Biomedical subjects

Roland Seifert

Publications and source records attributed to Roland Seifert.

30 records · Page 2Linked to original sources

PubMed2003

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

The formyl peptide receptor (FPR) is expressed in neutrophils, couples to G(i)-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 G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells and measured high-affinity agonist binding and guanosine 5'- O-(3-thiotriphosphate) (GTPgammaS) binding. The B(max) 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 G(i)-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 G(i)-proteins activated by FPR-26. We conclude that (i) FPR isoforms 98 and G6 exhibit a partial G(i)-protein coupling defect relative to FPR-26 and that (ii) E346 critically determines constitutive activity, G(i)-protein coupling and physical state of FPR-26.

Amino Acid Sequence↗

PubMed2003

Multiple differences in agonist and antagonist pharmacology between human and guinea pig histamine H1-receptor.

Species isoforms of histamine H2-, H3-, and H4-receptors differ in their pharmacological properties. The study aim was to dissect differences between the human H1R (hH1R) and guinea pig H1R (ghH1R). We coexpressed hH1R and gpH1R with regulators of G-protein signaling in Sf9 insect cells and analyzed the GTPase activity of Gq-proteins. Small H1R agonists showed similar effects at hH1R and gpH1R, whereas bulkier 2-phenylhistamines and histaprodifens were up to approximately 10-fold more potent at gpH1R than at hH1R. Most 2-phenylhistamines and histaprodifens were more efficacious at gpH1R than at hH1R. Several first-generation H1R antagonists were approximately 2-fold, and arpromidine-type H1R antagonists up to approximately 10-fold more potent at gpH1R than at hH1R. [3H]Mepyramine competition binding studies confirmed the potency differences of the GTPase studies. Phe-153-->Leu-153 or Ile-433-->Val-433 exchange in hH1R (hH1R-->gpH1R) resulted in poor receptor expression, low [3H]mepyramine affinity, and functional inactivity. The Phe-153-->Leu-153/Ile-433-->Val-433 double mutant expressed excellently but only partially changed the pharmacological properties of hH1R. Small H1R agonists and 2-phenylhistamines interacted differentially with human and guinea pig H2R in terms of potency and efficacy, respectively. Our data show the following: 1) there are differences in agonist- and antagonist-pharmacology of hH1R and gpH1R encompassing diverse classes of bulky ligands. These differences may be explained by higher conformational flexibility of gpH1R relative to hH1R; 2) Phe-153 and Ile-433 are critical for proper folding and expression of hH1R; and 3) H2R species isoforms distinguish between H1R agonists.

Amino Acid Sequence↗

PubMed2003

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.

Asparagine↗

PubMed2003

2'(3')-O-(N-methylanthraniloyl)-substituted GTP analogs: a novel class of potent competitive adenylyl cyclase inhibitors.

2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTP gamma S) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on G alpha(s)- and G alpha(i)-protein-mediated signaling. MANT-GTP gamma S/MANT-GppNHp had lower affinities for G alpha(s) and G alpha(i) than GTP gamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G alpha fusion proteins. MANT-GTP gamma S was much less effective than GTP gamma S at disrupting the ternary complex between the formyl peptide receptor and G alpha(i2). MANT-GTP gamma S/MANT-GppNHp non-competitively inhibited GTP gamma S/GppNHp-, AlF(4)(-)-, beta(2)-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G alpha(s)-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP gamma S/MANT-GppNHp was not due to G alpha(s) inhibition because it was also observed in G alpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked AC inhibition by GTP gamma S/GppNHp in S49 cyc(-) membranes but enhanced the potency of MANT-GTP gamma S/MANT-GppNHp at inhibiting AC by approximately 4-8-fold. MANT-GTP gamma S and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 and 160 nm, respectively. The K(i) value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTP gamma S/MANT-GppNHp bind to G alpha(s)- and G alpha(i)-proteins with low affinity and are ineffective at activating G alpha. Instead, MANT-GTP gamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.

Adenylyl Cyclase Inhibitors↗

PubMed2003

Properties of Arg389-beta1-adrenoceptor-Gsalpha fusion proteins: comparison with Gly389-beta1-adrenoceptor-Gsalpha fusion proteins.

