Folding of G? Subunits: Implications for Disease States.
ABSTRACT: G-proteins play a central role in signal transduction by fluctuating between "on" and "off" phases that are determined by a conformational change. cAMP is a secondary messenger whose formation is inhibited or stimulated by activated Gi?1 or Gs? subunit. We used tryptophan fluorescence, UV/vis spectrophotometry, and circular dichroism to probe distinct structural features within active and inactive conformations from wild-type and tryptophan mutants of Gi?1 and Gs?. For all proteins studied, we found that the active conformations were more stable than the inactive conformations, and upon refolding from higher temperatures, activated wild-type subunits recovered significantly more native structure. We also observed that the wild-type subunits partially regained the ability to bind nucleotide. The increased compactness observed upon activation was consistent with the calculated decrease in solvent accessible surface area for wild-type Gi?1. We found that as the temperature increased, G? subunits, which are known to be rich in ?-helices, converted to proteins with increased content of ?-sheets and random coil. For active conformations from wild-type and tryptophan mutants of Gi?1, melting temperatures indicated that denaturation starts around hydrophobic tryptophan microenvironments and then radiates toward tyrosine residues at the surface, followed by alteration of the secondary structure. For Gs?, however, disruption of secondary structure preceded unfolding around tyrosine residues. In the active conformations, a ?-cation interaction between essential arginine and tryptophan residues, which was characterized by a fluorescence-measured red shift and modeled by molecular dynamics, was also shown to be a contributor to the stability of G? subunits. The folding properties of G? subunits reported here are discussed in the context of diseases associated to G-proteins.
Project description:Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.
Project description:BACKGROUND AND PURPOSE: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands. EXPERIMENTAL APPROACH: We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays. KEY RESULTS: Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did. CONCLUSIONS AND IMPLICATIONS: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.
Project description:Gi?1 is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of Gi?1 bound to GDP•AMF or GTP?S with that of the inactive, GPD-bound protein indicates that a conformational change occurs in the activation step centered on three switch regions. The contribution of each tryptophan residue (W211 in the switch II region, W131 in the ?-helical domain, and W258 in the GTPase domain) toward the intrinsic protein fluorescence was evaluated by using W211F, W131F, and W258F mutants. All three tryptophan residues contributed significantly toward the emission spectra regardless of the conformation. When activated by either GDP•AMF or GTP?S, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP•AMF and GTP?S, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a ?-cation interaction that results in a red shift in the emission spectra of WT, and W131F and W258F mutants, but a blue shift for the W211F mutant. The observed shifts did not show a relationship with the span of the W211-R208 bridge, but rather with changes in the total interaction energies. Trypsin digestion of the active conformations only occurred for the W211F mutant indicating that the electrostatic ?-cation interaction blocks access to R208, which was consistent with the molecular dynamics simulations. We conclude that solvent accessibility and interaction energies account for the fluorescence features of Gi?1 .
Project description:G-protein levels were assessed in liver and brain membranes of lean and obese mice. ADP-ribosylation and immunodetection studies revealed a decrease in the abundance of Gs and Gi alpha-subunits in the liver membranes of obese mice compared with lean mice. In contrast, in brain membranes, the abundance of these proteins was not significantly different between lean and obese mice. Studies at the mRNA level in both liver and brain revealed no difference in gene expression between lean and obese mice. Protein and mRNA studies both showed that Gs, Gi alpha 1, Gi alpha 2, Go alpha and G beta subunits are present in brain membranes, and Gi alpha 3 is barely detectable. In liver, Ga alpha, Gi alpha 2 and G beta subunits are the major constituents, whereas Gi alpha 1, Gi alpha 3 and Go alpha are barely detectable. It is possible that the differences observed at the protein level are due to different rates of translation of the mRNA. Different rates of release of the alpha-subunits from the membrane and/or different rates of degradation would also explain these results.
Project description:Coupling between G-protein-coupled receptors (GPCRs) and the G proteins is a key step in cellular signaling. Despite extensive experimental and computational studies, the mechanism of specific GPCR-G protein coupling remains poorly understood. This has greatly hindered effective drug design of GPCRs that are primary targets of ?1/3 of currently marketed drugs. Here, we have employed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method to decipher the mechanism of the GPCR-G protein interactions. Adenosine receptors (ARs) were used as model systems based on very recently determined cryo-EM structures of the A1AR and A2AAR coupled with the Gi and Gs proteins, respectively. Changing the Gi protein to the Gs led to increased fluctuations in the A1AR and agonist adenosine (ADO), while agonist 5'-N-ethylcarboxamidoadenosine (NECA) binding in the A2AAR could be still stabilized upon changing the Gs protein to the Gi. Free energy calculations identified one stable low-energy conformation for each of the A1AR-Gi and A2AAR-Gs complexes as in the cryo-EM structures, similarly for the A2AAR-Gi complex. In contrast, the ADO agonist and Gs protein sampled multiple conformations in the A1AR-Gs system. GaMD simulations thus indicated that the A1AR preferred to couple with the Gi protein to the Gs, while the A2AAR could couple with both the Gs and Gi proteins, being highly consistent with experimental findings of the ARs. More importantly, detailed analysis of the atomic simulations showed that the specific AR-G protein coupling resulted from remarkably complementary residue interactions at the protein interface, involving mainly the receptor transmembrane 6 helix and the G? ?5 helix and ?4-?6 loop. In summary, the GaMD simulations have provided unprecedented insights into the dynamic mechanism of specific GPCR-G protein interactions at an atomistic level.
