The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding, activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a(65-74), Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gin (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-x(L)

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the death domain and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue BcI-x(L). These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus.

Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-RI) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis, In previous studies, me have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis, We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Pour of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors, (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-RI and more so for the TRAIL decoy receptors TRAIL-R3 and -R4, Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-RI and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-RI and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

V4 region of small subunit rDNA indicates polyphyly of the Fellodistomidae (Digenea) which is supported by morphology and life-cycle data

There is no morphological synapomorphy for the disparate digeneans, the Fellodistomidae Nicoll, 1909. Although all known life-cycles of the group include bivalves as first intermediate hosts, there is no convincing morphological synapomorphy that can be used to unite the group. Sequences from the V4 region of small subunit (18S) rRNA genes were used to infer phylogenetic relationships among 13 species of Fellodistomidae from four subfamilies and eight species from seven other digenean families: Bivesiculidae; Brachylaimidae; Bucephalidae; Gorgoderidae; Gymnophallidae; Opecoelidae; and Zoogonidae. Outgroup comparison was made initially with an aspidogastrean. Various species from the other digenean families were used as outgroups in subsequent analyses. Three methods of analysis indicated polyphyly of the Fellodistomidae and at least two independent radiations of the subfamilies, such that they were more closely associated with other digeneans than to each other. The Tandanicolinae was monophyletic (100% bootstrap support) and was weakly associated with the Gymnophallidae (< 50-55% bootstrap support). Monophyly of the Baccigerinae was supported with 78-87% bootstrap support, and monophyly of the Zoogonidae + Baccigerinae received 77-86% support. The remaining fellodistomid species, Fellodistomum fellis, F. agnotum and Coomera brayi (Fellodistominae) plus Proctoeces maculatus and Complexobursa sp. (Proctoecinae), formed a separate clade with 74-92% bootstrap support. On the basis of molecular, morphological and life-cycle evidence, the subfamilies Baccigerinae and Tandanicolinae are removed from the Fellodistomidae and promoted to familial status. The Baccigerinae is promoted under the senior synonym Faustulidae Poche, 1926, and the Echinobrevicecinae Dronen, Blend & McEachran, 1994 is synonymised with the Faustulidae. Consequently, species that were formerly in the Fellodistomidae are now distributed in three families: Fellodistomidae; Faustulidae (syn. Baccigerinae Yamaguti, 1954); and Tandanicolidae Johnston, 1927. We infer that the use of bivalves as intermediate hosts by this broad range of families indicates multiple host-switching events within the radiation of the Digenea.

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using H-1-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.

Association of a glucocorticoid receptor gene marker with human essential hypertension.

Recently, a bi-allelic polymorphism in the glucocorticoid receptor gene (GRL) has been shown to be associated with individuals at high risk of developing hypertension and accumulation of abdominal visceral fat, a known risk factor for cardiovascular disease. The evaluate the role of GRL in essential hypertension and obesity, case-control studies were conducted using 88 hypertensive, 123 normotensive, 150 lean and 94 obese subjects. Genotypes for a highly polymorphic microsatellite marker (D5S207) located within 200 kb of the glucocorticoid receptor gene, were determined by PCR. Allele frequencies between hypertensive and normotensive groups were significantly (P = 0.0005) different whereas no significant differences were observed between lean and obese populations. In conclusion, the results suggest that the glucocorticoid receptor gene or perhaps another gene located in close proximity and in linkage disequilibrium with D5S207, is involved in hypertension development

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

Identification of residues involved in binding of IL5 to beta(com) using beta(IL3) and beta(com) chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, beta(com) and beta(IL3). beta(com) is used by the IL3, ILS and GM-CSF receptors whereas Pns is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta(IL3) that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the beta(IL3) B'-C' loop were replaced with beta(com) sequence a form of beta(IL3) was produced that was able to respond to IL5. This region is also responsible for IL3 binding to beta(IL3) in the absence of alpha chain. It is therefore an important structural motif of beta(com) and beta(IL3) responsible for both ligand interaction and specificity. (C) 1999 Federation of European Biochemical Societies.

GABA receptors inhibited by benzodiazepines mediate fast inhibitory transmission in the central amygdala

The amygdala is intimately involved in emotional behavior, and its role in the generation of anxiety and conditioned fear is well known. Benzodiazepines, which are commonly used for the relief of anxiety, are thought to act by enhancing the action of the inhibitory transmitter GABA. We have examined the properties of GABA-mediated inhibition in the amygdala. Whole-cell recordings were made from neurons in the lateral division of the central amygdala. Application of GABA evoked a current that reversed at the chloride equilibrium potential. Application of the GABA antagonists bicuculline or SR95531 inhibited the GABA-evoked current in a manner consistent with two binding sites. Stimulation of afferents to neurons in the central amygdala evoked an IPSC that was mediated by the release of GABA. The GABA(A) receptor antagonists bicuculline and picrotoxin failed to completely block the IPSC. The bicuculline-resistant IPSC was chloride-selective and was unaffected by GABA(B)-receptor antagonists. Furthermore, this current was insensitive to modulation by general anesthetics or barbiturates. In contrast to their actions at GABA(A) receptors, diazepam and flurazepam inhibited the bicuculline-resistant IPSC in a concentration-dependent manner. These effects were fully antagonized by the benzodiazepine site antagonist Ro15-1788. We conclude that a new type of ionotropic GABA receptor mediates fast inhibitory transmission in the central amygdala. This receptor may be a potential target for the development of new therapeutic strategies for anxiety disorders.

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

Implication of the visual system in the regulation of activity cycles in the absence of solar light: 2-[I-125]iodomelatonin binding sites and melatonin receptor gene expression in the brains of demersal deep-sea gadiform fish

Relative eye size, gross brain morphology and central localization of 2-[I-125]iodomelatonin binding sites and melatonin receptor gene expression were compared in six gadiform fish living at different depths in the north-east Atlantic Ocean: Phycis blennoides (capture depth range 265-1260 m), Nezumia aequalis (445-1512 m), Coryphaenoides rupestris (706-1932 m), Trachyrincus murrayi (1010-1884 m), Coryphaenoides guentheri (1030 m) and Coryphaenoides (Nematonurus) armatus (2172-4787 m). Amongst these, the eye size range was 0.15-0.35 of head length with a value of 0.19 for C.(N.) armatus, the deepest species. Brain morphology reflected behavioural differences with well-developed olfactory regions in P.blennoides, T.murrayi and C. (N.) armatus and evidence of olfactory deficit in N. aequalis, C. rupestris and C. guentheri. All species had a clearly defined optic tectum with 2-[I-125] iodomelatonin binding and melatonin receptor gene expression localized to specific brain regions in a similar pattern to that found in shallow-water fish. Melatonin receptors were found throughout the visual structures of the brains of all species. Despite living beyond the depth of penetration of solar light these fish have retained central features associated with the coupling of cycles of growth, behaviour and reproduction to the diel light-dark cycle. How this functions in the deep sea remains enigmatic.