Receptor Recognition Site Research Articles

Receptor Recognition Sites of Cytokines Are Organized as Exchangeable Modules: TRANSFER OF THE LEUKEMIA INHIBITORY FACTOR RECEPTOR-BINDING SITE FROM CILIARY NEUROTROPHIC FACTOR TO INTERLEUKIN-6

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.

Amino acid substitutions in a conserved region in the stalk of the Newcastle disease virus HN glycoprotein spike impair its neuraminidase activity in the globular domain.

The ectodomain of the paramyxovirus haemagglutinin-neuraminidase (HN) glycoprotein spike can be divided into two regions: a membrane-proximal, stalk-like structure and a terminal globular domain. The latter contains all the antibody recognition sites of the protein, as well as its receptor recognition and neuraminidase (NA) active sites. These two activities of the protein can be separated by monoclonal antibody functional inhibition studies and mutations in the globular domain. Herein, we show that mutation of several conserved residues in the stalk of the Newcastle disease virus HN protein markedly decrease its NA activity without a significant effect on receptor recognition. Thus, mutations in the stalk, distant from the NA active site in the globular domain, can also separate attachment and NA. These results add to an increasing body of evidence that the NA activity of this protein is dependent on an intact stalk structure.

Binding of rat alpha 1-inhibitor-3-methylamine to the alpha 2-macroglobulin signaling receptor induces second messengers.

Binding of receptor-recognized forms of tetrameric human alpha 2-macroglobulin (alpha 2M*) to a macrophage signaling receptor induces cAMP synthesis, increases in inositol 1,4,5-triphosphate (IP3) synthesis, and a concomitant rise in cytosolic free calcium ([Ca2+]i). The alpha 2M* signaling receptor is coupled to a pertussis-toxin insensitive G protein. Binding of alpha 2M* also occurs to the low density lipoprotein receptor-related protein/alpha 2M receptor (LRP/alpha 2MR), but this binding does not induce signal transduction. Rat alpha 1-inhibitor-3 (alpha 1I3) is a monomeric member of the alpha-macroglobulin/complement superfamily. Like alpha 2M, it can react with proteinases or methylamine which induces a conformational change causing activated alpha 1I3 to bind to LRP/alpha 2MR. We now report that alpha 1I3-methylamine binds to the macrophage alpha 2M* signaling receptor inducing a rapid rise in the synthesis of IP3 with a subsequent 1.5- to 3-fold rise in [Ca2+]i. alpha 1I3-methylamine binding to macrophages also caused a statistically significant elevation in cAMP. Native alpha 1I3, like alpha 2M, was unable to induce signal transduction. alpha 1I3 forms a complex with alpha 1-microglobulin, which has a distinct conformation from alpha 1I3 and is recognized by LRP/alpha 2MR. This complex also induces an increase in [Ca2+]i comparable to the effect of alpha 1I3-methylamine on macrophages. It is concluded that activation of alpha 1I3 by methylamine or binding of alpha 1-microglobulin causes similar conformational changes in the inhibitor, exposing the receptor recognition site for the alpha 2M* signaling receptor, as well as for LRP/alpha 2MR.

Selective labelling of 5-HT 7 receptor recognition sites in rat brain using [ 3H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT 7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (±)-pindolol (10 μM)-insensitive [ 3H]5-CT ([ 3H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 μM) displayed a pharmacological profile similar to the recombinant 5-HT 7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (±)-pindolol (10 μM)-insensitive [ 3H]5-CT recognition sites also resembled, pharmacologically, the 5-HT 7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [ 3H]5-CT binding to residual, possibly, 5-HT 1A sites. Competition for this [ 3H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT 7 receptor. Saturation studies also indicated that (±)-pindolol (10 μM)/WAY 100635 (100 nM)-insensitive [ 3H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity ( B max=33.2±0.7 fmol mg −1 protein, p K d=8.78±0.05, mean±S.E.M., n=3). The development of this 5-HT 7 receptor binding assay will aid investigation of the rat native 5-HT 7 receptor.

Taste preference in nonhuman primates to compounds sweet in man.

