Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.     

Topology of ligand binding sites on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of both alpha subunits there exist the binding sites for agonists such as the neurotransmitter acetylcholine (ACh) and for competitive antagonists such as d-tubocurarine. Agonists trigger the channel opening upon binding while competitive antagonists compete for the former ones and inhibit its pharmacological action. Identification of all residues involved in recognition and binding of agonist and competitive antagonists is a primary objective in order to understand which structural components are related to the physiological function of the AChR. The picture for the localisation of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are mainly located on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are sequentially identical, the observed high and low affinity for agonists on the receptor is conditioned by the interaction of the alpha subunit with the delta or the gamma chain, respectively. This relationship is opposite for curare-related drugs. This molecular interaction takes place probably at the interface formed by the different subunits. The principal component for the agonist/competitive antagonist binding sites involves several aromatic residues, in addition to the cysteine pair at 192-193, in three loops-forming binding domains (loops A-C). Other residues such as the negatively changed aspartates and glutamates (loop D), Thr or Tyr (loop E), and Trp (loop F) from non-alpha subunits were also found to form the complementary component of the agonist/competitive antagonist binding sites. Neurotoxins such as alpha-, kappa-bungarotoxin and several alpha-conotoxins seem to partially overlap with the agonist/competitive antagonist binding sites at multiple point of contacts. The alpha subunits also carry the binding site for certain acetylcholinesterase inhibitors such as eserine and for the neurotransmitter 5-hydroxytryptamine which activate the receptor without interacting with the classical agonist binding sites. The link between specific subunits by means of the binding of ACh molecules might play a pivotal role in the relative shift among receptor subunits. This conformational change would allow for the opening of the intrinsic receptor cation channel transducting the external chemical signal elicited by the agonist into membrane depolarisation. The ion flux activity can be inhibited by non-competitive inhibitors (NCIs). For this kind of drugs, a population of low-affinity binding sites has been found at the lipid-protein interface of the AChR. In addition, several high-affinity binding sites have been found to be located at different rings on the M2 transmembrane domain, namely luminal binding sites. In this regard, the serine ring is the locus for exogenous NCIs such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222, phencyclidine, and trifluoromethyliodophenyldiazirine. Trifluoromethyliodophenyldiazirine also binds to the valine ring, which is the postulated site for cembranoids. Additionally, the local anaesthetic meproadifen binding site seems to be located at the outer or extracellular ring. Interestingly, the M2 domain is also the locus for endogenous NCIs such as the neuropeptide substance P and the neurotransmitter 5-hydroxytryptamine. In contrast with this fact, experimental evidence supports the hypothesis for the existence of other NCI high-affinity binding sites located not at the channel lumen but at non-luminal binding domains. (ABSTRACT TRUNCATED)     

Mutagenesis analysis of the membrane-proximal ligand binding site of the TGF-beta receptor type III extracellular domain

There are two TGF-beta binding subdomains in the extracellular domain of receptor type III (proximal and distal in relation to the transmembrane domain). Here we present an extension of our analysis of the proximal binding site of receptor type III. Due to the original deletion mutagenesis strategy, our proximal binding site contained 19 amino acids from the N-terminal part of the receptor. By deleting these, we demonstrated that they did not contribute to the binding ability of the proximal binding site. We also produced a soluble, secreted form of the proximal binding site and demonstrated that it was able to bind TGF-beta. Finally, we analyzed the role of the three asparagine residues (580, 591, 595) that are located in the region of the receptor that is necessary for expression of a functional proximal binding site, and found that mutation of these residues individually to alanine did not affect ligand binding.     

GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy

Two genes, mutations in which result in the phenotype of tuberous sclerosis (TSC), have recently been cloned. TSC2 on chromosome 16p13.3 encodes the protein tuberin, which appears to have growth regulating properties. TSC1 on chromosome 9q34 encodes hamartin which, as yet, has no specified cellular functions. Polyclonal antibodies were raised to synthetic peptides representing portions of tuberin and hamartin and used in immunoblots and immunohistochemical studies to localize the proteins in surgically resected neocortical tubers from four TSC patients. On Western blots of autopsy brain specimens, K-562 cell, and NT2 lysates, each antibody labelled a single band at the expected molecular weight. In immunohistochemical protocols on paraffin embedded tissue, antibodies to both tuberin and hamartin prominently labelled atypical and dysmorphic neuroglial cells that are a defining feature of TSC tubers. Some abnormal cells within cortical tuber sections were labelled with both tuberin and hamartin antisera. Our results suggest that tuberin and hamartin are both robustly expressed in similar populations of neuroglial cells of TSC tubers, even in the presence of TSC1 or TSC2 germline mutations. The roles of these gene products in normal and abnormal cortical development, tuber pathogenesis and the generation of seizures remain to be defined.     

