Measles virus recognizes its receptor, CD46, via two distinct binding domains within SCR1-2

Measles virus (MV) enters cells by attachment of the viral hemagglutinin to the major cell surface receptor CD46 (membrane cofactor protein). CD46 is a transmembrane glycoprotein whose ectodomain is largely composed of four conserved modules called short consensus repeats (SCRs). We have previously shown that MV interacts with SCR1 and SCR2 of CD46. (M. Manchester et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2303-2307) Here we report mapping the MV interaction with SCR1 and SCR2 of CD46 using a combination of peptide inhibition and mutagenesis studies. By testing a series of overlapping peptides corresponding to the 126 amino acid SCR1-2 region for inhibition of MV infection, two domains were identified that interacted with MV. One domain was found within SCR1 (amino acids 37-56) and another within SCR2 (amino acids 85-104). These results were confirmed by constructing chimeras with complementary regions from structurally similar, but non-MV-binding, SCRs of decay accelerating factor (DAF; CD55). These results indicate that MV contacts at least two distinct sites within SCR1-2.     

Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1

The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.     

Identification of residues within the 727-767 segment of human complement component C3 important for its interaction with factor H and with complement receptor 1 (CR1, CD35)

Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727-767 segment at the NH2 terminus of C3b alpha'-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738-767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545-552) which implicated residues within the 744-754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of alpha-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727-767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O).     

Analysis of the functional domains of complement receptor type 1 (C3b/C4b receptor; CD35) by substitution mutagenesis

The complement receptor type 1 (CR1; CD35), carrying 30 short consensus repeats (SCRs), has two sites. Site 1 contains SCR-1 and SCR-2 and binds C4b. Site 2 contains SCR-8 and SCR-9 and was reported to bind mainly C3b (Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A., and Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717). For the functional analysis we used two constructs, each with one site. CR1-4, composed of eight and one-half initial SCRs, carries site 1, binds C4b, and is cofactor for C4b cleavage. CR1-4(8,9), obtained from CR1-4 by converting site 1 to site 2, binds iC3/C3b and, unexpectedly, C4b. It is a cofactor for cleavage of both ligands. Its cofactor activity for C4b cleavage is greater than that of site 1. Analysis of the mutants constructed by interchanging homologous peptides between the two sites identified no sequences necessary for cofactor activity other than those required for binding. In site 2, peptides important for both ligands were found. Some modifications of either site led to higher activity for both ligands. Thus the activity of complement regulators can be increased by changing a few amino acids within SCRs, an important step toward the generation of more effective inhibitors of complement activation. Knowledge of the active sites of CR1 should be applicable to other SCR-containing proteins and should provide insights into the evolution of these proteins.     

Aseptic isolation of human complement component C3 for in vivo studies

A procedure for aseptic isolation of human complement component C3 is described. The principles may be useful in the preparation of other proteins for in vivo studies.     

Rapid purification of human complement receptor type 1 (CD35, CR1)

BACKGROUND: Migraine is a prevalent disorder whose relationship to other conditions remains poorly understood. METHODS: Associations between migraine and physiological, behavioral, and demographic characteristics were assessed in a retrospective cohort study of 79,588 enrollees in a large prepaid health maintenance organization who underwent a multiphasic preventive health checkup in 1971-1973. RESULTS: Migraine was found to be inversely associated with age and education and strongly associated with the female sex. The likelihood of migraine was significantly higher among blacks, smokers, those who drink more than six cups of coffee per day, those with Raynaud's syndrome, and those with a family history of migraine. The magnitude of associations between migraine and other factors was, in general, reduced among those with a self-reported physician diagnosis of migraine compared to those whose migraine status was defined on the basis of reported symptoms. CONCLUSIONS: Migraine prevalence was found to be higher in blacks and other unspecified minorities than in the white population. The magnitude of the associations between migraine and behavioral risk factors was strongly influenced by the method of migraine ascertainment. The inverse association with level of education suggests that social causation or drift may have been operating in this disease in the early 1970s, 15 to 20 years earlier than recent population-based studies would suggest. Further research is needed to fully appreciate the spectrum of disease and behaviors associated with migraine.     

Mutagenesis of human Mel1a melatonin receptor expressed in yeast reveals domains important for receptor function

Mechanisms of organization of the striatal compartments are poorly understood, although involvement of cell adhesion molecules in the compartmentalization has been suggested. Cadherin-8 distribution in the neonatal rat striatum was immunohistochemically studied using a rabbit anti-cadherin-8 antiserum. Intensity of cadherin-8 immunolabeling in the striatum was heterogeneous from postnatal day 0 to postnatal day 7. At postnatal day 9, cadherin-8 immunoreactivity was so weak that heterogeneity was no longer clearly seen. Cadherin-8 immunoreactivity was not detectable at postnatal day 14. Cadherin-8-rich and cadherin-8-poor areas were identical to calbindin-rich areas and tyrosine hydroxylase-rich patches, respectively, in allocation, indicating that cadherin-8 was predominantly expressed in the striatal matrix. These results suggest that cadherin-8 is involved in formation of the striatal compartmentalized structures during brain development.     

