Subcellular targeting of multiligand-binding protein gC1qR.

Abstract gC1q receptor, a protein originally described as the cell surface receptor for the globular heads of complement factor C1q, has been found to bind human H-kininogen with high affinity and specificity. Therefore, gC1qR has been considered candidate kininogen docking site on the surfaces of platelets, neutrophils and endothelial cells. Recent work demonstrating that gC1qR is an intracellular protein that is tightly associated with mitochondria rather than targeted to the cell surface has challenged this view. To further probe cellular trafficking routes of gC1qR, we overexpressed human gC1qR in a mammalian cell and monitored cell surface exposure of recombinant gC1qR by virtue of its…

Human kininogens interact with M protein, a bacterial surface protein and virulence determinant.

Streptococcus pyogenes, the most significant streptococcal species in clinical medicine, expresses surface proteins with affinity for several human plasma proteins. Here we report that kininogens, the precursors to the vasoactive kinins, bind to the surface of S. pyogenes. M protein, a surface molecule and a major virulence factor-in these bacteria, occurs in > 80 different serotypes. Among 49 strains of S. pyogenes, all of different M serotypes, 41 bound radiolabelled kininogens, whereas 6 M protein-negative mutant strains showed no affinity. M protein of most serotypes bind fibrinogen, and among the 55 strains tested, binding of kininogens was closely correlated to fibrinogen bindi…

Endothelin-1 and endothelin receptor status in kidney transplants undergoing acute rejection.

Abstract Endothelin-1 (ET-1) is a potent vasoconstrictor with vasopressor and mitogenic effects. Blood samples were collected from 21 renal transplant patients undergoing acute rejection at the time of diagnostic kidney biopsy: there were 20 men and one woman, mean age 35.6 years. All patients were on triple immunosuppressive therapy with cyclosporine A, azathioprine and methylprednisolone. Twenty living kidney donors pre-uninephrectomy (11 men and nine women, mean age 34 years) served as controls. Control kidney was obtained from fresh autopsy material and normal kidney tissue from nephrectomies for malignancy. Mean plasma ET-1 was significantly increased at 1.56±0.2 pg ml −1 during acute …

Cellular visualization of tissue prokallikrein in human neutrophils and myelocytes

The vasoactive peptides bradykinin and kallidin (lysyl-bradykinin) have been implicated in diapedesis, a cellular process by which neutrophils migrate through endothelial cell gap junctions. The kinin peptides are released from their precursor moiety, kininogen, by the specific action of endoproteinases, the kallikreins. Kininogens have been demonstrated on the surface of neutrophils, and the presence of a competent processing enzyme such as tissue prokallikrein in neutrophils has been postulated, but firm evidence for this is still lacking. We have raised antibodies to a synthetic peptide that is a sequence copy of the activation segment of human TK and demonstrated that the anti-peptide a…

Different protein turnover of interleukin-6-type cytokine signalling components.

Interleukin (IL)-6 and IL-6-type cytokines signal through the gp130/Jak/STAT signal transduction pathway. The key components involved are the signal transducing receptor subunit gp130, the Janus kinases Jak1, Jak2 and Tyk2, STAT1 and STAT3 of the family of signal transducers and activators of transcription, the protein tyrosine phosphatase SHP2 and the suppressors of cytokine signalling SOCS1, SOCS2 and SOCS3. Whereas considerable information has been accumulated concerning the time-course of activation for the individual signalling molecules, data on the availability of the proteins involved in IL-6-type cytokine signal transduction are scarce. Nevertheless, availability of these molecules…

Two distinct Ca2+ influx pathways activated by the bradykinin B2 receptor.

The hormone-induced depletion of cellular Ca stores provides a signal for the Ca2+ influx into electrically non-excitable cells; however, the underlying molecular mechanisms remain elusive. Therefore, we analyzed bradykinin-activated Ca2+ influx into human foreskin fibroblast cells, HF-15, by fura-2 and 45Ca labeling to discriminate between Ca2+ influx into the fura-sensitive compartment and Ca uptake into fura-insensitive Ca stores. Bradykinin-activated CaZt influx into the fura-sensitive compartment was blocked by inhibitors of NO synthases. These inhibitors also suppressed bradykinin-activated increases in cGMP, indicating that the NO-dependent increase in cGMP is involved in the activat…

ACE inhibitor potentiation of bradykinin-induced venoconstriction

1. Angiotensin-converting enzyme (ACE) inhibitors exert their cardiovascular effects not only by preventing the formation of angiotensin II (AII), but also by promoting the accumulation of bradykinin in or at the vessel wall. In addition, certain ACE inhibitors have been shown to augment the vasodilator response to bradykinin, presumably by an interaction at the level of the B2 receptor. We have investigated whether this is a specific effect of the ACE inhibitor class of compounds in isolated endothelium-denuded segments of the rabbit jugular vein where bradykinin elicits a constrictor response which is exclusively mediated by activation of the B2 receptor. 2. Moexiprilat and ramiprilat (< …

Correlations in palmitoylation and multiple phosphorylation of rat bradykinin B2 receptor in Chinese hamster ovary cells.

