Biomedical subjects

K Wenzel-Seifert

Publications and source records attributed to K Wenzel-Seifert.

At least 19 recordsLinked to original sources

PubMed2001

Defective Gi protein coupling in two formyl peptide receptor mutants associated with localized juvenile periodontitis.

The formyl peptide receptor (FPR) is a prototypical chemoattractant receptor expressed in neutrophils. It is well known that the FPR couples to G(i) proteins to activate phospholipase C, chemotaxis, and cytotoxic cell functions, but the in vivo role of the FPR in man has remained elusive. Recently, F110S and C126W mutations of the FPR have been associated with localized juvenile periodontitis. We studied FPR-F110S and FPR-C126W in comparison with wild-type FPR (FPR-WT) by coexpressing epitope-tagged versions of these receptors with the G protein Galpha(i2)beta(1)gamma(2) in Sf9 insect cells. FPRs were efficiently expressed in Sf9 membranes as assessed by immunoblotting using the beta(2)-adrenoreceptor as a standard. FPR-C126W differed from FPR-WT and FPR-F110S in migration on SDS-polyacrylamide gels and tunicamycin-sensitive glycosylation. FPR-WT efficiently reconstituted high-affinity agonist binding and agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Galpha(i2)beta(1)gamma(2). In contrast, FPR-F110S only weakly reconstituted agonist-stimulated GTPgammaS binding, and FPR-C126W was completely inefficient. Collectively, our data show almost complete and complete loss of G(i) protein coupling in FPR-F110S and FPR-C126W, respectively. The severe functional defects in FPR-F110S and FPR-C126W contrast with the discrete clinical symptoms associated with these mutations, indicating that loss of FPR function in host defense is, for the most part, readily compensated.

Aggressive Periodontitis↗

PubMed2001

The olfactory G protein G(alphaolf) possesses a lower GDP-affinity and deactivates more rapidly than G(salphashort): consequences for receptor-coupling and adenylyl cyclase activation.

The olfactory G protein G(alphaolf) differs from the short splice variant of G(salpha) (G(salphaS)) in 80 amino acids, but little is known about biochemical differences between G(alphaolf) and G(salphaS). We addressed this question by analyzing fusion proteins of the beta2-adrenoceptor (beta2AR) and G(alphaolf) and G(salphaS), respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High-affinity agonist binding studies showed that G(alphaolf) possesses a lower GDP-affinity than G(salphaS) As a result, the agonist-free beta2AR and the beta2AR occupied by partial agonists were more efficient at promoting GDP-dissociation from G(alphaolf) than from G(salphaS) a assessed by guanosine 5'-O-(3-thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing beta2AR-G(alphaolf) than in membranes expressing beta2AR-G(salphaS) at similar levels, reflecting the lower abundance of G(alphaolf-GDP) relative to G(salphaS-GDP). The maximum agonist-stimulated AC activity with beta2AR-G(salphaS) was more than twofold higher than with beta2AR-G(alphaolf), but the relative agonist-stimulation of AC with beta2AR-G(alphaolf) was much greater than with beta2AR-G(salphaS). The difference in maximum AC activity can be explained by more rapid deactivation of G(alphaolf-GTP) by GTP hydrolysis and GTP dissociation relative to G(salphaS-GTP). Taken together, there are biochemical differences between G(alphaolf) and G(salphaS), supporting different roles of these G proteins in vivo.

Adenylyl Cyclases↗

PubMed2001

Distinct interaction of human and guinea pig histamine H2-receptor with guanidine-type agonists.

