Roland Seifert

Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors

Abstract. The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1–3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na+ that stabilize the R state, and pertussis toxin that uncouples GPCRs from Gi/Go-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to Gs-, Gi- and Gq-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.

GPCR–Gα fusion proteins: molecular analysis of receptor–G-protein coupling

Abstract The efficiency of interactions between G-protein-coupled receptors (GPCRs) and heterotrimeric guanine nucleotide-binding proteins (G proteins) is greatly influenced by the absolute and relative densities of these proteins in the plasma membrane. The study of these interactions has been facilitated by the use of GPCR–Gα fusion proteins, which are formed by the fusion of GPCR to Gα. These fusion proteins ensure a defined 1:1 stoichiometry of GPCR to Gα and force the physical proximity of the signalling partners. Thus, fusion of GPCR to Gα enhances coupling efficiency can be used to study aspects of receptor–G-protein coupling that could not otherwise be examined by co-expressing GPCRs and G proteins as separate proteins. The results of studies that have made use of GPCR–Gα fusion proteins will be discussed in this article, along with the strengths and limitations of this approach.

Quantitative Analysis of Formyl Peptide Receptor Coupling to Giα1, Giα2, and Giα3

The human formyl peptide receptor (FPR) is a prototypical Gi protein-coupled receptor, but little is known about quantitative aspects of FPR-Gi protein coupling. To address this issue, we fused the FPR to Giα1, Giα2, and Giα3 and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Gα ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding and GTP hydrolysis and photolabeling of Gα, we demonstrate highly efficient coupling of the FPR to fused Giα1, Giα2, and Giα3without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three Giα isoforms. The K d values of high affinity agonist binding were ∼100-fold lower than the EC50 (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on theB max values of agonist saturation binding and ligand-regulated GTPγS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several Gi proteins. Analysis of agonist saturation binding, ligand-regulated GTPγS saturation binding and quantitative immunoblotting with membranes expressing FPR-Giα fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to Giα1, Giα2, and Giα3 with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate Gi proteins linearly and not catalytically.

Structural Basis for the Inhibition of Mammalian Membrane Adenylyl Cyclase by 2 ′(3′)-O-(N-Methylanthraniloyl)-guanosine 5 ′-Triphosphate

Membrane-bound mammalian adenylyl cyclase (mAC) catalyzes the synthesis of intracellular cyclic AMP from ATP and is activated by stimulatory G protein α subunits (Gαs) and by forskolin (FSK). mACs are inhibited with high potency by 2 ′(3′)-O-(N-methylanthraniloyl) (MANT)-substituted nucleotides. In this study, the crystal structures of the complex between Gαs·GTPγS and the catalytic C1 and C2 domains from type V and type II mAC (VC1·IIC2), bound to FSK and either MANT-GTP·Mg2+ or MANT-GTP·Mn2+ have been determined. MANT-GTP coordinates two metal ions and occupies the same position in the catalytic site as P-site inhibitors and substrate analogs. However, the orientation of the guanine ring is reversed relative to that of the adenine ring. The MANT fluorophore resides in a hydrophobic pocket at the interface between the VC1 and IIC2 domains and prevents mAC from undergoing the “open” to “closed” domain rearrangement. The Ki of MANT-GTP for inhibition of VC1·IIC2 is lower in the presence of mAC activators and lower in the presence of Mn2+ compared with Mg2+, indicating that the inhibitor binds more tightly to the catalytically most active form of the enzyme. Fluorescence resonance energy transfer-stimulated emission from the MANT fluorophore upon excitation of Trp-1020 in the MANT-binding pocket of IIC2 is also stronger in the presence of FSK. Mutational analysis of two non-conserved amino acids in the MANT-binding pocket suggests that residues outside of the binding site influence isoform selectivity toward MANT-GTP.

Generation of a homology model of the human histamine H(3) receptor for ligand docking and pharmacophore-based screening.

The human histamine H3 receptor (hH3R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH3R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH3R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH3R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH3R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [3H]Nα-methylhistamine binding assay. The compounds tested showed affinities at hH3R with Ki values ranging from 0.079 to 6.3xa0μM.

