Erratum to: "The agonist SR 146131 and the antagonist SR 27897 occupy different sites on the human CCK(1) receptor"
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Journal Article↗
Biomedical subjects
P Ferrara
Publications and source records attributed to P Ferrara.
Folding simulations of a three-stranded antiparallel beta -sheet peptide.
Protein folding is a grand challenge of the postgenomic era. In this paper, 58 folding events sampled during 47 molecular dynamics trajectories for a total simulation time of more than 4 micros provide an atomic detail picture of the folding of a 20-residue synthetic peptide with a stable three-stranded antiparallel beta-sheet fold. The simulations successfully reproduce the NMR solution conformation, irrespective of the starting structure. The sampling of the conformational space is sufficient to determine the free energy surface and localize the minima and transition states. The statistically predominant folding pathway involves the formation of contacts between strands 2 and 3, starting with the side chains close to the turn, followed by association of the N-terminal strand onto the preformed 2-3 beta-hairpin. The folding mechanism presented here, formation of a beta-hairpin followed by consolidation, is in agreement with a computational study of the free energy surface of another synthetic three-stranded antiparallel beta-sheet by Bursulaya and Brooks [(1999) J. Am. Chem. Soc. 121, 9947-9951]. Hence, it might hold in general for antiparallel beta-sheets with short turns.
Models, Molecular↗
Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB(2) receptor.
We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2. 1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)- pyrazo le-3-carboxamide) on the human cannabinoid CB(2) receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser(161) and Ser(165), were singly mutated to the cognate cannabinoid CB(1) receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1R,3R,4R)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 (R)-(+)[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB(2) receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB(2) receptor-specific residue, Cys(175) resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.
Amino Acid Sequence↗
The agonist SR 146131 and the antagonist SR 27897 occupy different sites on the human CCK(1) receptor.
1-[2-(4-(2-Chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid (SR 27897) is an effective CCK(1) receptor antagonist, while the structurally related molecule 2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid (SR 146131) is a highly potent and specific agonist for the same receptor. To discover how the two molecules interact with the human cholecystokinin (CCK) CCK(1) receptor, we have carried out binding and activity studies with 33-point mutated receptors. Only six mutants showed altered [3H]SR 27897 binding properties, Lys(115), Lys(187), Phe(198), Trp(209), Leu(214) and Asn(333). In contrast, numerous mutations throughout the receptor either reduced SR 146131 agonist potency, Phe(97), Gly(122), Phe(198), Trp(209), Ile(229), Asn(333), Arg(336) and Leu(356) or increased it, Tyr(48), Cys(94), Asn(98), Leu(217) and Ser(359). Only mutations of Phe(198), Trp(209) and Asn(333) affected both SR 27897 and SR 146131 binding or activity. The collated information was used to construct molecular models of SR 27897 and SR 146131 bound to the human CCK(1) receptor. The clear difference in the binding sites of SR 27897 and SR 146131 offers a molecular explanation for their contrasting pharmacological characteristics.
Amino Acid Sequence↗
Computer simulations of protein folding by targeted molecular dynamics.
We have performed 128 folding and 45 unfolding molecular dynamics runs of chymotrypsin inhibitor 2 (CI2) with an implicit solvation model for a total simulation time of 0.4 microseconds. Folding requires that the three-dimensional structure of the native state is known. It was simulated at 300 K by supplementing the force field with a harmonic restraint which acts on the root-mean-square deviation and allows to decrease the distance to the target conformation. High temperature and/or the harmonic restraint were used to induce unfolding. Of the 62 folding simulations started from random conformations, 31 reached the native structure, while the success rate was 83% for the 66 trajectories which began from conformations unfolded by high-temperature dynamics. A funnel-like energy landscape is observed for unfolding at 475 K, while the unfolding runs at 300 K and 375 K as well as most of the folding trajectories have an almost flat energy landscape for conformations with less than about 50% of native contacts formed. The sequence of events, i.e., secondary and tertiary structure formation, is similar in all folding and unfolding simulations, despite the diversity of the pathways. Previous unfolding simulations of CI2 performed with different force fields showed a similar sequence of events. These results suggest that the topology of the native state plays an important role in the folding process.
Computer Simulation↗
Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14.
Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.
Amino Acid Sequence↗
Cutting edge: human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production.