Primates have stimulated more interest than any other group as humans are ranked in this same mammalian order. Gustatory responses of human and nonhuman primates have already been compared for compounds such as monosaccharides, oligosaccharides, polyols, amino acids, dipeptides, proteins, dihydrochalcones, sulfamates, saccharin, acesulfame, diterpenes or urea derivatives, all known to be sweet in man. But no rational comparison in primates has been attempted. Using a structure-activity relationship study in primates, it is now possible to classify the primate sweetness receptors into four types according to the behavioral responses observed from various selected compounds sweet in humans. The four types are represented by (1) the Callitrichidae and (2) the Cebidae, both from the infraorder Platyrrhini (New World monkeys), (3) the Lemuridae and Lorisidae, from the suborder Prosimii (prosimians), and (4) the Cercopithecidae (Old World monkeys), Hylobatidae (lesser apes), Pongidae (great apes), and Hominidae (humans) from the infraorder Catarrhini (Old World simians). By a comparative study of the putative receptor recognition sites postulated for each type of receptor, it is inferred that the Callitrichidae (marmosets and tamarins) have retained the most primitive sweetness receptor among primates. As we believe that the evolution of the sweetness receptor is a key factor involved in the raising of nonhuman primates from a 'primitive grade' towards a more 'advanced' or 'simian grade,' the possible phylogenetic implications of these findings will be discussed.

Comparison of the effects of hydrogen peroxide, 4-hydroxy-2-nonenal and β-amyloid (25–35) upon calcium signalling

The neurotoxic β-amyloid (Aβ) peptide fragment Aβ(25–35) has been suggested to exert its deleterious effects on cells via production of hydrogen peroxide. In human platelets and in the presence of DMSO to prevent production of hydroxyl radicals from hydrogen peroxide, both Aβ(25–35) and hydrogen peroxide were found to increase intracellular calcium levels. Hydrogen peroxide in addition reduced the calcium response to thrombin, whereas this was not seen with Aβ(25–35). A similar pattern of effects to those seen with hydrogen peroxide were also seen with the neurotoxic aldehyde lipid peroxidation product 4-hydroxy-2-nonenal (HNE). The initial increase in calcium produced by hydrogen peroxide was not affected by EGTA, but was partially prevented by dithiothreitol. The calcium response to Aβ(25-35) which was also seen with Aβ(1- 40) and Aβ(1-42) but not with the inactive peptide Aβ(40-1) consisted of an EGTA-sensitive and an EGTA-resistant component, of which the latter was also sensitive to DTT. Hydrogen peroxide increased basal phosphoinositide breakdown in rat brain miniprisms and decreased the responses to nor- adrenaline, carbachol and veratrine. The specific binding of 3 H inositol -1,4,5 -trisphosphate ( 3 H Ins(1,4,5)P3 ) to its receptor recognition site in human platelet membranes was increased by Aβ(25-35) but remained unchanged following hydrogen peroxide treatment. It is concluded that under conditions where production of hydroxyl radicals from hydrogen peroxide is blocked, hydrogen peroxide and Aβ(25-35) produce their effects on calcium by affecting the mobilisation of intracellular calcium. The qualitative differences in the calcium responses of these two agents can be explained (a) by an additional effect of Aβ(25-35) upon calcium entry and (b) by differences in their effects upon the Ins(1,4,5)P3 receptor. ©1998 Elsevier Science Ltd. All rights reserved.

Differential gene induction by glucocorticoid and progesterone receptors

The question of how glucocorticoid, progesterone, androgen, and mineralocorticoid receptors can generate distinct patterns of gene expression despite similar, if not identical, DNA sequence recognition properties is a central question in steroid biology. This study addresses the hypothesis that glucocorticoid and progesterone receptors can differentially utilize the promoter context created by a series of receptor recognition sites. This hypothesis predicts that for different receptors an individual (often suboptimal) binding site contributes to the overall response element activity to a different degree. Therefore, mutations to an individual binding site of a multipartite hormone response element may differentially affect the response to different steroids. This study tests this hypothesis by introducing mutations into a receptor recognition site that is part of a hormone responsive promoter derived from the mouse mammary tumor virus. We find that weakening one site of a multipartite element differentially affects glucocorticoid and progestin signaling both in fibroblasts and in mammary carcinoma cells. A similar test was done in a simplified promoter context comprised only of a TATA box and a pair of receptor recognition sites. Mutations introduced into one of these sites impaired glucocorticoid response more than the progestin response. However, high receptor expression can partially overcome the effect of some target site mutations. Thus both receptor expression levels and the inherent ability to utilize the context created by a multipartite response element contribute to quantitative differences in the response to receptors that share DNA sequence recognition properties.