Enhanced generation of O2- by human neutrophils via a complement iC3b/Mac-1 interaction

There is evidence for a tumor necrosis factor alpha (TNF alpha)-initiated and CD11b/CD18-dependent burst of superoxide anion (O2-) and hydrogen peroxide production by human polymorphonuclear leukocytes which are adherent to surfaces bearing a variety of proteins. In the current studies neutrophils were stimulated with opsonized (by fresh human serum) zymosan particles in the presence of cytochalasin B, to prevent internalization of particles and to simulate the interaction of neutrophils with protein-bearing surfaces. Under these conditions, the cells demonstrated 2.9-fold greater production of O2- when compared to nonopsonized zymosan particles. Heat inactivation or cobra venom factor treatment of human serum prior to opsonization resulted in 98% and 66% reductions, respectively, in O2- responses. C3 and factor B were required for this response, since sera deficient in either component caused 56 and 68% reductions, respectively, in O2- production. Sera deficient in Clq, C2, C4, C5, C6, C7 or C9 showed no defect in their ability to enhance O2- responses to zymosan particles. Monoclonal antibody to iC3b, but not monoclonal antibodies to C3c or C3d, caused a 29% reduction (p < 0.01) in O2- generation. Antibodies to CD18 (R15.7) or CD11b (CL44 and 60.1) reduced the incremental production of O2- by 76, 71 and 77%, respectively. Two antibodies directed against CD11a as well as the isotype-matched control (MOPC 21) were without effects. These data suggest that, in this model of neutrophil activation, the pathway for O2- generation is a Mac-1 (but not LFA-1)-dependent pathway and also requires iC3b. These findings may be relevant to complement-mediated, neutrophil-dependent vascular injury in vivo.     

Identification of complement receptor type 1-related proteins on primate erythrocytes

The purpose of this study was to characterize the structure and function of the immune adherence receptor (CR1, CD35, C3b/C4b receptor) of primates. Western blotting, immunoprecipitation, ELISA, and affinity chromatography with homologous C3b and C4b were utilized. The major cross-reactive E membrane protein of ten species of primates tested was lower in m.w. than was human CR1 and fell into two size groups of 55 to 75 and 130 to 165 kDa. There was 10- to 100-fold more CR1 per primate E than human E. Five species also expressed lesser quantities of a protein similar in m.w. (approximately 200 kDa) to human CR1. In contrast to E, the major cross-reactive protein on PBMC was similar in size to human CR1. Four species also expressed lesser amounts of a lower m.w. protein on their PBMC of the same M(r) as that found on their E. Affinity chromatography demonstrated that the approximately 200-kDa form, if present, was recovered with a similar efficiency to that of human CR1. Three patterns of binding, however, were identified among the lower m.w. proteins: 1) C3b > or = C4b; 2) C4b > C3b; and C3b only or predominantly. The fact that these E proteins cross-react with Ab to human CR1, bind homologous C3b and, in most cases, C4b, and for some species represent the only such protein expressed on their E identifies them as immune adherence receptors. The 70-kDa CR1 of the chimpanzee E seems to arise by alternative splicing of the mRNA encoding the 200-kDa protein. These data raise interesting questions relative to the evolution of CR1 in primates and provide a basis for analysis of structure-function relationships among these size forms of CR1.     

Comparative haemodynamic studies of endothelin-1, -2 and -3 on conscious SHRSP and WKY

1. Depressor and pressor effects of endothelin-1, -2 and -3 in relation to hypertension were investigated in conscious WKY and SHRSP. 2. Changes of systolic arterial pressure to both depressor and pressor responses caused by three doses of endothelin-1, -2 or -3 (0.1, 0.3 and 1 nmol/kg) occurred to a similar extent between WKY and SHRSP. These data showed that endothelins may not exert an important role on the pathogenesis of hypertension. 3. Endothelin-1 decreased the cardiac index more in SHRSP than in WKY, indicating the dominance of ETA receptors in SHRSP compared with WKY. 4. ET-1 was the most potent vasodepressor and vasodilator of three endothelin peptides in rats. 5. During the pressor responses to endothelin-1 and -3, cardiac arrhythmia was observed with high frequency in the animals of both groups, indicating the arrhythmogenic effect of ET.     

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.     

Comparison of the signaling abilities of the cytoplasmic domains of the insulin receptor and the insulin receptor-related receptor in 3T3-L1 adipocytes

In the present work a chimeric receptor containing the intracellular domain of the insulin receptor-related receptor (IRR) and the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor was expressed in 3T3-L1 adipocytes and compared with the parallel chimeric receptor containing the cytoplasmic domain of the insulin receptor (IR). Both chimeric receptors exhibited CSF-stimulated tyrosine kinase activity when assayed in vitro after in vivo activation comparable to that of the endogenous IR present in these cells. No cross-activation of the expressed chimeric and endogenous receptors was observed. The cytoplasmic domain of the IRR was found to 1) mediate activation of the Ser/Thr kinase Akt/PKB, 2) stimulate glucose uptake, 3) inhibit lipolysis, and 4) stimulate glycogen synthase, all with a potency comparable to those of the expressed CSF-1R/IR chimera and the endogenous insulin receptors. These results indicate that despite the extensive differences in sequence between the cytoplasmic domains of the IRR and IR, the elements required for insulin-specific responses have been conserved in this distinct member of the insulin receptor family.     