High-affinity binding of the neonatal Fc receptor to its IgG ligand requires receptor immobilization

The neonatal Fc receptor (FcRn) binds maternal immunoglobulin G (IgG) during the acquisition of passive immunity by the fetus or newborn. In adult mammals, FcRn also binds IgG and returns it to the bloodstream, thus protecting IgG from a default degradative pathway. Biosensor assays have been used to characterize the interaction of a soluble form of FcRn with IgG. We use the statistical method of cross-validation to show that there are two classes of noninteracting binding sites, and these are sufficient to account for previously observed nonlinear Scatchard plots of FcRn/IgG binding data. We demonstrate that immobilization of FcRn on the biosensor surface reproduces the high-affinity IgG binding observed for membrane-bound FcRn, whereas immobilization of IgG results in lower affinity binding similar to that of the FcRn/IgG interaction in solution. The dependence of FcRn/IgG binding affinity on the coupled molecule provides further evidence in support of the previously hypothesized model that an FcRn dimer forms the high-affinity IgG binding site.     

Disruption of the first extracellular loop of thyrotropin receptor prevents ligand binding

In order to understand the function of the first extracellular loop of the human thyrotropin receptor (hTSHR), each of two peptides of nine amino acids was inserted into the first extracellular loop of hTSHR. hTSHR cDNA was subcloned into the eukaryotic expression vector, pRc/CMV (hTSHR/pRc/CMV). B-hTSHR/pRc/CMV, a mutant hTSHR cDNA which encodes a hydrophilic peptide insert (AGTTRRVAI) and C-hTSHR/pRc/CMV which encodes a hydrophobic peptide insert (ATVLVVPMI) between +486 Ileu and +487 Asp of hTSHR were transfected into Chinese hamster ovary cells to generate the B-1 and C-6 cell lines, respectively. Neither thyrotropin (TSH) nor thyroid stimulating antibody (TSAb) stimulated cAMP production by B-1 or C-6 cells. An 125I-TSH binding assay showed that neither cell line bound TSH. Our data demonstrated that these mutations impaired both TSH binding and cAMP production. This evidence suggests that the first extracellular loop of hTSHR may have a crucial role in the TSH- and TSAb-dependent signal transduction.     

Apparent thermodynamic parameters of ligand binding to the cloned rat mu-opioid receptor

This study examined the involvement of spinal dopamine D2 receptors in the cardiovascular effects induced by intravenous administration of the selective dopamine D2 receptor agonist quinpirole, as has been previously reported for the hypotensive action of systemic bromocriptine. In normotensive pentobartitone-anaesthetised rats, intravenous injection of quinpirole (25 to 1000 microg/kg) decreased mean aortic pressure and heart rate in a dose-related manner. The intravenous (0.5 mg/kg) or intrathecal (40 microg/rat at T9-T10) pretreatment with domperidone, a dopamine D2 receptor antagonist that does not cross the blood-brain barrier, significantly reduced the maximal hypotensive and bradycardic responses to intravenous quinpirole (1000 microg/kg). In contrast, the latter effects were fully abolished either by intravenous metoclopramide (5 mg/kg) or combined pretreatment with intravenous and intrathecal domperidone. In addition, when injected intrathecally at the T9-T10 level of the spinal cord, quinpirole (7.7 to 61.4 microg/rat) also produced dose-dependent depressor and bradycardic effects which could be blocked by intrathecal, but not intravenous, domperidone pretreatment. This suggests that, in anaesthetised normotensive rats, the hypotensive and bradycardic responses to intravenous quinpirole are fully mediated by dopamine D2 receptors, some of which are located in the peripheral circulation and some of which are located within the spinal cord. The latter finding is novel, suggesting that partial spinal mediation may not be peculiar to bromocriptine, as was previously thought. Rather, partial spinal mediation may be common to most dopamine D2 receptor agonists.     

Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.     

A system of immunoenzyme analysis using biotinylated monoclonal antibodies for typing hantavirus antigens

Enzyme immunoassay with biotin-labeled monoclonal antibodies to Puumala virus was used for typing hantavirus antigens. The system is type-specific, that is, it detects Puumala virus only, which was demonstrated by typing 9 hantavirus antigens in infected Vero E5 cells and by epizootological analysis of an HFRS outbreak in the Yegoryevsk district of the Moscow region in November-December 1995. The suggested system may be used as an element of EIA screening of natural samples in analysis of the screening results.     