It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H2-receptor (H2R) agonists are lower at the H2R of human neutrophils than at the H2R of the guinea pig atrium. To elucidate these differences, we analyzed fusion proteins of the human H2R (hH2R) and guinea pig H2R (gpH2R), respectively, and the short splice variant of Gsalpha (GsalphaS) expressed in Sf9 cells. The potencies and efficacies of small H2R agonists in the GTPase assay and the potencies of antagonists at inhibiting histamine-stimulated GTP hydrolysis by hH2R-GsalphaS and gpH2R-GsalphaS were similar. In contrast, the potencies and efficacies of guanidines were lower at hH2R-GsalphaS than at gpH2R-G(salphaS). Guanidines bound to hH2R-GsalphaS with lower affinity than to gpH2R-GsalphaS, and high-affinity binding of guanidines at gpH2R-GsalphaS was more resistant to disruption by GTPgammaS than binding at hH2R-GsalphaS. Molecular modeling suggested that the nonconserved Asp-271 in transmembrane domain 7 of gpH2R (Ala-271 in hH2R) confers high potency to guanidines. This hypothesis was confirmed by Ala-271-->Asp-271 mutation in hH2R-GsalphaS. Intriguingly, the efficacies of guanidines at the Ala-271-->Asp-271 mutant and at hH2R/gpH2R chimeras were lower than at gpH2R. Our model suggests that a Tyr-17/Asp-271 H-bond, present only in gpH2R-GsalphaS but not the other constructs studied, stabilizes the active guanidine-H2R state. Collectively, our data show 1) distinct interaction of H2R species isoforms with guanidines, 2) that a single amino acid in transmembrane domain 7 critically determines guanidine potency, and 3) that an interaction between transmembrane domains 1 and 7 is important for guanidine efficacy.

Alternative Splicing↗

PubMed2001

Functional differences between full and partial agonists: evidence for ligand-specific receptor conformations.

The interaction of an agonist-bound G-protein-coupled receptor (GPCR) with its cognate G-protein initiates a sequence of experimentally quantifiable changes in both the GPCR and G-protein. These include the release of GDP from G(alpha), the formation of a ternary complex between the nucleotide-free G-protein and the GPCR, which has a high affinity for agonist, followed by the binding of GTP to G(alpha), the dissociation of the GPCR/G-protein complex, and the hydrolysis of GTP. The efficacy of an agonist is a measure of its ability to activate this cascade. It has been proposed that efficacy reflects the ability of the agonist to stabilize the active state of the GPCR. We examined a series of beta(2)-adrenoceptor (beta(2)AR) agonists (weak partial agonists to full agonists) for their efficacy at promoting two different steps of the G-protein activation/deactivation cycle: stabilizing the ternary complex (high-affinity, GTP-sensitive agonist binding), and steady-state GTPase activity. We obtained results for the wild-type beta(2)AR and a constitutively active mutant of the beta(2)AR (beta(2)AR(CAM)) using fusion proteins between the GPCRs and G(salpha) to facilitate GPCR/G-protein interactions. There was no correlation between efficacy of ligands in activating GTPase and their ability to stabilize the ternary complex at beta(2)AR(CAM). Our results suggest that the GPCR state that optimally promotes the GDP release and GTP binding is different from the GPCR state that stabilizes the ternary complex. By strongly stabilizing the ternary complex, certain partial agonists may reduce the rate of G-protein turnover relative to a full agonist.

Adrenergic beta-2 Receptor Agonists↗

PubMed2001

Unmasking different constitutive activity of four chemoattractant receptors using Na+ as universal stabilizer of the inactive (R) state.

Neutrophils express receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) complement C5a, leukotriene B4 (LTB4) and platelet-activating factor (PAF). The aim of this study was to analyze the constitutive activity of chemoattractant receptors by studying binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to the G-protein Gi alpha 2 beta 1 gamma 2 expressed in Sf9 cells. We used Na+ as modulator of constitutive activity because there are no known inverse agonists for the C5a receptor (C5aR), LTB4 receptor (BLTR) and PAF receptor (PAFR). In the absence of NaCl, PAF and LTB4 exhibited larger relative stimulatory effects on GTP gamma S binding than fMLP and C5a. NaCl showed larger inhibitory effects on basal GTP gamma S binding in membranes expressing the formyl peptide receptor (FPR) and C5aR than in membranes expressing BLTR and PAFR. The order of potency of NaCl at inhibiting basal GTP gamma S binding was FPR > C5aR approximately BLTR > PAFR. As a result of the inhibitory effect of NaCl on basal GTP gamma S binding, the relative stimulatory effects of agonists were increased. By quantitatively analyzing the expression levels of chemoattractant receptors and Gi alpha 2 and the stoichiometry of receptor/G-protein coupling we obtained no evidence for structural instability of constitutively active receptors and catalytical G-protein activation. Taken together, the FPR and C5aR exhibit higher constitutive activity than the BLTR and PAFR. Na+ acts as a universal stabilizer of the inactive (R) state in chemoattractant receptors. The different potencies of NaCl at suppressing basal G-protein activity with different receptors indicate that chemoattractant receptors differ from each other in their Na(+)-affinity.