Rat bradykinin B2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B2 receptor was isolated on oligo(dT)-cellulose using N-(epsilon-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA)30-mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isola…

Subtype-Specific Desensitization of Human Endothelin ETA and ETB Receptors Reflects Differential Receptor Phosphorylation

Endothelins regulate blood pressure in mammals through G protein-coupled receptors. Two receptor subtypes, ETA and ETB, exist which differ by their agonist profiles. Here we show subtype-specific differences in the inactivation of these endothelin receptors. Using a modified inositol phosphate accumulation assay, we found that stimulation of ETA by endothelin-1 results in sustained activation of the subtype, retaining >30% of its initial activity even 20 min after agonist administration, whereas the ETB rapidly deactivated after agonist stimulation, losing >80% of its initial activity within 5 min after endothelin application. The discrepancy in receptor inactivation is reflected by subtype…

Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis.

Neutrophil-borne heparin-binding protein (HBP) is a multifunctional protein involved in the progression of inflammation. HBP is stored in neutrophil granules and released upon stimulation of the cells in proximity to endothelial cells. HBP affects endothelial cells in multiple ways; however, the molecular and cellular mechanisms underlying the interaction of HBP with these cells are unknown. Affinity isolation and enzymatic degradation demonstrated that HBP released from human neutrophils binds to endothelial cell-surface proteoglycans, such as syndecans and glypican. Flow cytometry indicated that a significant fraction of proteoglycan-bound HBP is taken up by the endothelial cells, and we …

Anti-Idiotypic Antibodies Against the Kinin Receptor

Three sets of monoclonal antibodies against bradykinin (MBK1, MBK2, and MBK3) were generated by somatic cell fusion, characterized by their peptide specificity, and compared with the known ligand specificity of the kinin receptor subtypes. By these criteria, the paratope of MBK3 resembled the B 2 receptor binding site, whereas MBK1 shared principal binding characteristics with the B 1 receptor. Anti-idiotypic antibodies against MBK1, MBK2, and MBK3 were raised in rabbit and sheep

Probing human beta1- and beta2 -adrenoceptors with domain-specific fusion protein antibodies.

In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta 1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonis…

Na+ ions binding to the bradykinin B2 receptor suppress agonist-independent receptor activation.

Control of the balance between receptor activation and inactivation is a prerequisite for seven transmembrane domain (7TM) receptor function. We asked for a mechanism to stabilize the inactive receptor conformation which prevents agonist-independent receptor activation. Na+ ions have reciprocal effects on agonist versus antagonist interaction with various 7TM receptors. To investigate the Na+ dependence of receptor activation we chose the bradykinin B2 receptor as a prototypic 7TM receptor. Decrease of the intracellular Na+ content from 40 mM to 10 mM of COS-1 cells transiently expressing rat B2 receptors activated the B2 receptor in the absence of agonist as shown by a 3-fold increase in t…

Ligand-induced phosphorylation/dephosphorylation of the endogenous bradykinin B2 receptor from human fibroblasts.

We have studied the ligand-induced phosphorylation/dephosphorylation of the bradykinin B2 receptor endogenously expressed in human HF-15 fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36), derived from the extreme carboxyl terminus of the human B2 receptor, precipitated the receptor from solubilized membranes of HF-15 cells that had been labeled with [32P]orthophosphate. A low basal level of B2 receptor phosphorylation was found in the absence of a ligand. Stimulation of the cells with the B2 receptor agonists bradykinin, [Lys0,Hyp3]bradykinin, kallidin, and T-kinin resulted in a rapid and efficient phosphorylation of the receptor. The B2 receptor antagonist HOE140 and the B1 …

Mapping the cell binding site on high molecular weight kininogen domain 5.

Investigations mapped the region(s) on the light chain of high molecular weight kininogen (HK) that participates in cell binding. Sequential and overlapping peptides of domain 5 (D5H) were synthesized to determine its cell binding site(s). Three peptides from non-overlapping regions on D5H were found to inhibit biotin-HK binding to endothelial cells. Peptides GKE19 and HNL 21 weakly inhibited biotin-HK binding with IC50 of 792 and 215 microM, respectively. Peptide HKH20 inhibited biotin-HK binding with an IC50 of 0.2 microM. Two peptides, GGH18 and HVL24, which overlapped HKH20, also inhibited biotin-HK binding to endothelial cells with IC50 values of 108 and 0.8 microM, respectively. Bioti…

Angiotensin-Converting Enzyme Inhibitor Ramiprilat Interferes With the Sequestration of the B 2 Kinin Receptor Within the Plasma Membrane of Native Endothelial Cells

Background —ACE (kininase II) inhibitors have been shown to exert their beneficial cardiovascular effects via the inhibition of both angiotensin II formation and bradykinin breakdown. Because recent evidence suggests that ACE inhibitors may also interfere with B 2 kinin receptor signaling and thus enhance the vascular response to bradykinin, we examined whether the distribution of B 2 kinin receptors within the plasma membrane of native endothelial cells is affected by an ACE inhibitor. Methods and Results —Localization of the B 2 kinin receptor in membranes prepared from native porcine aortic endothelial cells was evaluated by means of specific [ 3 H]bradykinin binding and immunoprecipita…

Identification of a plasminogen-binding motif in PAM, a bacterial surface protein.

Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33, 41, 52, 53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the…

Subtype-specific endothelin-A and endothelin-B receptor desensitization correlates with differential receptor phosphorylation.

In the rat cardiovascular system endothelin-1 (ET-1) elicits prolonged physiologic responses mediated by the ET A receptor, whereas the effects mediated by the ET B receptor are transient. The molecular mechanisms for the subtype-specific responses are not yet clear. However, post-translational modifications such as phosphorylation and palmitoylation may play an important role. In Sf9 cells overexpressing the human ET A and ET B receptors, both subtypes are palmitoylated. However, only the ET B but not the ET A receptor is phosphorylated in a ligand-dependent manner. Because phosphorylation is believed to play an important role in ligand-dependent receptor inactivation, we analyzed whether …

Rat fetuin: distribution of protein and mRNA in embryonic and neonatal rat tissues

Fetuin is a serum protein widely distributed in the animal kingdom and found in all mammalian species so far investigated. It is mainly a fetal protein, in the sense that the highest concentrations are found in serum and body fluids of embryos and fetuses. In order to elucidate possible biological functions of fetuin, we have studied its synthesis and distribution during the prenatal development of the rat with immunohistochemistry and in situ hybridization. We have isolated fetuin from rat serum and produced an antibody against this protein. In situ hybridization was performed using a 375-nucleotides-long digoxigenin-labeled riboprobe. Fetuin was unevenly distributed in all organ systems d…

Acrosin, the peculiar sperm-specific serine protease.

The sperm enzyme acrosin has long been known as one of the key enzymes in the mammalian fertilization process. Elucidation of primary structures of preproacrosin from various species have allowed a deeper insight into the structural organization and the complex evolution of the sperm proteinase acrosin. In addition to the typical elements of serine proteases, the acrosin molecule possesses one novel domain that might convey DNA-binding properties.

Palmitoylation of Endothelin Receptor A

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ETA) incorporates [3H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ETA into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ETA mutants was essentially unchanged as compared to the palmitoylated wild type ETA suggesting that the replacement of the cysteine residues did not alter the overall structure o…

Absorption of kininogen from human plasma by Streptococcus pyogenes is followed by the release of bradykinin.

H-kininogen (high-molecular-mass kininogen, HK) is the precursor of the vasoactive peptide hormone bradykinin (BK). Previous work has demonstrated that HK binds to Streptococcus pyogenesthrough M-proteins, fibrous surface proteins and important virulence factors of these bacteria. Here we find that M-protein-expressing bacteria absorb HK from human plasma. The HK bound to the bacteria was found to be cleaved, and analysis of the degradation pattern suggested that the cleavage of HK at the bacterial surface is associated with the release of BK. Moreover, addition of activated plasma prekallikrein to bacteria preincubated with human plasma, resulted in BK release. This mechanism, by which a p…

Identification of an endothelial cell binding site on kininogen domain D3

High and low molecular mass kininogen, two multidomain plasma proteins, bind to endothelial cells, platelets, and neutrophils in the intravascular compartment. The specific cell attachment site on their common heavy chain is mediated by domain-3, a cystatin-like structure with inhibitory capacity for papain-like proteinases (Jiang, Y., Müller-Esterl, W., and Schmaier, A. H. (1992) J. Biol. Chem. 267, 3712-3717). In this report, the domain-3 cell binding site is determined by an antibody-directed strategy. The epitope of monoclonal antibody HKH15, which binds to domain-3 and blocks the binding of kininogens to platelets and endothelial cells, was mapped using seven synthetic peptides, which …

The N-terminal Amino Group of [Tyr8]Bradykinin Is Bound Adjacent to Analogous Amino Acids of the Human and Rat B2 Receptor

To obtain data of the bradykinin B2 receptor's agonist binding site, we used a combined approach of affinity labeling and "immunoidentification" of receptor fragments generated by cyanogen bromide cleavage. Domain-specific antibodies to the various extracellular receptor domains were applied to detect receptor fragments with covalently attached [125I-Tyr8]bradykinin. As a cross-linker we used the homobifunctional reagent disuccinimidyl tartarate (DST), which reacts preferentially with primary amines. With this technique a [125I-Tyr8]bradykinin-labeled receptor fragment derived from the third extracellular domain was identified. The epsilon-amino group of lysine (Lys172) of the human B2 rece…