Receptor-mediated recognition and uptake of iron from human transferrin by Staphylococcus aureus and Staphylococcus epidermidis.

Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea-polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin.

Probing the receptor recognition site of the FimH adhesin by fimbriae-displayed FimH-FocH hybrids.

Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces. This binding is conferred by the minor fimbrial component FimH. The binding domain of the FimH adhesin has been studied by constructing hybrids of FimH and a homologous protein, FocH, originating from F1C fimbriae. F1C fimbriae do not bind to D-mannosides or confer agglutination of any known types of erythrocytes or yeast. It was previously shown that the FocH protein can be readily substituted by the FimH adhesin, resulting in hybrid fimbriae with the same binding characteristics as type 1 fimbriae. The receptor binding of fimbriae-presented chimeric FimH-FocH hybrids was studied. FimH-FocH fusions encompassing 72% of the N-terminus of FimH fused to the complementary sector of FocH conferred agglutination of erythrocytes and yeast cells at a comparable level to FimH. Surprisingly, it was also found that similar fusions containing between 56 and 66% FimH still conferred binding to yeast cells, D-mannose-BSA and D-mannose-beads but did not give rise to agglutination. The receptor binding capacity of fusions containing 50% or less of the FimH N-terminal region was virtually abolished. The results point to the presence of a D-mannose-receptor-binding core domain in FimH, the affinity of which is modulated by other sectors of the protein to enable binding to extended mannose-containing targets.

Characterization of GABA Receptors

AbstractDescribed in this unit are ligand binding assays for GABAA, GABAB, and GABAC receptor recognition sites in brain tissue. These assays may also be used to determine the affinity of an unlabeled compound for the GABA binding sites. Not included in this unit are ligand binding assays for other components of the GABAA, receptor complex, such as the benzodiazepine or ion channel binding sites.

Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae.

Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.

Cellular localization of AT1 receptor mRNA and protein in normal placenta and its reduced expression in intrauterine growth restriction. Angiotensin II stimulates the release of vasorelaxants.

Angiotensin II (ANG II) is a potent vasoconstrictor and growth promoter. Quantitative receptor autoradiography using the nonselective radioligand [125I]ANG II and subtype-selective competing compounds demonstrated the presence of both ANG II receptor (AT)1 and AT2 receptor recognition sites. In addition, a relatively small population of apparently non-AT1/non-AT2 sites was identified that may represent a novel high affinity ANG II recognition site in human placenta. Using placental membrane preparations, the AT2 receptor antagonist PD123177 failed to compete for [3H]ANG II binding at relevant concentrations, whereas the AT1 receptor antagonist losartan competed in a monophasic manner for all the specific binding, suggesting that the non-AT1/non-AT2 recognition site identified using autoradiography may be a cytosolic binding site. AT1 receptor binding was significantly reduced (P < 0. 02) in intraeuterine growth restriction (IUGR) pregnancies. Western blot analysis confirmed this showing a reduction in AT1 receptor protein. In situ hybridization and immunocytochemistry revealed that AT1 receptor mRNA and protein were localized throughout pregnancy in the cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast, as well as in or around the blood vessels of placental villi. The intensity of the hybridization signal for AT1 receptor mRNA over the syncytium was reduced in IUGR. ANG II evoked a rapid and concentration-dependent release of NO in first trimester cytotrophoblast-like cells that was abolished by the inclusion of the competitive NOS inhibitor NG-monomethyl-L-arginine. Neither losartan nor PD123177 alone significantly inhibited ANG II-evoked NO release, and when cells were stimulated with ANG II in the presence of losartan (10 microM) and PD123177 (10 microM) in combination, NO release was significantly inhibited (P < 0.05). These observations also suggest, for the first time, the existence of a cross-talk between AT1 or AT2 receptors in trophoblast and that the reduction in placental AT1 receptors in IUGR may, in part, account for poor placental function in this disorder.