The human beta1-adrenoceptor (beta1AR) exists in several isoforms and activates adenylyl cyclase (AC) via Gs-proteins. The Arg389-isoform of the beta1AR (beta1AR-R389) expressed in CHW cells is much more efficient than the Gly389 isoform of the beta1AR (beta1AR-G389) at stabilizing the ternary complex and activating AC (Mason et al. 1999). The beta1AR-G389 fused to the Gsalpha splice variants GsalphaL or GsalphaS is efficient at stabilizing the ternary complex and activating AC (Wenzel-Seifert et al. 2002). Here, we show that beta1AR-R389-Gsalpha fusion proteins and beta1AR-G389-Gsalpha fusion proteins are similarly efficient at stabilizing the ternary complex and activating AC. In terms of agonist efficacies and agonist potencies in the [35S]guanosine 5'-O-(3-thiotriphosphate) binding assay, beta1AR-R389-Gsalpha fusion proteins and beta1AR-G389-Gsalpha fusion proteins are similar, too. Our present data fit to an increasing number of clinical studies that failed to detect physiology- or pathology-related functional differences between beta1AR-R389 and beta1AR-G389.

Adenylyl Cyclases↗

PubMed2002

GDP affinity and order state of the catalytic site are critical for function of xanthine nucleotide-selective Galphas proteins.

Xanthine nucleotide-selective small GTP-binding proteins with an Asp/Asn mutation are valuable for the analysis of individual GTP-binding proteins in complex systems. Similar applications can be devised for heterotrimeric G-proteins. However, Asp/Asn mutants of Galpha(o), Galpha(11), and Galpha(16) were inactive. An additional Gln/Leu mutation in the catalytic site, reducing GTPase activity and increasing GDP affinity, was required to generate xanthine nucleotide-selective unspecified G-protein alpha-subunit (Galpha). Our study aim was to generate xanthine nucleotide-selective mutants of Galpha(s), the stimulatory G-protein of adenylyl cyclase. The short splice variant of Galpha(s) (Galpha(sS)) possesses higher GDP affinity than the long splice variant (Galpha(sL)). Nucleoside 5'-[gamma-thio]triphosphates (NTPgammaSs) and nucleoside 5'-[beta,gamma-imido]triphosphates effectively activated a Galpha(sS) mutant with a D280N exchange (Galpha(sS)-N280), whereas nucleotides activated a Galpha(sL) mutant with a D295N exchange (Galpha(sL)-N295) only weakly. The Gln/Leu mutation enhanced Galpha(sL)-N295 activity. NTPgammaSs activated Galpha(sS)-N280 and a Galpha(sL) mutant with a Q227L and D295N exchange (Galpha(sL)-L227/N295) with similar potencies, whereas xanthosine 5'-triphosphate and xanthosine 5'-[beta,gamma-imido]triphosphate were more potent than GTP and guanosine 5'-[beta,gamma-imido]triphosphate, respectively. Galpha(sS)-N280 interacted with the beta(2)-adrenoreceptor and exhibited high-affinity XTPase activity. Collectively, (i) Galpha(sS)-N280 is the first functional xanthine nucleotide-selective Galpha with the Asp/Asn mutation alone; (ii) sufficiently high GDP affinity is crucial for Galpha Asp/Asn mutant function; (iii) with nucleoside 5'-triphosphates and nucleoside 5'-[beta,gamma-imido]triphosphates, Galpha(s)-N280 and Galpha(sL)-L227/N295 exhibit xanthine nucleotide selectivity, whereas NTPgammaSs sterically perturb the catalytic site of Galpha and annihilate xanthine selectivity.

Animals↗

PubMed2002

Efficient adenylyl cyclase activation by a beta2-adrenoceptor-G(i)alpha2 fusion protein.

The G-protein G(i)alpha can activate adenylyl cyclase (AC), but the relevance of this AC activation is unknown. We used receptor-G protein co-expression and receptor-G protein fusion proteins to investigate G(i)alpha(2) regulation of AC in Sf9 cells. G(i)alpha(2) was fused to the beta(2)-adrenoceptor (beta(2)AR), a preferentially G(s)-coupled receptor, or the formyl peptide receptor (FPR), a G(i)-coupled receptor. The FPR co-expressed with, or fused to, G(i)alpha(2), reduced AC activity. In contrast, the beta(2)AR fused to G(i)alpha(2) was a highly efficient AC activator, while the beta(2)AR co-expressed with G(i)alpha(2) was not. Agonist efficiently stimulated incorporation of [alpha-32P]GTP azidoanilide into beta(2)AR-G(i)alpha(2). We explain AC activation by beta(2)AR-G(i)alpha(2) by a model in which there is interaction of the beta(2)AR and AC, preventing tethered G(i)alpha(2) from interacting with the inhibitory G(i)alpha site of AC. The postulated beta(2)AR/AC interaction brings G(i)alpha(2) into close proximity of the G(s)alpha site of AC, enabling G(i)alpha(2) to activate AC.