The nucleotide and deduced amino acid structures of sheep and pig fetuin. Common structural features of the mammalian fetuin family

This study was initiated to gain further insight into the structural features of the mammalian fetuin family. The cDNA structures of sheep and pig fetuin were determined. The cDNA insert encoding sheep (pig) fetuin comprised 1550 (1470) nucleotides, including 54 (46) nucleotides encoding a signal peptide of 18 (15) residues and 1038 (1041) nucleotides encoding the 346 (347) amino acids of the mature plasma protein. The predicted amino-terminal sequence of the mature pig fetuin was confirmed by the amino-terminal sequence of the purified protein. However, two alternative sheep amino-terminal sequences were found in fetuin purified from the plasma of a single sheep fetus; the minor product wa…

Isolation and Characterization of the Kininogen-binding Protein p33 from Endothelial Cells

Abstract Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5H that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 (“LDC27”) and 20 residues of D5H (“HKH20”), respectively (Herwald, H., Hasan, A. A. K., Godovac-Zimmermann, J., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Muller-Esterl, W. (1995) J. B…

Activation of the contact-phase system on bacterial surfaces--a clue to serious complications in infectious diseases.

Fever, hypotension and bleeding disorders are common symptoms of sepsis and septic shock. The activation of the contact-phase system is thought to contribute to the development of these severe disease states by triggering proinflammatory and procoagulatory cascades; however, the underlying molecular mechanisms are obscure. Here we report that the components of the contact-phase system are assembled on the surface of Escherichia coli and Salmonella through their specific interactions with fibrous bacterial surface proteins, curli and fimbriae. As a consequence, the proinflammatory pathway is activated through the release of bradykinin, a potent inducer of fever, pain and hypotension. Absorpt…

Kinin receptors in human vascular tissue: their role in atheromatous disease

Using samples of many human blood vessels, obtained at autopsy and specific antibodies directed to peptide sequences of the kinin B1 and B2 receptors, we demonstrate the localisation of these receptors within the human vascular system using standard immunolabelling techniques. In large elastic arteries and veins, kinin receptors are present only in the endothelial cells whereas in all muscular arteries and arterioles, these receptors are present in both the endothelial and smooth muscle cells. The identification of kinin receptors in human blood vessels confirms that kinins may modulate both vascular permeability and contractility. The incidental finding at histology, of patchy atheromatous…

Cloning and Targeted Deletion of the Mouse Fetuin Gene

We proposed that the alpha2-Heremans Schmid glycoprotein/fetuin family of serum proteins inhibits unwanted mineralization. To test this hypothesis in animals, we cloned the mouse fetuin gene and generated mice lacking fetuin. The gene consists of seven exons and six introns. The cystatin-like domains D1 and D2 of mouse fetuin are encoded by three exons each, whereas a single terminal exon encodes the carboxyl-terminal domain D3. The promoter structure is well conserved between rat and mouse fetuin genes within the regions shown to bind transcription factors in the rat system. Expression studies demonstrated that mice homozygous for the gene deletion lacked fetuin protein and that mice heter…

Kininogen binding protein p33/gC1qR is localized in the vesicular fraction of endothelial cells

AbstractThe endothelial protein p33/gC1qR is thought to mediate the assembly of components of the kinin-forming and complement-activating pathways on the surface of cardiovascular cells. FACS analysis of intact human umbilical vein endothelial cells using specific antibodies to p33 revealed a minor fluorescence on the cell surface whereas permeabilized cells showed a bright fluorescence indicative of an intracellular localization of p33. Immunostaining of fixed cells confirmed the predominant intracellular localization of p33. Fractionation studies demonstrated that the vesicular but not the membrane fraction of EA.hy926 cells is rich in p33. We conclude that externalization of p33 must pre…

Receptor phosphorylation does not mediate cross talk between muscarinic M(3) and bradykinin B(2) receptors.

This study examined cross talk between phospholipase C-coupled muscarinic M3and bradykinin B2receptors coexpressed in Chinese hamster ovary (CHO) cells. Agonists of either receptor enhanced phosphoinositide signaling (which rapidly desensitized) and caused protein kinase C (PKC)-independent, homologous receptor phosphorylation. Muscarinic M3but not bradykinin B2receptors were also phosphorylated after phorbol ester activation of PKC. Consistent with this, muscarinic M3receptors were phosphorylated in a PKC-dependent fashion after bradykinin B2receptor activation, but muscarinic M3receptor activation did not influence bradykinin B2receptor phosphorylation. Despite heterologous phosphorylatio…

Cytoplasmic STAT proteins associate prior to activation

The commonly accepted model of STAT factor activation at the cytoplasmic part of the receptor assumes that signal transducers and activators of transcription (STATs) are recruited from a cytoplasmic pool of monomeric STAT proteins. Based on a previous observation that non-phosphorylated STAT3-Src homology 2 domains dimerize in vitro, we investigated whether the observed dimerization is of physiological relevance within the cellular context. We show that STAT1 and STAT3 are pre-associated in non-stimulated cells. Apparently, these complexes are not able to translocate into the nucleus. We provide evidence that the event of STAT activation is more complex than previously assumed.