Broad Specificity of Mammalian Adenylyl Cyclase for Interaction with 2,3-Substituted Purine- and Pyrimidine Nucleotide Inhibitors

Membrane adenylyl cyclases (mACs) play an important role in signal transduction and are therefore potential drug targets. Earlier, we identified 2′,3′-O-(N-methylanthraniloyl) (MANT)-substituted purine nucleotides as a novel class of highly potent competitive mAC inhibitors (Ki values in the 10 nM range). MANT nucleotides discriminate among various mAC isoforms through differential interactions with a binding pocket localized at the interface between the C1 and C2 domains of mAC. In this study, we examine the structure/activity relationships for 2′,3′-substituted nucleotides and compare the crystal structures of mAC catalytic domains (VC1:IIC2) bound to MANT-GTP, MANT-ATP, and 2′,3′-(2,4,6-trinitrophenyl) (TNP)-ATP. TNP-substituted purine and pyrimidine nucleotides inhibited VC1:IIC2 with moderately high potency (Ki values in the 100 nM range). Elongation of the linker between the ribosyl group and the MANT group and substitution of N-adenine atoms with MANT reduces inhibitory potency. Crystal structures show that MANT-GTP, MANT-ATP, and TNP-ATP reside in the same binding pocket in the VC1:IIC2 protein complex, but there are substantial differences in interactions of base, fluorophore, and polyphosphate chain of the inhibitors with mAC. Fluorescence emission and resonance transfer spectra also reflect differences in the interaction between MANT-ATP and VC1:IIC2 relative to MANT-GTP. Our data are indicative of a three-site mAC pharmacophore; the 2′,3′-O-ribosyl substituent and the polyphosphate chain have the largest impact on inhibitor affinity and the nucleotide base has the least. The mAC binding site exhibits broad specificity, accommodating various bases and fluorescent groups at the 2′,3′-O-ribosyl position. These data should greatly facilitate the rational design of potent, isoform-selective mAC inhibitors.

The human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors

According to the two-state model of G-protein-coupled receptor (GPCR) activation, GPCRs isomerize from an inactive (R) state to an active (R*) state. In the R* state, GPCRs activate G-proteins. Agonist-independent R/R* isomerization is referred to as constitutive activity and results in an increase in basal G-protein activity, i.e. GDP/GTP exchange. Agonists stabilize the R* state and further increase, whereas inverse agonists stabilize the R state and decrease, basal G-protein activity. Constitutive activity is observed in numerous wild-type GPCRs and disease-causing GPCR mutants with increased constitutive activity. The human formyl peptide receptor (FPR) exists in several isoforms (FPR-26, FPR-98 and FPR-G6) and activates chemotaxis and cytotoxic cell functions of phagocytes through G(i)-proteins. Studies in HL-60 leukemia cell membranes demonstrated inhibitory effects of Na(+) and pertussis toxin on basal G(i)-protein activity, suggesting that the FPR is constitutively active. However, since HL-60 cells express several constitutively active chemoattractant receptors, analysis of constitutive FPR activity was difficult. Sf9 insect cells do not express chemoattractant receptors and G(i)-proteins and provide a sensitive reconstitution system for FPR/G(i)-protein coupling. Such expression studies showed that FPR-26 is much more constitutively active than FPR-98 and FPR-G6 as assessed by the relative inhibitory effects of Na(+) and of the inverse agonist cyclosporin H on basal G(i)-protein activity. Site-directed mutagenesis studies suggest that the E346A exchange in the C-terminus critically determines dimerization and constitutive activity of FPR. Moreover, N-glycosylation of the N-terminus seems to be important for constitutive FPR activity. Finally, we discuss some future directions of research.