Project description:BACKGROUND:Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant G? subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels, measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration. RESULTS:Of the opsins tested, wild-type human rod opsin had the highest activity for chimeric Gs proxies for Gi and Gt (Gsi and Gst) and was matched in Go proxy (Gso) activity only by a human rod opsin/scallop opsin chimera. Rod opsin drove roughly equivalent responses via Gsi, Gso, and Gst, while cone opsins showed much lower activities with Gso than Gsi or Gst, and a human rod opsin/amphioxus opsin chimera demonstrated higher activity with Gso than with Gsi or Gst. We failed to detect activity for opsin chimeras bearing three intracellular fragments of mGluR6, and observed unexpectedly complex response profiles for scallop and amphioxus opsins thought to be specialized for Go. CONCLUSIONS:These results identify rod opsin as the most potent non-selective Gi/o/t-coupled opsin, long-wave sensitive cone opsin as the best for selectively activating Gi/t over Go, and a rod opsin/amphioxus opsin chimera as the best choice for selectively activating Go over Gi/t.
Project description:G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished ?-arrestin-mediated desensitization, resulting in increased Gs signaling.
Project description:The enzymatic activity of Bacillus subtilis glutamine synthetase (GS), which catalyzes the synthesis of glutamine from ammonium and glutamate, is regulated by glutamine feedback inhibition. The feedback-inhibited form of B. subtilis GS regulates the DNA-binding activities of the TnrA and GlnR nitrogen transcriptional factors. Bacterial GS proteins contain a flexible seven-residue loop, the Glu304 flap, that closes over the glutamate entrance to the active site. Amino acid substitutions in Glu304 flap residues were examined for their effects on gene regulation, enzymatic activity, and feedback inhibition. Substitutions in five of the Glu304 loop residues resulted in constitutive expression of both TnrA- and GlnR-regulated genes, indicating that this flap is important for regulating the activity of these transcription factors. The residues in the highly conserved Glu304 flap appear to be optimized for glutamate binding because mutant enzymes with substitutions in five of the flap residues had increased glutamate Km values compared to that for wild-type GS. The E304A and E304D substitutions increased the ammonium Km values compared to that for wild-type GS and conferred high-level resistance to inhibition by glutamine, glycine, and methionine sulfoximine. A model for the role of the Glu304 residue in glutamine feedback inhibition is proposed.
Project description:The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.
Project description:PURPOSE: delta-Crystallin is a soluble structural protein in found in avian eye lenses; it shares high amino acid sequence identity with argininosuccinate lyase. E294 is the only residue located at the double dimer interface and it performs hydrogen bonding with the active site residues of H160 and K323 in the neighboring and diagonal subunits, respectively. H160 is reported to play an important role in catalysis due to its H-bond interaction with the fumarate moiety of the substrate. In order to clarify the function of E294 in either stabilization of the quaternary structure or in catalysis, we carried out site-directed mutagenesis and functional analysis. METHODS: The structure of both wild-type and mutant proteins were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, and analytical ultracentrifugation. Structural stability was measured by CD and tryptophan fluorescence. A modeled structure of the E294L mutant was built and optimized with energy minimization. RESULTS: No gross structural changes were observed when E294 was substituted with leucine, as judged by circular dichroism, tryptophan fluorescence, ANS fluorescence, and sedimentation velocity analyses. However, this mutant enzyme had only about 10% of the activity of a wild-type enzyme and its secondary structure was more easily denatured by increased temperature than that of a wild-type enzyme. The mutant protein also underwent its first unfolding transition at a lower concentration of guanidinium-hydrochloride than the wild-type protein. CONCLUSIONS: These results indicate that the interactions offered by E294 in the dimer-dimer interface of delta-crystallin are required to maintain the hydrogen bonding network in the active site for catalysis. Disruption of the interaction had no significant effect on the conformation and quaternary structure of delta-crystallin but it did lead to instability in the double dimer structure.