Antigens, CD↗

PubMed2001

Similar apparent constitutive activity of human histamine H(2)-receptor fused to long and short splice variants of G(salpha).

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta(2)-adrenoceptor (beta(2)AR) fused to the long splice variant of G(salpha) (G(salphaL)) exhibits a higher apparent constitutive activity than the beta(2)-adrenoceptor fused to the short splice variant of G(salpha) (G(salphaS)). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the beta(2)AR fused to G(salphaL) relative to the beta(2)AR fused to G(salphaS), indicating that the agonist-free beta(2)AR and the beta(2)AR occupied by partial agonists promote GDP dissociation from G(salphaL) more efficiently than from G(salphaS). In fact, the GDP affinity of G(salphaS) fused to the beta(2)AR is higher than the GDP affinity of G(salphaL) fused to the beta(2)AR. We asked the question whether the histamine H(2)-receptor (H(2)R) exhibits similar coupling to G(salpha) splice variants as the beta(2)AR. To address this question, we studied H(2)R-G(salpha) fusion proteins expressed in Sf9 cells. In contrast to beta(2)AR-G(salpha) fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H(2)R fused to G(salphaL) and G(salphaS) as assessed by guanosine-5'-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5'-O-(3-thio)triphosphate binding indicated that G(salphaS) fused to the H(2)R possesses a higher GDP-affinity than G(salphaL) fused to the H(2)R. Our data show that the H(2)R fused to G(salphaL) and G(salphaS) possesses similar constitutive activity and is insensitive to differences in GDP affinity of G(salpha) splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.

Alternative Splicing↗

PubMed2000

Molecular analysis of beta(2)-adrenoceptor coupling to G(s)-, G(i)-, and G(q)-proteins.

The beta(2)-adrenoceptor (beta(2)AR) couples to the G-protein G(s) to activate adenylyl cyclase. Intriguingly, several studies have demonstrated that the beta(2)AR can also interact with G-proteins of the G(i)- and G(q)-family. To assess the efficiency of beta(2)AR interaction with various G-protein alpha-subunits (G(xalpha)), we expressed fusion proteins of the beta(2)AR with the long (G(salphaL)) and short (G(salphaS)) splice variants of G(salpha), the G(i)-proteins G(ialpha2) and G(ialpha3), and the G(q)-proteins G(qalpha) and G(16alpha) in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to G(xalpha) because of the defined 1:1 stoichiometry of receptor and G-protein and the efficient coupling. Here, we show that the beta(2)AR couples to G(s)-, G(i)-, and G(q)-proteins as assessed by ternary complex formation and ligand-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding. The combined analysis of ternary complex formation, GTPgammaS binding, agonist efficacies, and agonist potencies revealed substantial differences in the interaction of the beta(2)AR with the various classes of G-proteins. Comparison of the coupling of the beta(2)AR and formyl peptide receptor to G(ialpha2) revealed receptor-specific differences in the kinetics of GTPgammaS binding. We also detected highly efficient stimulation of GTPgammaS dissociation from G(salphaL), but not from G(qalpha) and G(16alpha), by a beta(2)AR agonist. Moreover, we show that the 1:1 stoichiometry of receptor to G-protein in fusion proteins reflects the in vivo stoichiometry of receptor/G-protein coupling more closely than was previously assumed. Collectively, our data show 1) that the beta(2)AR couples differentially to G(s)-, G(i)-, and G(q)-proteins, 2) that there is ligand-specific coupling of the beta(2)AR to G-proteins, 3) that receptor-specific G-protein conformational states may exist, and 4) that nucleotide dissociation is an important mechanism for G-protein deactivation.

Adrenergic Agonists↗

PubMed1999

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3).

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha 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) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha 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 G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) 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 G(i) proteins linearly and not catalytically.

Animals↗

PubMed1999

GPCR-Galpha fusion proteins: molecular analysis of receptor-G-protein coupling.

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-Galpha fusion proteins, which are formed by the fusion of GPCR to Galpha. These fusion proteins ensure a defined 1:1 stoichiometry of GPCR to Galpha and force the physical proximity of the signalling partners. Thus, fusion of GPCR to Galpha 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-Galpha fusion proteins will be discussed in this article, along with the strengths and limitations of this approach.