The mechanism(s) controlling activation of naive B cells, their proliferation, Ag receptor affinity maturation, isotype switching, and their fate as memory or plasma cells is not fully elucidated. Here we show that between 24 and 60% of CD19+ cells in PBMC bind soluble CD14 (sCD14). Tonsillar B cells also bind sCD14, but preferentially the CD38-ve/low cells. Interaction of sCD14 with B cells resulted in higher levels of IgG1 and marked inhibition of IgE production by activated tonsillar B cells and Ag-stimulated PBMC. We found that sCD14 interfered with CD40 signaling in B cells, inhibited IL-6 production by activated B cells, and increased the kinetics and magnitude of CD40 ligand expression on T cells. Together with the previously reported effects on T cells, these findings define sCD14 as a novel soluble regulatory factor capable of modulating cellular and humoral immune responses by interacting directly with T and B cells.
B-Lymphocyte Subsets↗
Essential role of extracellular charged residues of the human CCK(1) receptor for interactions with SR 146131, SR 27897 and CCK-8S.
We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor.
Amino Acid Sequence↗
High-level synthesis of human prolactin in Chinese-Hamster ovary cells.
Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA. Without methotrexate (MTX) amplification, p658-hPRL-transfected stable cell lines, secreting up to approximately 10 microg of hPRL/10(6) cells per day, could be rapidly obtained; production by pEDdc-hPRL-transfected cells was about 10-fold lower. However, a three-step MTX amplification of the latter led to clones secreting up to approximately 30 microg of hPRL/10(6) cells per day. A pilot production using a hollow-fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 microg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained approximately 10% glycosylated PRL. Chromatographically purified non-glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay). This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.
Animals↗
Urinary excretion of glycosaminoglycans in patients with isolated nocturnal enuresis or combined with diurnal incontinence.
OBJECTIVE: To determine variations in the amount of glycosaminoglycans (GAGs) excreted by patients with nocturnal enuresis and/or diurnal incontinence. PATIENTS, SUBJECTS AND METHODS: The study included 27 patients (aged 5-15 years) with nocturnal enuresis and/or diurnal incontinence, and 27 healthy age-matched children. Their urinary GAG excretion was assessed over 24 h using the sodium tetraborate-carbazole method. RESULTS: Patients with nocturnal enuresis and/or diurnal incontinence had higher mean values of urinary GAG excretion than age-matched controls. There were significant differences in GAG excretion between those with nocturnal enuresis and diurnal incontinence and those with nocturnal enuresis alone. CONCLUSIONS: GAG excretion in patients with nocturnal enuresis and/or diurnal incontinence was significantly higher than in normal children, suggesting that measuring urinary GAGs may be useful in evaluating physiopathological conditions of the bladder wall, and hence in monitoring potential damage in the bladder mucosa.
Adolescent↗
Two different desmopressin (DDAVP) regimens in enuretic monozygotic twins with allergic diathesis.
Primary monosymptomatic nocturnal enuresis is a common complaint in paediatrics. Treatment with desmopressin nasal spray is one of the most often used pharmacological approaches, but concerning therapy interruption, to date no guidelines have yet been established. The aim of this study was to report the use of desmopressin in two monozygotic twin sisters to establish if tolerability and efficacy of treatment can be affected by different modalities of therapy interruption.
Administration, Intranasal↗
Potential role of peripheral benzodiazepine receptors in inflammatory responses.
This study investigates the anti-inflammatory effects of 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide and 7-chloro-5-(4-chlorophenyl)-1, 3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2-one in paw oedema induced by carrageenan in mice. Pretreatment (24 h) with both ligands inhibited oedema formation in at different doses (0.00001-10 mg/kg, i.p.) with range of inhibition of 25% to 70%, in animals with or without adrenal glands. These results demonstrate, for the first time, an in vivo anti-inflammatory property of peripheral benzodiazepine receptor ligands.
Adrenalectomy↗
Human 76p: A new member of the gamma-tubulin-associated protein family.
The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.
Amino Acid Sequence↗
Contrasting roles of leu(356) in the human CCK(1) receptor for antagonist SR 27897 and agonist SR 146131 binding.
A new highly specific, potent non-peptide agonist for the cholecystokinin subtype 1 receptor (CCK(1)), SR 146131 (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid) was recently described [Bignon, E., Bachy, A., Boigegrain, R., Brodin, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Molimard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O.Thurneyssen, O., Soubrié, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, orally active and selective non-peptide cholecystokinin subtype I receptor agonist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742-751]. From binding and activity assays with chimeric constructs of human CCK(1) and the cholecystokinin subtype 2 receptor (CCK(2)) and receptors carrying point mutations, we show that Leu(356), situated in transmembrane domain seven in the CCK(1) receptor, is a putative contact point for SR 146131. In contrast, Leu(356) is probably not in contact with the CCK(1) receptor specific antagonist SR 27897 (1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl]acetic acid), a compound structurally related to SR 146131, since its replacement by alanine, histidine or asparagine gave receptors having wild-type CCK(1) receptor SR 27897 binding affinity. Previous mutational analysis of His(381), the cognate position in the rat CCK(2) receptor, had implicated it as being involved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK(2) receptor. Moreover, binding and activity assays with the natural CCK receptor agonist, CCK-8S, show that CCK-8S is more susceptible to the mutations in that position in the CCK(1) receptor than in the CCK(2) receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype.