Effects of NMDA receptor glycine recognition site antagonism on cerebral metabolic rate for glucose and cerebral blood flow in the conscious rat

Glycine is a requisite cofactor for glutamatergic activation of the N-methyl- d-aspartate (NMDA) receptor. Antagonism of glutamate at the NMDA receptor has been shown to cause substantial changes in regional cerebral metabolic rate for glucose utilization (CMRglu) and blood flow (CBF). This study examined CMRglu and CBF changes caused by antagonism of glycine at the NMDA receptor recognition site. Rats were anesthetized with halothane and vascular access was obtained. The animals were then awakened. One hour later, either vehicle (control) or ACEA 1021 (5 mg/kg followed by 3.5 mg·kg −1·h −1 or 10 mg/kg followed by 7 mg·kg −1·h −1) was infused intravenously. CMRglu and CBF were then determined. Autoradiographic analysis of 25 regions revealed effects of ACEA 1021 on CMRglu in the frontal, sensory, parietal and auditory cortices and the anteroventral and subthalamic nuclei. These changes deviated less than 15% from control. Effects on CBF were also small. The CMRglu and CBF effects of ACEA 1021 are substantially less than those previously observed for either competitive or non-competitive glutamate NMDA antagonists. We conclude that inhibition of the NMDA glycine recognition site has little or no effect on CMRglu or CBF at the doses examined. This is consistent with the absence of psychotomimetic effects observed for this class of drugs.

The connection between absence-like seizures and hypothermia induced by penicillin: possible implication on other animal models of petit mal epilepsy

In this study we investigated the relationship between penicillin-induced hypothermia and petit mal epilepsy induced by this proconvulsant antibiotic. In order to find a possible dose-dependent relationship, we used two doses: 1500.000 and 1000.000 U/kg b.wt., both known as being sufficient to induce absence-like attacks with subsequent spike and wave discharges (SWD) in electrocorticogram (ECoG). Because of experimental data suggesting penicillin binding to benzodiazepine receptor recognition site, we also studied penicillin-induced changes in body temperature after diazepam pretreatment. Results of this study clearly show that penicillin in doses known to induce petit mal-like epilepsy concomitantly induces statistically significant dose-dependent decrease in body temperature. Pretreatment with diazepam completely prevents both penicillin-induced hypothermia and SWDs. On the other hand, both the diazepam and mixed diazepam+penicillin treatments did not significantly alter body temperature. These results suggest, however, that at least some of the penicillin effects described could be assigned to its binding to the benzodiazepine receptor recognition site at GABA A ionophore. This may have an important clinical implication because the inhibitory action of penicillin at the benzodiazepine receptor recognition site could account for the mechanism of penicillin-induced unspecific encephalopathies in humans. The relationship between petit mal epilepsy and hypothermia sheds new light on the action mechanisms of penicillin-induced absence seizures.

Receptor recognition sites reside in both lobes of human serum transferrin.

The binding of iron by transferrin leads to a significant conformational change in each lobe of the protein. Numerous studies have shown that the transferrin receptor discriminates between iron-saturated and iron-free transferrin and that it modulates the release of iron. Given these observations, it seems likely that there is contact between each lobe of transferrin and the receptor. This is the case with chicken transferrin, in which it has been demonstrated unambiguously that both lobes are required for binding and iron donation to occur [Brown-Mason and Woodworth (1984) J. Biol. Chem. 259, 1866-1873]. Further support to this contention is added by the ability of both N- and C-domain-specific monoclonal antibodies to block the binding of a solution containing both lobes [Mason, Brown and Church (1987) J. Biol. Chem. 262, 9011-9015]. In the present study a similar conclusion is reached for the binding of human serum transferrin to the transferrin receptor. With the use of recombinant N- and C-lobes of human transferrin produced in a mammalian expression system, we show that both lobes are required to achieve full binding. (Production of recombinant C-lobe in the baby hamster kidney cell system is reported here for the first time.) Each lobe is able to donate iron to transferrin receptors on HeLa S3 cells in the presence of the contralateral lobe. The results are not identical with the chicken system, because the C-lobe alone shows a limited ability to bind to receptors and to donate iron. Further complications arise from the relatively weak re-association between the two lobes of human transferrin compared with the re-association of the ovotransferrin lobes. However, domain-specific monoclonal antibodies to either lobe block the binding of N- and C-lobe mixtures in the human system, thus substantiating the need for both.

