covariation Evolutionary Trace, to infer potential structure-function couplings and roles in mGluRs. We found a subset of evolutionarily important residues at known functional sites and evidence of coupling among distinct structural clusters in mGluR. In addition, experimental mutagenesis and functional assays confirmed that some highly covariant residues are coupled, revealing their synergy. Collectively, these findings inform a critical step toward understanding the molecular and structural basis of amino acid variation patterns within mGluRs and provide insight for drug development, protein engineering, and analysis of naturally occurring variants

Co-expression of Gbeta 5 Enhances the Function of Two Ggamma Subunit-like Domain-containing Regulators of G Protein Signaling Proteins

Regulators of G protein signaling (RGS) stimulate the GTPase activity of G protein Galpha subunits and probably play additional roles. Some RGS proteins contain a Ggamma subunit-like (GGL) domain, which mediates a specific interaction with Gbeta 5. The role of such interactions in RGS function is unclear. RGS proteins can accelerate the kinetics of coupling of G protein-coupled receptors to G-protein-gated inwardly rectifying K+ (GIRK) channels. Therefore, we coupled m2-muscarinic acetylcholine receptors to GIRK channels in Xenopus oocytes to evaluate the effect of Gbeta 5 on RGS function. Co-expression of either RGS7 or RGS9 modestly accelerated GIRK channel kinetics. When Gbeta 5 was co-expressed with either RGS7 or RGS9, the acceleration of GIRK channel kinetics was strongly increased over that produced by RGS7 or RGS9 alone. RGS function was not enhanced by co-expression of Gbeta 1, and co-expression of Gbeta 5 alone had no effect on GIRK channel kinetics. Gbeta 5 did not modulate the function either of RGS4, an RGS protein that lacks a GGL domain, or of a functional RGS7 construct in which the GGL domain was omitted. Enhancement of RGS7 function by Gbeta 5 was not a consequence of an increase in the amount of plasma membrane or cytosolic RGS7 protein

Super-resolution Microscopy Reveals Photoreceptor-Specific Subciliary Location and Function of Ciliopathy-Associated Protein CEP290

Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multiorgan ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used super-resolution fluorescence microscopy and electron microscopy to localize CEP290 in the CC and in the primary cilia of cultured cells with subdiffraction resolution and to determine effects of CEP290 deficiency in 3 mutant models. Radially, CEP290 localizes in close proximity to the microtubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout the length of the CC whereas it is confined to the very base of primary cilia in human retinal pigment epithelium-1 cells. We found Y-shaped links, ciliary substructures between microtubules and membrane, throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliary components in early stages of degeneration. There were fewer cilia and no normal outer segments in the mutants, but the Y-shaped links were clearly present. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis

Loss of Class III Phosphoinositide 3-Kinase Vps34 Results in Cone Degeneration

The major pathway for the production of the low-abundance membrane lipid phosphatidylinositol 3-phosphate (PI(3)P) synthesis is catalyzed by class III phosphoinositide 3-kinase (PI3K) Vps34. The absence of Vps34 was previously found to disrupt autophagy and other membrane-trafficking pathways in some sensory neurons, but the roles of phosphatidylinositol 3-phosphate and Vps34 in cone photoreceptor cells have not previously been explored. We found that the deletion of Vps34 in neighboring rods in mouse retina did not disrupt cone function up to 8 weeks after birth, despite diminished rod function. Immunoblotting and lipid analysis of cones isolated from the cone-dominant retinas of the neural retina leucine zipper gene knockout mice revealed that both PI(3)P and Vps34 protein are present in mouse cones. To determine whether Vps34 and PI(3)P are important for cone function, we conditionally deleted Vps34 in cone photoreceptor cells of the mouse retina. Overall retinal morphology and rod function appeared to be unaffected. However, the loss of Vps34 in cones resulted in the loss of structure and function. There was a substantial reduction throughout the retina in the number of cones staining for M-opsin, S-opsin, cone arrestin, and peanut agglutinin, revealing degeneration of cones. These studies indicate that class III PI3K, and presumably PI(3)P, play essential roles in cone photoreceptor cell function and survival

Recurrent High-Impact Mutations at Cognate Structural Positions in Class a G Protein-Coupled Receptors Expressed in Tumors

G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or β-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics

Centriole and Transition Zone Structures in Photoreceptor Cilia Revealed by Cryo-Electron Tomography

Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the Y-shaped links, and the ciliary necklace. We have used cryo-electron tomography with subtomogram averaging and conventional transmission electron microscopy to elucidate the structures associated with the basal region of the connecting cilia of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules identified by genetics, proteomics, and superresolution fluorescence can be placed in our emerging model of photoreceptor sensory cilia

Modules in the photoreceptor RGS9-1•Gβ5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability

RGS (regulators of G protein signaling proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G gamma -like domains that bind G(beta5) proteins. Members of this subfamily play important roles in neuronal signaling, Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G gamma -like-G(beta 5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1, The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in, vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta 5S), and G(beta 5s), The dependence of RGS9-1 on Gp, co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis, These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(t alpha) inactivation

CovET: A Covariation-Evolutionary Trace Method That Identifies Protein Structure-Function Modules

Measuring the relative effect that any two sequence positions have on each other may improve protein design or help better interpret coding variants. Current approaches use statistics and machine learning but rarely consider phylogenetic divergences which, as shown by Evolutionary Trace studies, provide insight into the functional impact of sequence perturbations. Here, we reframe covariation analyses in the Evolutionary Trace framework to measure the relative tolerance to perturbation of each residue pair during evolution. This approach (CovET) systematically accounts for phylogenetic divergences: at each divergence event, we penalize covariation patterns that belie evolutionary coupling. We find that while CovET approximates the performance of existing methods to predict individual structural contacts, it performs significantly better at finding structural clusters of coupled residues and ligand binding sites. For example, CovET found more functionally critical residues when we examined the RNA recognition motif and WW domains. It correlates better with large-scale epistasis screen data. In the dopamine D2 receptor, top CovET residue pairs recovered accurately the allosteric activation pathway characterized for Class A G protein-coupled receptors. These data suggest that CovET ranks highest the sequence position pairs that play critical functional roles through epistatic and allosteric interactions in evolutionarily relevant structure-function motifs. CovET complements current methods and may shed light on fundamental molecular mechanisms of protein structure and function