Animals↗
IL-13 induces serine phosphorylation of cPLA2 in mouse peritoneal macrophages leading to arachidonic acid and PGE2 production and blocks the zymosan-induced serine phosphorylation of cPLA2 and eicosanoid production.
In a recent investigation, we demonstrated that long-term treatment of macrophages with IL-13 enhances cPLA2 expression and modulates zymosan-stimulated AA mobilization. In the present study, we examine the ability of IL-13 to modify the cPLA2 activity and the AA mobilization of macrophages after a short-period of treatment. We demonstrate that in resting macrophages, IL-13 induces, through a MAP kinase-dependent process, (1) an increase of free AA release within 15 min, followed by increased PGE2 production and (2) a time-dependent serine phosphorylation of cPLA2. Conversely, in macrophages stimulated by zymosan, IL-13 added 30 min before zymosan inhibited the AA release and the serine phosphorylation of cPLA2 induced by the phagocytic agonist. In conclusion, these findings show for the first time that a Th2-type cytokine can upregulate cPLA2 activity and downregulate zymosan-induced AA metabolism. Thus, establishment of the connection between these two events may help to understand the complex regulatory role of IL-13 on the macrophage AA metabolism.
Animals↗
Dual intracellular signaling pathways mediated by the human cannabinoid CB1 receptor.
It has long been established that the cannabinoid CB1 receptor transduces signals through a pertussis toxin-sensitive Gi/Go inhibitory pathway. Although there have been reports that the cannabinoid CB1 receptor can also mediate an increase in cyclic AMP levels, in most cases the presence of an adenylyl cyclase costimulant or the use of very high amounts of agonist was necessary. Here, we present evidence for dual coupling of the cannabinoid CB receptor to the classical pathway and to a pertussis toxin-insensitive adenylyl cyclase stimulatory pathway initiated with low quantities of agonist in the absence of any costimulant. Treatment of Chinese hamster ovary (CHO) cells expressing the cannabinoid CB1 receptor with the cannabinoid CP 55,940, {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hyd roxypropyl) cyclohexan-1-ol} resulted in cyclic AMP accumulation in a dose-response manner, an accumulation blocked by the cannabinoid CB1 receptor-specific antagonist SR 141716A, {N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride}. In CHO cells coexpressing the cannabinoid CB1 receptor and a cyclic AMP response element (CRE)-luciferase reporter gene system, CP 55,940 induced luciferase expression by a pathway blocked by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89). Under the same conditions the peripheral cannabinoid CB2 receptor proved to be incapable of inducing cAMP accumulation or luciferase activity. This incapacity allowed us to study the luciferase activation mediated by CB /CB2 chimeric constructs, from which we determined that the first and second internal loop regions of the cannabinoid CB1 receptor were involved in transducing the pathway leading to luciferase gene expression.
Animals↗
Soluble CD14 acts as a negative regulator of human T cell activation and function.
T cell activation is controlled by the coordination of stimulatory and negative regulatory signals which are not completely defined. In this study we tested for a possible direct effect of CD14 on the regulation of T cell activation and function. We show that soluble CD14 (sCD14) induces inhibition of antigen-mediated peripheral blood mononuclear cells (PBMC) proliferation and anti-CD3-mediated proliferation of CD4+CD8+, CD4+CD8+ and CD4+CD8+ Tcell clones. This effect is not due to cell death, but results from a marked inhibition of IL-2 production. Proliferation of T cell clones due to exogenous IL-2 is not affected by sCD14. We also found that sCD14 inhibits production of another Th1-like cytokine, IFN-gamma and a Th2-like cytokine, IL-4. Importantly, sCD14 induces a progressive accumulation of the inhibitory protein IkappaB-alpha. We show that sCD14 binds to activated T cells. Following cell activation, biotinylated sCD14 stains CD3+ PBMC, as well as human T cell clones with varying intensity. The binding is saturable, can be inhibited by excess of unlabeled sCD14 and, following binding, sCD14 is internalized. Collectively, these findings reveal a previously unrecognized function of sCD14, namely its capacity to negatively regulate T lymphocyte activation and function by interacting directly with activated T cells.
Biological Transport, Active↗
Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.
The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.
Amino Acid Sequence↗