Katharina Wenzel-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.

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.

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.

Functional differences between human formyl peptide receptor isoforms 26, 98, and G6

The formyl peptide receptor (FPR) is expressed in neutrophils, couples to Gi-proteins and activates phospholipase C, chemotaxis and cytotoxic cell functions. FPR isoforms 26, 98, and G6 differ from each other in amino acids 101, 192 and 346 (FPR-26: V101, N192, E346; FPR-98: L101, N192, A346; FPR-G6: V101, K192, A346), but the functional significance of those structural differences is unknown. In order to address this question, we analyzed FPR-26, FPR-98 and FPR-G6 by co-expressing recombinant FLAG epitope-tagged FPRs with the G-protein Giα2β1γ2 in Sf9 insect cells and measured high-affinity agonist binding and guanosine 5-O-(3-thiotriphosphate) (GTPγS) binding. The Bmax values of high-affinity agonist binding with FPR-98 and FPR-G6 were much lower than with FPR-26. FPR-98 and FPR-G6 activated considerably fewer Gi-proteins, and were much less constitutively active, than FPR-26. Whereas FPR-26 migrated as a monomer in SDS polyacrylamide electrophoresis, FPR-98 and FPR-G6 migrated as dimers and tetramers. In terms of immunoreactivity, FRP-98 and FPR-G6 were expressed at higher levels than FPR-26. Single amino acid exchanges at positions 101 (V→L), 192 (N→K) and 346 (E→A) in FPR-26 revealed that E346 accounts for FPR-26 migrating as a monomer and the high constitutive activity of FPR-26. The V101L, N192K and E346A exchanges all reduced high-affinity agonist binding and the number of Gi-proteins activated by FPR-26. We conclude that (i) FPR isoforms 98 and G6 exhibit a partial Gi-protein coupling defect relative to FPR-26 and that (ii) E346 critically determines constitutive activity, Gi-protein coupling and physical state of FPR-26.