Similarities and differences in the coupling of human β1- and β2-adrenoceptors to Gsα splice variants

Abstract The human β 1 -adrenoceptor (β 1 AR) and β 2 -adrenoceptor (β 2 AR) couple to G s -proteins to activate adenylyl cyclase (AC). There are differences in desensitization between the β 2 AR and the originally cloned Gly389-β 1 AR, but with respect to ternary complex formation, constitutive activity, and AC activation the picture is unclear. To learn more about the similarities and differences between the β 1 AR and β 2 AR, we analyzed coupling of the Gly389-β 1 AR to the G s α splice variants G s α L and G s α S using β 1 AR-G s α fusion proteins expressed in Sf9 cells and compared the data with previously published data on β 2 AR-G s α fusion proteins (Seifert et al ., J Biol Chem 1998;273:5109–16). Fusion ensures defined receptor/G-protein stoichiometry and efficient coupling. The agonist (−)-isoproterenol stabilized the ternary complex at β 1 AR-G s α S , β 1 AR-G s α L , β 2 AR-G s α S , and β 2 AR-G s α L with similar efficiency. β 1 AR-G s α L but not β 1 AR-G s α S showed the hallmarks of constitutive activity as assessed by increased potencies and efficacies of partial agonists and AC activation by the agonist-free receptor. Similar differences were observed previously for β 2 AR-G s α S and β 2 AR-G s α L . β 1 AR-G s α S and β 2 AR-G s α S were similarly efficient at activating AC, but β 1 AR-G s α L was ∼4-fold more efficient at activating AC than β 2 AR-G s α L . Our data show that (i) the β 1 AR and β 2 AR are similarly efficient at stabilizing the ternary complex with G s α splice variants, (ii) G s α L confers constitutive activity to the β 1 AR and β 2 AR, and (iii) the β 1 AR coupled to G s α L is more efficient at activating AC than the β 2 AR coupled to G s α L . These data help us understand some of the discrepancies regarding similarities and differences between the β 1 AR and β 2 AR.

Co-expression of the β2-adrenoceptor and dopamine D1-receptor with Gsα proteins in Sf9 insect cells: limitations in comparison with fusion proteins

The G-protein G(salpha) exists in three isoforms, the G(salpha) splice variants G(salphashort) (G(salphaS)) and G(salphalong) (G(salphaL)), and the G-protein G(alphaolf) that is not only involved in olfactory signaling but also in extrapyramidal motor regulation. Studies with beta(2)-adrenoceptor (beta(2)AR)-G(salpha) fusion proteins showed that G(salpha) proteins activate adenylyl cyclase (AC) in the order of efficacy G(salphaS)>G(salphaL) approximately G(alphaolf) and that G(salpha) proteins confer the hallmarks of constitutive activity to the beta(2)AR in the order of efficacy G(salphaL)>G(alphaolf)>G(salphaS). However, it is unclear whether such differences between G(salpha) proteins also exist in the nonfused state. In the present study, we co-expressed the beta(2)AR and dopamine D(1)-receptor (D(1)R) with G(salpha) proteins at different ratios in Sf9 insect cells. In agreement with the fusion protein studies, nonfused G(alphaolf) was less efficient than nonfused G(salphaS) and G(salphaL) at activating AC, but otherwise, we did not observe differences between the three G(salpha) isoforms. Thus, it is much easier to dissect differences between G(salpha) isoforms using beta(2)AR-G(salpha) fusion proteins than nonfused G(salpha) isoforms.

Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor.

The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G(i)-proteins. The FPR expressed with G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells exhibits high constitutive activity as assessed by strong inhibitory effects of an inverse agonist and Na(+) on basal guanosine 5()-O-(3-thiotriphosphate) (GTPgammaS) binding. The aim of our study was to analyze the role of N-glycosylation in FPR function. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in Sf9 membranes. However, in terms of high-affinity agonist binding, kinetics of GTPgammaS binding, number of G(i)-proteins activated, and constitutive activity, non-glycosylated FPR was much less active than native FPR. FPR-Asn4Gln/Asn10Gln/Asn179Gln and FPR-Asn4Gln/Asn10/Gln exhibited similar defects. Our data indicate that N-glycosylation of N-terminal Asn4 and Asn10 but not of Asn179 in the second extracellular loop is essential for proper folding and, hence, function of FPR. FPR deglycosylation by bacterial glycosidases could be a mechanism by which bacteria compromise host defense.