Molecular Co-operation between Protein PAM and Streptokinase for Plasmin Acquisition by Streptococcus pyogenes

Bacterial surface-associated plasmin formation is believed to contribute to invasion, although the underlying molecular mechanisms are poorly understood. To define the components necessary for plasmin generation on group A streptococci we used strain AP53 which exposes an M-like protein ("PAM") that contains a plasminogen-binding sequence with two 13-amino acid residues long tandem repeats (a1 and a2). Utilizing an Escherichia coli-streptococcal shuttle vector, we replaced a 29-residue long sequence segment of Arp4, an M-like protein that does not bind plasminogen, with a single (a1) or the combined a1a2 repeats of PAM. When expressed in E. coli, the purified chimeric Arp/PAM proteins both …

Expression and cellular localization of kininogens in the human kidney

Expression and cellular localization of kininogens in the human kidney. Human high (H) and low (L) molecular weight kininogens are encoded by distinct mRNAs derived by alternative splicing from a single kininogen gene. Previous studies have demonstrated the presence of L-kininogen but not of H-kininogen in the distal nephron structures of the kidney. Using the highly sensitive reverse trancriptase-polymerase chain reaction (RT-PCR) we have been able to demonstrate the expression of both H-kininogen mRNA and L-kininogen mRNA in kidney and liver. The presence of H- and L-kininogen antigen was shown immunohistochemically by applying specific antibodies that discriminate between the two types o…

Structural dissection of the multidomain kininogens. Fine mapping of the target epitopes of antibodies interfering with their functional properties.

Kininogens, the large precursor molecules of the vasoactive kinin peptides, are prototypic multidomain proteins serving numerous functions. To investigate their structure-function relationships, we have raised a panel of monoclonal antibodies against human H-kininogen and L-kininogen and fragments thereof and characterized them with respect to their target epitopes. Of 35 antibodies, 12 were directed to the amino-terminal domains (D1 to D3) of cystatin-like structure, 3 recognized domain D4 bearing the kinin segment, 17 bound to the carboxyl-terminal domains of H-kininogen (D5H and D6H), and 3 bound to the carboxyl-terminal domain D5L of L-kininogen. At least 14 distinct epitopes spread ove…

Mapping of the Discontinuous H-kininogen Binding Site of Plasma Prekallikrein

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K(D) = 1.2 x 10(-8) M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 approximately A…

Rat tyrosine kinase inhibitor shows sequence similarity to human α2-HS glycoprotein and bovine fetuin

Human alpha 2-HS glycoprotein and bovine fetuin, abundant proteins of fetal plasma, are structural members of the fetuin family within the cystatin superfamily. They are characterized by the presence of two N-terminally located cystatin-like units and a unique C-terminal sequence segment not present in the other members of the cystatin superfamily. Search for related sequences revealed that the natural inhibitor of the insulin receptor tyrosine kinase [Auberger, Falquerho, Contreres, Pages, Le Cam, Rossi &amp; Le Cam (1989) Cell (Cambridge, Mass.) 58, 631-640] shows sequence similarity to the mammalian fetuins. The sequence identity between rat tyrosine kinase inhibitor, human alpha 2-HS gl…

Anti-Idiotypic Antibodies Against the Kinin Receptor

Three sets of monoclonal antibodies against bradykinin (MBK1, MBK2, MBK3) were generated by somatic cell fusion, characterized by their peptide specificity and compared to the known ligand specificity of the kinin receptor subtypes. By these criteria the paratope of MBK3 resembled the B2 receptor binding site whereas MBK1 shared principal binding characteristics with the B1 recrptor. Anti-idiotypic antibodies against MBK1, MBK2 and MBK3 were raised in rabbit and sheep. Specificity of the network components was verified by inhibition experiments on the level of peptide, idiotype and anti-idiotype. Anti-idiotypic antibodies against MBK3 recognized a conformation-dependent epitope which was bi…

Mapping of the H-Kininogen Binding Site Exposed by the Prekallikrein Heavy Chain

Award ceremony of the E.K. Frey-E. Werle Foundation of the Henning L. Voigt family.