An RGD to LDV Motif Conversion within the Disintegrin Kistrin Generates an Integrin Antagonist That Retains Potency but Exhibits Altered Receptor Specificity: EVIDENCE FOR A FUNCTIONAL EQUIVALENCE OF ACIDIC INTEGRIN-BINDING MOTIFS

Integrin ligands almost invariably employ a variant of either the RGD or LDV motif as a key element of their receptor recognition site. These short acidic peptide sequences collaborate with specific nonhomologous flanking residues and spatially separate "synergy" sequences to determine receptor binding specificity. Although the consensus sequences for RGD and LDV motifs are quite different, their common use suggests that they might share a critical role in receptor-ligand engagement. To date, the effects of interconversion of the two motifs within a natural protein framework have not been tested; however, in this study, we have converted the natural RGD site found in the snake venom disintegrin kistrin into an LDV motif and examined the effects of the change on the specificity of integrin recognition and on disintegrin potency. While an assessment of receptor binding using cell adhesion and purified integrin solid-phase assays demonstrated recognition of recombinant RGD kistrin by alphaVbeta3 and alpha5beta1, a series of LDV kistrin chimeras did not bind to these integrins, but instead were recognized specifically by alpha4beta1. The minimal change to elicit this distinct switch in receptor specificity was found to involve alteration of only three residues within kistrin. Alanine scanning mutagenesis was used to provide further information on the functional contribution of the three residues. More important, the LDV kistrin chimeras also retained much of the characteristic potency of RGD kistrin, indicating that the kistrin scaffold is optimized for presentation of both RGD and LDV sequences. These findings provide evidence for similarities in motif pharmacophore and reinforce the hypothesis that RGD and LDV sites have an equivalent functional role in receptor binding. They also demonstrate the potential for other disintegrin-containing proteins, perhaps from the ADAM family, to employ LDV sequences for integrin binding.

Mutual interactions of the presynaptic histamine H 3 and prostaglandin EP 3 receptors on the noradrenergic terminals in the mouse brain

We studied whether interactions between the presynaptic histamine H 3 and prostaglandin EP 3 receptors on the noradrenergic neurons of the mouse brain cortex occur. Cerebral cortex slices from the mouse (and, in few experiments, from the rat) were preincubated with [ 3H]noradrenaline and then superfused with a physiological salt solution. Tritium overflow was evoked electrically, either at 0.3 or 3 Hz (2 min) (standard stimulation protocol) or at 100 Hz (eight pulses) (stimulation protocol under which almost no activation of the presynaptic α 2-adrenoceptors by endogenous noradrenaline occurs). In another set of experiments, Ca 2+ ions were introduced into Ca 2+-free K +-rich medium containing tetrodotoxin to evoke tritium overflow. The electrically-evoked tritium overflow (0.3 Hz) was inhibited by histamine or the H 3 receptor agonist imetit, acting via H 3 receptors, and by prostaglandin E 2 or the EP 3 receptor agonist sulprostone, acting via EP 3 receptors. When histamine or imetit was given first (at concentrations causing the maximum effect at H 3 receptors), the effect of prostaglandin E 2 on the evoked tritium overflow was attenuated by 5–10%. When prostaglandin E 2 or sulprostone was given first (at concentrations causing the maximum effect at EP 3 receptors), the effect of histamine or imetit on the evoked overflow was attenuated by almost 50%. The previous administration of prostaglandin E 2 also blunted the effect of histamine on the evoked tritium overflow evoked at 3 Hz; the degree of attenuation was identical when the current strength was 25 mA or was increased to 100 or 200 mA in order to partially compensate for the inhibitory effect of prostaglandin E 2 on the evoked overflow. In addition, prostaglandin E 2 attenuated the effect of histamine when tritium overflow was evoked (i) by 100 Hz, eight pulses or (ii) by Ca 2+ ions or (iii) when rat (instead of mouse) brain cortex slices were used. An interaction of prostaglandin E 2 or sulprostone with the H 3 receptor recognition site could be excluded since both prostanoids did not affect the specific binding of the H 3 agonist radioligand [ 3H]N α -methylhistamine to rat brain cortex membranes. In conclusion, mutual interactions occur between the presynaptic H 3 and EP 3 receptors involved in the inhibition of noradrenaline release in the mouse brain cortex. Pre-activation of the H 3 receptor slightly attenuates the EP 3 receptor-mediated effect whereas pre-activation of the EP 3 receptor more markedly attenuates the H 3 receptor-mediated effect. The interactions may occur between the receptors themselves or at a step behind the receptors (e.g., at the level of G proteins). The physiological significance of these interactions may be to limit the total extent of inhibition of noradrenaline release in a scenario under which both receptors are activated simultaneously.