Crystal structure of the ligand binding domain of the human nuclear receptor PPARgamma

The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor supergene family and are considered as key sensors of both lipid and glucose homeostasis. The role of the PPARgamma isoform in glucose metabolism is illustrated by the fact that anti-diabetic thiazolidinediones have been shown to be bona fide PPARgamma ligands. Here we report the crystal structure of apo-PPARgamma ligand binding domain (LBD) determined to 2.9-A resolution. Although the structure of apo-PPARgamma-LBD retains the overall fold described previously for other nuclear receptor LBDs, three distinct structural differences are evident. 1) The core AF-2 activation domain of apo-PPARgamma LBD is folded back toward the predicted ligand binding pocket similar to that observed in the holo-forms of other nuclear receptors. 2) The proposed ligand binding pocket of apo-PPARgamma-LBD is larger and more accessible to the surface in contrast to other LBDs. 3) The region of the LBD called the omega-loop is extended in PPARgamma and contains additional structural elements. Taken together, the apo-PPARgamma-LBD structure is in several aspects different from previously described LBDs. Given the central role of PPARgamma as a mediator in glucose regulation, the structure should be an important tool in the development of improved anti-diabetic agents.     

Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion

The GnRH receptor (GnRHR) is a G protein-coupled receptor expressed by gonadotropes in the anterior pituitary gland. In the past several years, much has been learned about the structure-function relationships that exist in this receptor with regard to ligand binding and signal transduction. However, the lack of specific antibodies has precluded any analyses of the behavior of the unbound form of this receptor. We have constructed a functional GnRHR in which enhanced green fluorescent protein is fused to the carboxyl-terminus of the murine GnRHR. This fusion receptor was expressed diffusely throughout the cell, with approximately 38% of the fusion receptors colocalized with a plasma membrane marker in the gonadotrope-derived alphaT3 cell line, and approximately 82% of the fusion receptors colocalized with a membrane marker in Chinese hamster ovary cells. Furthermore, the fusion receptor displayed a Kd of 0.8 nM for iodinated des-Gly10,D-Ala-6-GnRH N-ethyl amide in Chinese hamster ovary cells, which was similar to the Kd of the native GnRHR expressed in alphaT3 cells. The surface mobility of the fusion protein was examined by fluorescence photobleaching recovery methods. In the unbound state the majority of the receptors were laterally mobile and displayed a lateral diffusion rate of 1.2-1.6 x 10(-9) cm2/sec. Binding of GnRH reduced the rate of lateral diffusion over 3-fold and reduced the fraction of mobile receptors from approximately 76-91% to 44-61%. Like GnRH, the competitive GnRH antagonist antide slowed the rate of receptor diffusion approximately 3-fold. In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Thus, an intrinsically fluorescent GnRHR is trafficked to the plasma membrane of mammalian cells, is capable of ligand binding and signal transduction, and allows direct observation of the GnRHR in the nonligand-bound state. Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist.     

Investigation of the binding interactions of progesterone using muteins of the human progesterone receptor ligand binding domain designed on the basis of a three-dimensional protein model

The aim of this study was to investigate the binding interactions of the human progesterone receptor (hPR) with its natural ligand. Therefore, a homology-derived model of the hPR ligand binding domain has been constructed and used to predict residues potentially involved in interactions with progesterone. These residues and the free cysteines have been mutated (in total 13 residues with 15 mutations). All exchanges have been designed to preserve the three-dimensional structure of the protein. With respect to the binding characteristics towards progesterone, the muteins fall into three groups displaying no, reduced, or wildtype-like binding activity.     

Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit in endometrial cancer.     

Requirement of cysteine-rich repeats of the Fas receptor for binding by the Fas ligand

The Fas receptor is a member of a family of cell death receptors, including tumor necrosis factor receptor I (TNFR I), death receptor 3 and 4 (DR3 and DR4), and cytopathic avian receptor 1 (CAR1). The Fas receptor is composed of several discrete domains, including three cysteine-rich domains (CRDs), a transmembrane domain, and an intracellular domain responsible for transmitting an apoptotic signal. While the mechanism of Fas-mediated cell death has become elucidated, the requirements for Fas ligand binding to the receptor have not been fully defined. Using a series of chimeric Fc-receptor fusion proteins between the human Fas receptor and TNFR I, each cysteine-rich domain of Fas was found to be required for interaction with the Fas ligand. Interestingly, TNFR I CRD1 could partially substitute for the Fas CRD1. The importance of this domain was underscored by the analysis of a Fas extracellular mutation (C66R), which resulted in a complete loss of ligand binding. This mutation was cloned from a human patient suffering from Canale-Smith syndrome, which is characterized by autoimmunity resembling that observed in the lpr and lprcg mice. The localization of essential ligand binding domains in the Fas receptor correlated exactly with the ability of the Fas receptor fusion proteins to prevent cell death mediated by the Fas ligand.     