Distribution of bradykinin B2 receptors in sheep brain and spinal cord visualized by in vitro autoradiography

Bradykinin B2 receptors were localized in the sheep brain and spinal cord by quantitative in vitro autoradiography using a radiolabelled and specific bradykinin B2 receptor antagonist analogue, 3-4-hydroxyphenyl-propionyl-D-Arg0-[Hyp3,Thi5,D-Tic 7,Oic8]bradykinin, (HPP-HOE 140). This radioligand displays high affinity and specificity for bradykinin B2 receptors. The respective K(i) values of 0.32, 1.37 and 156 nM were obtained for bradykinin, HOE140 and D-Arg[Hyp3,D-Phe7,Leu8]bradykinin competing for radioligand binding to lamina II of sheep spinal cord sections. Using this radioligand, we have demonstrated the distribution of bradykinin B2 receptors in many brain regions which have not bee…

Mapping of the high molecular weight kininogen binding site of prekallikrein. Evidence for a discontinuous epitope formed by distinct segments of the prekallikrein heavy chain.

Prekallikrein, a glycoprotein involved in contact phase activation, circulates in plasma in the form of a binary complex with high molecular weight kininogen (H-kininogen). The binding to H-kininogen is mediated by the prekallikrein heavy chain consisting of four repetitive domains, A1-A4. To define more precisely the region(s) involved in kininogen binding, we have employed an affinity cross-linking strategy with a synthetic peptide of 31 residues which mimics the prekallikrein binding site of H-kininogen. Cross-linking of the radiolabeled peptide to (pre)kallikrein revealed a binding segment in the NH2-terminal portion of the prekallikrein heavy chain; another binding segment was located …

Overexpression and functional characterization of kinin receptors reveal subtype-specific phosphorylation.

G protein-coupled receptors such as the receptors for bradykinin are present in low copy numbers in most natural cells. To overcome the problems associated with the analysis of these receptors at the protein level, we used highly efficient expression systems such as the baculovirus/insect cell system. However, the structural and functional statuses of recombinant receptors have often remained elusive. We have expressed the two types of human kinin receptors, B1 and B2, in Sf9 cells. Both receptors are found on the surface of infected cells where they display the same pharmacological profiles as their cognate receptors of native cells. The functional analysis of kinin receptors coupled to th…

Posttranslational Processing of Human α2-HS Glycoprotein (Human Fetuin)

α2-HS glycoprotein (α2-HS) is a major protein occuring in human blood and calciferous tissues. Due to extensive sequence identity, α2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native α2-HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N-glycosylations and O-glycosylations, and sulfation of the carbohydrate side chains. Various two-chain forms of α2-HS have been prepared from human plasma, however, the single-chain precursor has not yet been isolated. Here, we have studied the biosynthesis of α2-HS by a human hepatoma cell line, HepG2. We demonstrate that a single-chain form…

Isolation of the Endothelin B Receptor from Bovine Lung Structure, Signal Sequence, and Binding Site

Bovine lung endothelin-B receptor has been isolated in good yield with a new procedure involving the use of endothelin-1 coupled to iminobiotin with a long spacer and avidin-agarose affinity chromatography. Contrary to previous reports, evidence has been obtained that the native form of this receptor corresponds to the full-length transcript expected on the basis of cDNA clones. The binding of endothelin to a variety of shortened fragments of the full receptor suggests that the long N-terminal sequence of this receptor has very little influence on the binding of endothelin and that the main determinants of the endothelin binding site might be constituted by residues in the sixth, and possib…

Kinine, Kallikreine und Kaskadensysteme

Die molekularen Grundlagen der Bioregulation sind bisher nur in Ansatzen verstanden. Steuerung und Regulation der vielfaltigen physiologischen Funktionen eines multizellularen Organismus sind namlich extrem komplex. Mit den modernen Methoden der Molekularbiologie, Zellbiologie und Immunologie versucht man, die molekularen Details regulatorischer Systeme Schritt fur Schritt aufzuspuren. Fur die Wissenschaft interessant sind dabei vor allem molekulare Netzwerke, die an Erkrankungen des Herz-Kreislauf-Systems und des Immunsystems, den haufigsten Todesursachen industrialisierter Lander beteiligt sind. Im folgenden wird anhand der Kinine, einer Gruppe von blutdrucksenkenden und entzundungsforder…

Bradykinin-induced Internalization of the Human B2Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail

The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the r…

Coupling of endothelin receptors to the ERK/MAP kinase pathway. Roles of palmitoylation and G(alpha)q.

Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ET(A) and ET(B). To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G(alpha) protein binding capacity. Endothelin-1 stimulation of wild-type ET(A) or ET(B) induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ET(A) and ET(B) mutants failed to …

NOSIP, a novel modulator of endothelial nitric oxide synthase activity.