Animals↗

PubMed1999

Examining the efficiency of receptor/G-protein coupling with a cleavable beta2-adrenoceptor-gsalpha fusion protein.

Reconstitution of high-affinity agonist binding at the beta2-adrenoceptor (beta2AR) expressed in Sf9 insect cells requires a large excess of the stimulatory G-protein of adenylyl cyclase, Gsalpha, relative to receptor [R. Seifert, T. W. Lee, V. T. Lam & B. K. Kobilka, (1998) Eur. J. Biochem. 255, 369-382]. In a fusion protein of the beta2AR and Gsalpha (beta2AR-Gsalpha), which has only a 1 : 1 stoichiometry of receptor and G-protein, high-affinity agonist binding and agonist-stimulated GTP hydrolysis, guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding and adenylyl cyclase (AC) activation are more efficient than in the nonfused coexpression system. In order to analyze the stability of the receptor/G-protein interaction, we constructed a fusion protein with a thrombin-cleavage site between beta2AR and Gsalpha (beta2AR-TS-Gsalpha). beta2AR-TS-Gsalpha efficiently reconstituted high-affinity agonist binding, agonist-stimulated GTP hydrolysis, GTP[S] binding and AC activation. Thrombin cleaves approximately 70% of beta2AR-TS-Gsalpha molecules in Sf9 membranes. Thrombin cleavage did not impair high-affinity agonist binding and GTP[S] binding but strongly reduced ligand-regulated GTPase activity and AC activity. We conclude that fusion of the beta2AR to Gsalpha promotes tight physical association of the two partners and that this association remains stable for a single activation/deactivation cycle even after cleavage of the link between the receptor and G-protein. Dilution of Gsalpha in the membrane and release of activated Gsalpha into the cytosol can both prevent cleaved beta2AR-TS-Gsalpha from undergoing multiple activation/deactivation cycles.

Adenylyl Cyclases↗

PubMed1999

Effects of guanine, inosine, and xanthine nucleotides on beta(2)-adrenergic receptor/G(s) interactions: evidence for multiple receptor conformations.

The aim of our study was to examine the effects of different purine nucleotides [GTP, ITP, and xanthosine 5'-triphosphate (XTP)] on receptor/G protein coupling. As a model system, we used a fusion protein of the beta(2)-adrenergic receptor and the alpha subunit of the G protein G(s). GTP was more potent and efficient than ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of several beta(2)-adrenergic receptor ligands on nucleotide hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The efficacy of agonists at promoting GTP hydrolysis correlated well with the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in the presence of ITP. The efficacy of ligands at stimulating AC in the presence of XTP differed considerably from the efficacies of ligands in the presence of GTP and ITP, and there was no evidence for receptor-regulated XTP hydrolysis. Our findings support the concept of multiple ligand-specific receptor conformations and demonstrate the usefulness of purine nucleotides as tools to study conformational states of receptors.

Adenosine Triphosphate↗

PubMed1998

Restricting mobility of Gsalpha relative to the beta2-adrenoceptor enhances adenylate cyclase activity by reducing Gsalpha GTPase activity.

The beta2-adrenoceptor (beta2AR) activates the G-protein Gsalpha to stimulate adenylate cyclase (AC). Fusion of the beta2AR C-terminus to the N-terminus of Gsalpha (producing beta2ARGsalpha) markedly increases the efficiency of receptor/G-protein coupling compared with the non-fused state. This increase in coupling efficiency can be attributed to the physical proximity of receptor and G-protein. To determine the optimal length for the tether between receptor and G-protein we constructed fusion proteins from which 26 [beta2AR(Delta26)Gsalpha] or 70 [beta2AR(Delta70)Gsalpha] residues of the beta2AR C-terminus had been deleted and compared the properties of these fusion proteins with the previously described beta2ARGsalpha. Compared with beta2ARGsalpha, basal and agonist-stimulated GTP hydrolysis was markedly decreased in beta2AR(Delta70)Gsalpha, whereas the effect of the deletion on binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was relatively small. Surprisingly, deletions did not alter the efficiency of coupling of the beta2AR to Gsalpha as assessed by GTP[S]-sensitive high-affinity agonist binding. Moreover, basal and ligand-regulated AC activities in membranes expressing beta2AR(Delta70)Gsalpha and beta2AR(Delta26)Gsalpha were higher than in membranes expressing beta2ARGsalpha. These findings suggest that restricting the mobility of Gsalpha relative to the beta2AR results in a decrease in G-protein inactivation by GTP hydrolysis and thereby enhanced activation of AC.