Adenylyl Cyclases↗

PubMed2002

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

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 G(i)/G(o)-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 G(s)-, G(i)- and G(q)-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.

Animals↗

PubMed2002

Distinct interactions of G(salpha-long), G(salpha-short), and G(alphaolf) with GTP, ITP, and XTP.

The G(s)-proteins G(salpha-short) (G(salphaS)) and G(salpha-long) (G(salphaL)), and the olfactory G(s) protein (G(alphaolf)) mediate activation of adenylyl cyclase by the beta(2)-adrenoceptor (beta(2)AR). Early studies showed that the purine nucleotides GTP, ITP, and XTP differentially support receptor-mediated adenylyl cyclase activation in various native membrane systems, but those findings have remained unexplained thus far. We systematically analyzed the effects of GTP, ITP, and XTP on the coupling of the beta(2)AR to G(salphaS), G(salphaL), and G(alphaolf), respectively, using fusion proteins expressed in Sf9 insect cells. Fusion proteins ensure defined receptor/G-protein stoichiometry and efficient coupling. At all three fusion proteins, GTP, ITP, and XTP exhibited unique profiles with respect to their potency and efficacy at disrupting high-affinity agonist binding and supporting adenylyl cyclase activation by partial and full agonists. Our data can be interpreted in two ways: (i) GTP, ITP, and XTP may stabilize different active conformations in various G(s)-proteins, or (ii) GTP, ITP, and XTP may differ from one another in the kinetics of interaction with various G(s)-proteins. Regardless of which of the two explanations is correct, our present data demonstrate that GTP, ITP, and XTP are highly efficient regulators of signal transduction mediated through a specific G-protein. Also discussed is the possibility that G-protein activation by ITP and XTP may be of relevance in Lesch-Nyhan syndrome, a defect of the purine salvage pathway associated with abnormalities in various neurotransmitter systems.

Adenylyl Cyclases↗

PubMed2002

Similarities and differences in the coupling of human beta1- and beta2-adrenoceptors to Gs(alpha) splice variants.

The human beta1-adrenoceptor (beta1AR) and beta2-adrenoceptor (beta2AR) couple to Gs-proteins to activate adenylyl cyclase (AC). There are differences in desensitization between the beta2AR and the originally cloned Gly389-beta1AR, 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 beta1AR and beta2AR, we analyzed coupling of the Gly389-beta1AR to the G(s(alpha)) splice variants Gs(alpha)L and Gs(alpha)S using beta1AR-Gs(alpha) fusion proteins expressed in Sf9 cells and compared the data with previously published data on beta2AR-Gs(alpha) 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 beta1AR-Gs(alpha)S, beta1AR-Gs(alpha)L, beta2AR-Gs(alpha)S, and beta2AR-Gs(alpha)L with similar efficiency. beta1AR-Gs(alpha)L but not beta1AR-Gs(alpha)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 beta2AR-Gs(alpha)S and beta2AR-Gs(alpha)L. beta1AR-Gs(alpha)S and beta2AR-Gs(alpha)S were similarly efficient at activating AC, but beta1AR-Gs(alpha)L was approximately 4-fold more efficient at activating AC than beta2AR-Gs(alpha)L. Our data show that (i) the beta1AR and beta2AR are similarly efficient at stabilizing the ternary complex with Gs(alpha) splice variants, (ii) Gs(alpha)L confers constitutive activity to the beta1AR and beta2AR, and (iii) the beta1AR coupled to Gs(alpha)L is more efficient at activating AC than the beta2AR coupled to Gs(alpha)L. These data help us understand some of the discrepancies regarding similarities and differences between the beta1AR and beta2AR.

Adenylyl Cyclases↗

PubMed2002

Distinct interactions of GTP, UTP, and CTP with G(s) proteins.

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.

Adenylyl Cyclases↗

PubMed2002

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling.

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.

Adenylyl Cyclases↗