Modulation of bradykinin receptor ligand binding affinity and its coupled G-proteins by nitric oxide

To determine whether nitric oxide (NO) can modulate bradykinin (BK) signaling pathways, we treated endothelial cells with an NO donor, S-nitrosoglutathione (GSNO), to determine its effect(s) on G-proteins (Gi and Gq) that are coupled to the type II kinin (BK2) receptor. Radioligand binding assays and Western analyses showed that GSNO (10-500 microM, 0-72 h) did not alter the expression of BK2 receptor, Gi, or Gq. However, GSNO caused a 6-fold increase in basal cGMP production and decreased high affinity BK bindings sites and GTPase activity by 74 and 85%, respectively. The cGMP analogue, dibutyryl-cGMP, also inhibited BK-stimulated GTPase activity by 74% suggesting that some of the effects of NO may be mediated through activation of guanylyl cyclase. The NO synthase inhibitor, Nomega-monomethyl-L-arginine, inhibited endogenous NO synthase activity and cGMP production by 91 and 76%, respectively, but increased BK-stimulated GTPase activity by 61%. To determine which G-proteins are affected by NO, we performed GTP binding assays with [35S]GTPgammaS followed by immunoprecipitation with specific G-protein antisera. Both GSNO and dibutyryl-cGMP increased basal G-protein GTP binding activities by 18-26%. However, GSNO decreased BK-stimulated Galphai2, Galphai3, and Galphaq/11 GTP binding activity by 93, 61, and 90%, respectively, whereas epinephrine-stimulated Galphas GTP binding activity was unaffected. These results suggest that NO can modulate BK signaling pathways by selectively inhibiting G-proteins of the Gi and Gq family.     

Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor beta

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RARbeta mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys228 is necessary to inhibit RA binding when RARbeta is modified by reagents which transfer large bulky groups while both Cys228 and Cys267 must be modified when a small functional group is transferred. These data suggest that both Cys228 and Cys267 but not Cys258 lie in the ligand binding pocket of RARbeta. However, Cys228 lies closer to the opening of the RARbeta ligand binding pocket whereas Cys267 lies more deeply buried.     

Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 x 10(-8) M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate > ibotenate >/= L-glutamate approximately (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.     

Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescei n (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83-87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054) and the photochemical cross-linking data.     

Changes in glomerular thromboxane A2 receptor expression and ligand binding following immune injury

Susceptibility of lipoproteins to oxidation is partly determined by their content in endogenous antioxidants, but also by the polyunsaturated fatty acids (PUFA)/monounsaturated fatty acids (MUFA) ratio. The aim of our study was to enrich human high-density lipoproteins (HDLs) with dioleoylphosphatidylcholine (DOPC) in order to modify the PUFA/MUFA ratio while maintainig the alpha-tocopherol/PUFA ratio constant and to appreciate the consequences of this enrichment before and after copper-induced oxidation. The enrichment of HDLs with DOPC was obtained by incubation of these lipoproteins with DOPC liposomes and further reisolation of HDLs. The consequent 40% HDL enrichment in MUFA was concomitant with a 35% loss in PUFA (MUFA/PUFA ratio = 1.43). The enrichment of HDLs with DOPC led to a 40% decrease in alpha-tocopherol content, which kept a constant alpha-tocopherol/PUFA ratio. The DOPC-HDLs exhibited a lower oxidizability by copper than the nonenriched HDLs (NE-HDLs), as shown by their twofold longer lag phase and the threefold lower propagation rate. Moreover, DOPC-HDLs led to a six- to sevenfold lower production of hydroperoxide molecular species from phosphatidylcholine and cholesteryl esters than NE-HDLs after 24 h copper oxidation. With regard to the cholesterol effluxing capacity, copper oxidation of HDLs led to a decrease of this property. However, our results clearly showed that DOPC enrichment of HDLs allowed us to keep a better effluxing capacity than in NE-HDLs after 24 h oxidation (22.3% vs 17.4%, respectively). Since apo A-I was degraded as well in DOPC-HDLs as in NE-HDLs, the better effluxing capacity of DOPC-HDLs could not come from a preserved integrity of apo A-I. It could be partly related to the improved fluidity of oxidized DOPC-HDLs compared to oxidized NE-HDLs, as shown by electron spin resonance data (correlation-relaxation time at 24 degreesC = 2.20 ns vs 3.00 ns after 24 h oxidation, in DOPC-HDLs and in NE-HDLs, respectively). Besides, it could also be hypothesized that the sevenfold lower content of phosphatidylcholine hydroperoxides in DOPC-HDLs than in NE-HDLs after 24 h copper oxidation could be involved in the better ability of oxidized DOPC-HDLs to mobilize cellular cholesterol.