Adenylyl Cyclases↗

PubMed1998

High constitutive activity of the human formyl peptide receptor.

The formyl peptide receptor (FPR) couples to pertussis toxin (PTX)-sensitive Gi-proteins to activate chemotaxis and exocytosis in neutrophils. PTX reduces not only formyl peptide-stimulated but also agonist-independent ("basal") Gi-protein activity, suggesting that the FPR is constitutively active. We aimed at identifying an inverse FPR agonist, i.e. a compound that suppresses constitutive FPR activity. In Sf9 insect cell membranes, the G-protein heterotrimer Gialpha2beta1gamma2 reconstituted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-stimulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTPgammaS-sensitive high affinity [3H]FMLP binding. The FPR "antagonist" cyclosporin H (CsH) potently and efficiently reduced basal GTPgammaS binding in Sf9 membranes. Another FPR antagonist, N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L- phenylalanine did not inhibit basal GTPgammaS binding but blocked the inhibitory effect of CsH on GTPgammaS binding. Na+ reduced basal GTPgammaS binding and eliminated the inhibitory effect of CsH. Similar effects of FMLP, CsH, and Na+ as in Sf9 membranes were observed with FPR expressed in the mammalian cell line HEK293. Our data show that the human FPR possesses high constitutive activity. CsH is an inverse FPR agonist and stabilizes the FPR in an inactive state. Na+ also stabilizes the FPR in an inactive state and, thereby, diminishes inverse agonist efficacy.

Animals↗

PubMed1998

Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.

The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gs alpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gs alpha (Gs alphaL) binds GDP with lower affinity than the short splice variant (Gs alphaS), but the impact of this difference on the interaction of Gs alpha with the beta 2AR is not known. We studied the beta2 AR/Gs alpha interaction using receptor/G-protein fusion proteins (beta2 AR Gs alphaS and beta2 AR Gs alphaL) expressed in Sf9 cells. Fusion of the beta2 AR to Gs alpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2 AR or Gs alpha. The beta2 AR in beta2 AR Gs alphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2 AR in beta2 AR Gs alphaS. The apparent constitutive activity of the beta2 AR in beta2 AR Gs alphaL may be due to the lower GDP affinity of Gs alphaL compared with Gs alphaS, i.e. Gs alphaL is more often nucleotide-free than Gs alphaS and, therefore, more frequently available to stabilize the beta2 AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

Adrenergic beta-Agonists↗

PubMed1998

Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.

The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsalpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gsalpha (GsalphaL) binds GDP with lower affinity than the short splice variant (GsalphaS), but the impact of this difference on the interaction of Gsalpha with the beta2AR is not known. We studied the beta2AR/Gsalpha interaction using receptor/G-protein fusion proteins (beta2ARGsalphaS and beta2ARGsalphaL) expressed in Sf9 cells. Fusion of the beta2AR to Gsalpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2AR or Gsalpha. The beta2AR in beta2ARGsalphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2AR in beta2ARGsalphaS. The apparent constitutive activity of the beta2AR in beta2ARGsalphaL may be due to the lower GDP affinity of GsalphaL compared with GsalphaS, i.e. GsalphaL is more often nucleotide-free than GsalphaS and, therefore, more frequently available to stabilize the beta2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

Adenylyl Cyclases↗

PubMed1997

Complex regulation of human neutrophil activation by actin filaments: dihydrocytochalasin B and botulinum C2 toxin uncover the existence of multiple cation entry pathways.