Receptor-receptor interactions of complement receptor type 3 in neutrophil membranes

The leukocyte integrin CR3 (CD11b/CD18) is known to participate in a variety of cell functions. Recent studies have indicated that CR3 may communicate with other plasma membrane receptors to carry out several cell functions. In this review we discuss these potential receptor-receptor interactions of CR3 and present a unifying model of CR3's diverse functions.     

1H and 13C NMR studies of conformational behaviour of Leu-enkephalin

The presence of secondary sensory cells in the Octopus gravity receptor system has been demonstrated. In serial thin sections of the receptor cells (hair cells) no axons were found leaving the cells. Instead, synapses were observed with synaptic vesicles lying inside the receptor cells. Both data clearly indicate that the receptor hair cells represent secondary sensory cells. In addition, efferent contacts to the receptor cells could be confirmed.     

1H-NMR studies on thymopoietin-type oligopeptides--assignment of the proton resonances and investigation of conformational preferences

The thymopoietin-type tripeptides TP3 (HArg-Lys-AspOH), TP(D-Asp)3(HArg-Lys-D-AspOH) and tetrapeptide TP4 (HArg-Lys-Asp-ValOH) were studied by one- and two-dimensional, 500 MHz 1H-NMR spectroscopy in H2O and D2O solutions at four different pH values. All proton resonances of the three oligopeptides were assigned by two-dimensional phase-sensitive TOCSY experiments at pH 12.2, 9.1, 5.9 and 3.6. At these pH-values well-defined stages of protonation and concomitant molecular charges exist, allowing different possibilities for intra-molecular and inter-residual orientations. Conformation-sensitive rotating frame nuclear Overhauser enhancement (ROESY) two-dimensional experiments were also performed at the above pH values. These experiments indicated no definite solution conformation of any of the molecules at any pH. Standard one-dimensional experiments were also carried out and three-bond coupling constants were measured for the NH--CH and the Asp CH--CH moieties. The coupling constants provided evidence that non-statistical orientations of the functional groups exist which are changed upon protonation of the basic sites.     

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.     

Intracellular storage and regulated plasma membrane expression of human complement receptor type 1 in rat basophil leukemia cell transfectants

Polymorphonuclear neutrophils (PMN) contain multiple distinct secretory compartments that are sequentially mobilized during cell activation. Complement receptor type 1 (CR1) is a marker for a readily mobilizable secretory vesicle compartment, which can undergo exocytic fusion with the plasma membrane independently of secretion of traditional granule contents. The basis for the formation of these distinct compartments is incompletely understood. Primary and secondary granules are generated directly from the Golgi complex during different stages of development of the cell, obviating the need for sorting signals for proper packaging of their constituents. To determine whether the secretory vesicles are formed in a similar manner, we studied a stable rat basophilic leukemia cell line (RBL-CR1) transfected with a plasmid containing the cDNA of human CR1 driven by a viral promoter. The CR1 was present primarily intracellularly in small vesicles resembling the CR1 storage pools in resting PMN. Activation of RBL-CR1 resulted in translocation of intracellular CR1 to the plasma membrane, with mobilization requirements different from those of the classical RBL granules. Thus, in RBL-CR1, continuously synthesized CR1 is stored and upregulated in much the same way as in PMN. This suggests that differential timing of gene expression is not essential for proper storage of CR1 and that other sorting mechanisms are involved, which can be studied in RBL-transfectants.     

14-3-3beta protein associates with insulin receptor substrate 1 and decreases insulin-stimulated phosphatidylinositol 3'-kinase activity in 3T3L1 adipocytes

The 14-3-3 protein family has been implicated in growth factor signaling. We investigated whether 14-3-3 protein is involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the immunoprecipitate with anti-14-3-3beta antibody at the basal condition. 100 nM insulin increased the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nM wortmannin. An in vitro binding study revealed that glutathione S-transferase-14-3-3beta fusion protein directly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Because the recombinant IRS-1 was not phosphorylated on its tyrosine residues, the results suggest that serine/threonine phosphorylation of IRS-1 is responsible for the association. When the cells are treated with insulin, phosphatidylinositol 3'-kinase (PI3K) is supposed to complex either 14-3-3beta-IRS-1 or IRS-1. The 14-3-3beta-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation, first with anti-14-3-3beta and then with anti-IRS-1 antibodies. The specific activity of the PI3K in the former was approximately half of that in the latter, suggesting that 14-3-3beta protein bound to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K in 3T3L1 adipocytes.     

Structure-function analysis of the active sites of complement receptor type 1

Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.