The Contact Zones in Human α2‐Macroglobulin

A functional domain termed the contact zone, which is the region of a subunit interacting with another non-covalently bound subunit, is suggested to play a decisive role in the trapping mechanism of human alpha2-macroglobulin. Tetrameric alpha2-macroglobulin can be dissociated into stable dimers with intact thiol esters by sodium thiocyanate, whereby the contact zones are disrupted. The dissociation leads to significant conformational changes, as studied by ultraviolet-difference spectroscopy, CD, fluorescence and affinity partitioning. The conformation of the dimers is similar to that of MeNH2-treated alpha2-macroglobulin, in which the thiol esters are cleaved, a conformational state with a closed trap occurs, and receptor-recognition sites are exposed. The receptor-binding domain is at least partly exposed in the dimer, as judged by binding of specific mAbs. The bait region in the dimers can be cleaved by proteases, and activation of the thiol esters ensues without binding of the protease. When the dimers were treated with MeNH2, no conformational changes could be detected by ultraviolet-difference spectroscopy or CD. The conformational changes occurring on dissociation into dimers are suggested to be related to trap closure and receptor-recognition-site exposure without cleavage of the thiol esters. The model presented here suggests that two separate conformational changes occur in alpha2-macroglobulin upon activation. The first involves changes at the contact zones as a result of the thiol-ester cleavage, and the second causes exposure of the receptor-recognition sites and closure of the trap.

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes. 1983.

Saturable binding of (±)‐[3H]‐baclofen and [3H]‐γ‐aminobutyric acid ([3H]‐GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. The binding of [3H]‐baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+‐dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. The saturable portion of bound [3H]‐baclofen formed 20.3 ± 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 μm), (–)‐baclofen (0.04 μm) and to a much lesser extent by (+)‐baclofen (33 μm). Isoguvacine, piperidine‐4‐sulphonic acid and bicuculline methobromide were inactive (up to 100 μm) and muscimol was only weakly active (IC50 = 12.3 μm). Saturable binding of [3H]‐GABA increased on adding CaCl2 or MgSO4 (up to 2.5 Mm and 5.0 Mm respectively) to the Tris‐HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline‐sensitive binding of GABA (GABAA site binding) and could be completely displaced by (–)‐baclofen (IC50 = 0.13 μm). Increasing the Ca2+ concentration (0 to 2.5 Mm) increased the binding capacity of the membranes without changing their affinity for the ligand. The binding of [3H]‐GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non‐ionic detergent Triton‐X‐100 (0.05% v/v) reduced the binding capacity by 50%. The pharmacological profile of displacers of [3H]‐GABA from GABAB sites correlated well with that for [3H]‐baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. It is concluded that [3H]‐baclofen or [3H]‐GABA are both ligands for the same bicuculline‐insensitive, divalent cation‐dependent binding sites in the rat brain.

Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.

Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.