In human neutrophils, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenalalanine (fMLP), the Ca(2+)-ATPase inhibitor, thapsigargin, and the lectins, concanavalin A (Con A) and mistletoe lectin I (ML I), stimulate the entry of Ca2+ and Na+ with subsequent activation of exocytosis and superoxide anion (O2-) formation. We studied the role of actin in neutrophil activation. The actin filament-disrupting substances, dihydrocytochalasin B (dhCB) and botulinum C2 toxin (C2 toxin) potentiated fMLP- and lectin-stimulated Ca(2+)- and Na+ entry. Lectin-induced Mn2+ entry was enhanced by actin disruption, whereas fMLP-triggered Mn2+ entry was unaffected. dhCB and C2 toxin inhibited fMLP- and lectin-stimulated Ba2+ influx. The actin disrupters also inhibited fMLP- and ML I-induced Sr2+ influx, whereas Con A-stimulated Sr2+ entry was not influenced by dhCB and C2 toxin. Thapsigargin-stimulated cation entry was not altered by actin disruption. DhCB and botulinum C2 toxin potentiated lysozyme release induced by all four stimuli. Con A and ML I per se activated O2- formation only in the presence and not in the absence of dhCB. Con A potentiated the stimulatory effects of ML I on O2- formation in the presence of dhCB and primed neutrophils to respond to ML I in the absence of dhCB. Our data indicate the following: (1) dhCB and C2 toxin uncover the existence of multiple cation entry pathways in neutrophils; (2) actin disruption facilitates exocytosis and O2- formation by enhancement of Ca(2+)- and Na+ entry and by altering the function of proteins involved in activation of secretion and O2- formation; and (3) Con A and ML I, which possess different sugar specificities, activate different signaling pathways in neutrophils.

Actins↗

PubMed1997

In U-937 promonocytes, misteltoe lectin I increases basal [Ca2+]i, enhances histamine H1- and complement C5a-receptor-mediated rises in [Ca2+]i, and induces cell death.

Mistletoe lectin I (ML I) from Viscum album inhibits cell growth and induces apoptosis (programmed cell death) in several cell types. Because increases in cytosolic Ca2+ concentration ([Ca2+]i) constitute a signal for the induction of apoptosis, we studied the effects of ML I on basal [Ca2+]i, receptor-mediated rises in [Ca2+]i and cell viability, using human U-937 promonocytes as model system. Treatment of U-937 cells with ML I (30-100 ng/ml) significantly increased basal [Ca2+]i. ML I (10-30 ng/ml) enhanced histamine-induced rises in [Ca2+]i up to five-fold. The effect of histamine was inhibited by clemastine but not by famotidine, indicative for its mediation via H1-receptors. ML I additionally enhanced the stimulatory effect of complement C5a on [Ca2+]i, whereas the effect of ATP was unaffected. ML I did not induce responsiveness of U-937 cells towards a bacteria-derived chemotactic peptide. ML I up to 10 ng/ml did not affect cell viability and growth of U-937 cells. ML I at 30 ng/ml moderately inhibited cell growth and reduced cell viability. At 100 ng/ml, ML I was strongly cytotoxic. Our data support the view that Ca2+ plays a role as intracellular signal molecule in the induction of apoptosis and point to an accelerating role of H1- and C5a-receptors in the regulation of this process.

Antigens, CD↗

PubMed1996

Thapsigargin activates univalent- and bivalent-cation entry in human neutrophils by a SK&F I3 96365- and Gd3+-sensitive pathway and is a partial secretagogue: involvement of pertussis-toxin-sensitive G-proteins and protein phosphatases 1/2A and 2B in the signal-transduction pathway.

The Ca2+-ATPase inhibitor thapsigargin (TG) activates bivalent-cation early in human neutrophils via depletion of intracellular Ca2+ stores bu little is known about the underlying mechanism and the functional role of TG-induced cation entry. We studied the effects of TG on univalent- and bivalent cation entry, lysozyme release and superoxide-anion (O2-) formation in human neutrophils. TG, like the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), stimulated entry of Ca2+, Mn2+, Ba2+, Sr2+ and Na+ in a 1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365)- and Gd3+-sensitive manner. The inhibitors of protein phosphates 1/2A, calyculin A and okadaic acid, diminished TG-induced cation influxes, whereas the inhibitors of protein phosphatase 2B, cyclosporin A and FK-506, were potentiators. Pertussis toxin (PTX) partially inhibited the effects of TG on Ca2+ and Mn2+ entry. TG and fMLP activated inward currents with a linear current-voltage relationship and a reversal potential at about 0 mV. TG activated lysozyme release and potentiated fMLP-induced O2- formation. TG-induced lysozyme release was inhibited by SK&F 96365, PTX and the removal of extracellular Ca2+ or Na+. Our data show that TG activates a non-selective and SK&F 96365- and Gd3+-sensitive cation entry pathway and is a partial secretagogue. TG-stimulated cation entry involves PTX-sensitive G-proteins and protein phosphatases, with protein phosphatases 1/2A and 2B playing opposite roles.

Cations↗