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds …

Localization of Bradykinin B2Receptors in the Endometrium and Myometrium of Rat Uterus and the Effects of Estrogen and Progesterone1

In the uterus, bradykinin is a potent inducer of smooth muscle contraction, which is mediated by the bradykinin B2 receptor subtype. However, little is known about the distribution or regulation of this receptor in this tissue. The aim of this study was to localize the B2 receptor in the uterus and determine whether the levels of this receptor were altered during the estrous cycle and modulated by estrogen and/or progesterone in ovariectomized rats. At diestrus, uterine B2 receptors were localized to both the circular and longitudinal smooth muscle layers of the myometrium, the endometrial stroma, the glandular epithelium, and the layer subjacent to the luminal epithelium. B2 receptor level…

Purification and Characterization of the Soluble Interleukin-6 Receptor from Human Plasma and Identification of An Isoform Generated through Alternative Splicing

The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein α1-antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglyc…

Cloning and Functional Characterization of the Ornithokinin Receptor

Kinins are proinflammatory peptides that dilate vessels, increase vascular permeability, contract smooth muscles, and provoke pain. The known mammalian kinin receptors are classified as two subtypes, i.e. the B1 receptor triggered by [des-Arg9]bradykinin and inhibited by [des-Arg9,Leu8]bradykinin, and the B2 receptor stimulated by bradykinin and antagonized by HOE140. Here we report the cloning of a non-mammalian kinin receptor gene amplified from genomic chicken DNA. The protein predicted from the open reading frame shows 31 and 49% sequence identity to the human B1 and B2 receptors, respectively, suggesting that it represents a G protein-coupled receptor of the kinin receptor family. The …

Mapping of the Discontinuous Kininogen Binding Site of Prekallikrein

Prekallikrein, the precursor to the serine proteinase kallikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 nM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the distal…

Immunological Probes for the Bradykinin B2 Receptor. A Toolbox

Publisher Summary This chapter provides an overview of the immunological tools for bradykinin (BK) B 2 receptor. Receptors for kinins are classified as two major subtypes, B 1 and B 2 , although other subtypes may exist. B 1 receptors are activated by carboxyterminally truncated kinins, whereas BK and kallidin (Lys-BK) are B 2 receptor agonists. Molecular cloning has revealed the primary structures of B 1 and B 2 receptors and identified them as members of the G protein-coupled receptor family, characterized by seven membrane-spanning α-helices. In some tissues, B 1 receptor expression is induced by cytokines, such as interleukin-1, whereas the B 2 receptor is thought to be expressed consti…

Activation of mitogen-activated protein kinase by the bradykinin B2receptor is independent of receptor phosphorylation and phosphorylation-triggered internalization

Recent evidence suggests that serine/threonine phosphorylation and internalization of beta2-adrenergic receptors play critical roles in signalling to the mitogen-activated protein kinase cascade. To investigate whether this represents a general mechanism employed by G protein-coupled receptors, we studied the requirement of these processes in the activation of mitogen-activated protein kinase by G alpha(q)-coupled bradykinin B2 receptors. Mutant B2 receptors impaired in receptor phosphorylation and internalization are fully capable to activate mitogen-activated protein kinase. Bradykinin-induced long-term effects on mitogenic signalling monitored by measuring the transcriptional activity of…

Limited Proteolysis of Human α2-HS Glycoprotein/Fetuin

alpha2-HS glycoprotein is a major protein of human plasma whose function is still obscure. A proteolytically processed form of alpha2-HS glycoprotein lacking a segment of 40 amino acid residues bridging its heavy and light chain portions ("connecting peptide") has been described suggesting that this peptide is released by post-translational processing to fulfill biological role(s) of alpha2-HS glycoprotein. To test this hypothesis we investigated how the connecting peptide is released from the parental molecule by limited proteolysis. We developed monoclonal antibodies to various portions of the connecting peptide and its NH2-terminal flanking region which cross-react with the native alpha2…

Kinin receptor status in normal and inflammed gastric mucosa

No documented studies have been reported on the presence of B1 and B2 kinin receptors in the mammalian gastric mucosa. This first study aimed to immunolocalise sites of B1 and B2 kinin receptors in the human pyloric gastric mucosa and to evaluate its role in gastritis. Biopsies were obtained from patients with dyspepsia during endoscopic examination of the patient. The diagnosis and grading of the gastritis was performed on histological examination. Sections were immunostained for both B1 and B2 receptors using rabbit anti-human B1 and B2 kinin receptor antibodies. Control tissue was obtained from partial gastrectomy specimens, following surgical excision of the antrum for duodenal ulcers. …

Inhibition of B2 receptor internalization delays its dephosphorylation

Extracellular Domains of the Bradykinin B2 Receptor Involved in Ligand Binding and Agonist Sensing Defined by Anti-peptide Antibodies

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised. The antibodies strongly reacted with their corresponding antigens and cross-reacted both with the denatured and the native B2 receptor. Affinity-purified antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140. Antibodies to extracellular domain 3 (second loop) efficiently interfered, in a concentration-dependent manner, with…

An NMR Study of the Interaction of 15N-Labelled Bradykinin with an Antibody Mimic of the Bradykinin B2 Receptor

