Theodore G. Wensel

RGS9, a GTPase Accelerator for Phototransduction

The rod outer segment phototransduction GAP (GTPase-accelerating protein) has been identified as RGS9, a member of the RGS family of G alpha 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 (G alpha(t)), and this acceleration is enhanced by the gamma subunit of the phototransduction effector cGMP phosphodiesterase (PDEgamma). 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.

Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen‐combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.

RGS expression rate-limits recovery of rod photoresponses.

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.

Instability of GGL domain-containing RGS proteins in mice lacking the G protein β-subunit Gβ5

RGS (regulator of G protein signaling) proteins containing the G protein γ-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein β-subunit family, Gβ5. This interaction is necessary for the stability of both the RGS protein and for Gβ5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that Gβ5 levels may be limiting. To examine further the interactions of Gβ5 and the GGL domain-containing RGS proteins, we inactivated the Gβ5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of Gγ2 and RGS4 proteins remain normal in the absence of Gβ5. The homozygous Gβ5 knockout (Gβ5–/–) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the Gβ5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of Gβ5 and all four RGS proteins that form protein complexes with Gβ5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.

Structure of TRPV1 channel revealed by electron cryomicroscopy

The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of full-length rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-Å structure of TRPV1 determined by using single-particle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N- and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.

Evolutionary Trace of G Protein-coupled Receptors Reveals Clusters of Residues That Determine Global and Class-specific Functions

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

The regulator of G protein signaling (RGS)-9-1⋅Gβ5 complex forms the GTPase accelerating protein for Gαt in vertebrate photoreceptors. Although the complex is soluble when expressed in vitro, extraction of the endogenous protein from membranes requires detergents. The detergent extracts contain a complex of RGS9-1, Gβ5, Gαt, and a 25-kDa phosphoprotein, R9AP (RGS9-1-Anchor Protein). R9AP is encoded by one intronless gene in both human and mouse. Full or partial cDNA or genomic clones were obtained from mice, cattle, human, zebrafish, and Xenopus laevis. R9AP mRNA was detected only in the retina, and the protein only in photoreceptors. R9AP binds to the N-terminal domain of RGS9-1, and anchors it to the disk membrane via a C-terminal transmembrane helix.

A GTPase-accelerating factor for transducin, distinct from its effector cGMP phosphodiesterase, in rod outer segment membranes

Hydrolysis of GTP by the photoreceptor G protein transducin (Gt alpha) was found to occur with kinetics identical to the inactivation of its effector cGMP phosphodiesterase (PDE), but was too slow (tens of seconds) in dilute rod outer segment (ROS) suspensions to account for subsecond recovery of the light response. Raising the concentration of ROS membranes increased the rates of GTP hydrolysis and PDE inactivation in parallel as much as 6-fold. Holo-PDE and its gamma subunit had weak effects on GTPase kinetics (< 1.6-fold and < 1.3-fold, respectively). ROS membranes stripped of PDE retained approximately 90% of a GTPase accelerating activity that was protease sensitive, indicating that they contain a GTPase-accelerating factor distinct from PDE.

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin.

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.

Prediction and confirmation of a site critical for effector regulation of RGS domain activity.

A critical challenge of structural genomics is to extract functional information from protein structures. We present an example of how this may be accomplished using the Evolutionary Trace (ET) method in the context of the regulators of G protein signaling (RGS) family. We have previously applied ET to the RGS family and identified a novel, evolutionarily privileged site on the RGS domain as important for regulating RGS activity. Here we confirm through targeted mutagenesis of RGS7 that these ET-identified residues are critical for RGS domain regulation and are likely to function as global determinants of RGS function. We also discuss how the recent structure of the complex of RGS9, Gt/i1α–GDP–AlF4− and the effector subunit PDEγ confirms their contact with the effector–G protein interface, forming a structural pathway that communicates from the effector-contacting surface of the G protein and RGS catalytic core domain to the catalytic interface between Gα and RGS. These results demonstrate the effectiveness of ET for identifying binding sites and efficiently focusing mutational studies on their key residues, thereby linking raw sequence and structure data to functional information.