An isotope-edited NMR study of the peptide hormone bradykinin (RPPGFSPFR) bound to the Fab fragment of a monoclonal antibody against bradykinin (MBK3) is reported. MBK3 was previously shown to provide a binding site model of the B2 bradykinin receptor [Haasemann, M., Buschko, J., Faussner, A., Roscher, A. A., Hoebeke, J., Burch, R. M. & Muller-Esterl, W. (1991) Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Immunol. 147, 3882-3892]. Bradykinin was obtained in a uniformly 15N-labelled form using recombinant expression of a fusion protein consisting of the glutathione-binding domain of glutathione S-transferase fused to residues 354-375 of the high-molecular-…

Posttranslational processing of human alpha 2-HS glycoprotein (human fetuin). Evidence for the production of a phosphorylated single-chain form by hepatoma cells.

alpha 2-HS glycoprotein (alpha 2-HS) is a major protein occurring in human blood and calciferous tissues. Due to extensive sequence identity, alpha 2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native alpha 2-HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N-glycosylations and O-glycosylations, and sulfation of the carbohydrate side chains. Various two-chain forms of alpha 2-HS have been prepared from human plasma, however, the single-chain precursor has not yet been isolated. Here, we have studied the biosynthesis of alpha 2-HS by a human hepatoma cell line, HepG2. We demon…

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: A possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75–80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [3H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca2+]i) in fura 2-AM loaded sf21 cells, and promoted [35S]-GTPγS binding to membranes.…

Parallel reduction of plasma levels of high and low molecular weight kininogen in patients with cirrhosis

SummaryLittle is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 μg/ml [range 22-99 μg/ml]) and LK (58 μg/ml [15…

Tissue kallikrein and kininogen in human sweat glands and psoriatic skin

The cellular localization of immunoreactive tissue kallikrein and kininogen was studied in normal and psoriatic human skin. Immunoreactivity to both enzyme and substrate was observed in secretory granules of the dark cells in the secretory fundus (acinus) of the sweat glands. Double immunostaining revealed a segmental distribution of the two antigens. Each acinar section contained either tissue kallikrein or kininogen. However, there appeared to be a junctional zone in which both were present, but in separate dark cells. Immunoreactivity for both antigens was also observed in close apposition to the luminal microvilli of the duct cells. No specific immunostaining was seen in sebaceous gland…

The Serum Protein α2-HS Glycoprotein/Fetuin Inhibits Apatite Formation in Vitro and in Mineralizing Calvaria Cells

We present data suggesting a function of alpha2-HS glycoproteins/fetuins in serum and in mineralization, namely interference with calcium salt precipitation. Fetuins occur in high serum concentration during fetal life. They accumulate in bones and teeth as a major fraction of noncollagenous bone proteins. The expression pattern in fetal mice confirms that fetuin is predominantly made in the liver and is accumulated in the mineralized matrix of bones. We arrived at a hypothesis on the molecular basis of fetuin function in bones using primary rat calvaria osteoblast cultures and salt precipitation assays. Our results indicate that fetuins inhibit apatite formation both in cell culture and in …

Localization of the bradykinin B2 receptor in uterus, bladder and Madin-Darby canine kidney cells

Kinins are biologically active peptides that act through specific receptors, B1 and B2. Here we describe the localization of the bradykinin B2 receptor in Madin-Darby canine kidney cells and in the uterus and urinary bladder of rat or human origin. We discuss the suitability of anti-peptide antibodies to assess the tissue distribution of bradykinin B2 receptors.

The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor.

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin rece…

Structural features of the human bradykinin B2 receptor probed by agonists, antagonists, and anti-idiotypic antibodies

The human bradykinin B2 receptor belongs to the family of G-protein-coupled receptors. To characterize the receptor protein, we have solubilized the membranes of cultured human foreskin fibroblasts bearing the B2 receptor. Affinity cross-linking of the solubilized receptor with the labeled agonist, 125I-Tyr0-bradykinin, or the labeled antagonist, 125I-(4-hydroxy-phenyl-propionyl)-HOE140, revealed major bands of apparent molecular mass of 69 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and of 59 kDa under non-reducing conditions. A 1000-fold molar excess of each of the unlabeled ligands quenched the specific labeling suggesting that the agonist and the antagonist …

Assembly of human contact phase proteins and release of bradykinin at the surface of curli-expressing Escherichia coli.

Previous work has demonstrated that most strains of the human pathogen Streptococcus pyogenes bind kininogens through M protein, a fibrous surface protein and virulence determinant. Here we find that strains of several other pathogenic bacterial species, both Gram-positive and Gram-negative, isolated from patients with sepsis, also bind kininogens, especially kininogen (HK). The most pronounced interaction was seen between HK and Escherichia coli. Among clinical isolates of E. coli, the majority of the enterohaemorrhagic, enterotoxigenic, and sepsis strains, but none of the enteroinvasive and enteropathogenic strains, bound HK. Binding of HK to E. coli correlated with the expression of curl…

Search for autoantibodies to the human bradykinin B2 receptor.