A complement receptor locus: genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to 1q32

The alternative or classical pathways for complement system component C3 may be triggered by microorganisms and antigen-antibody complexes. In particular, an activated fragment of C3, C3b, covalently attaches to microorganisms or antigen-antibody complexes, which in turn bind to the C3b receptor, also known as complement receptor 1. The genes encoding the proteins that constitute the C3-activating enzymes have been cloned and mapped to a "complement activation" locus in the major histocompatibility complex, and we demonstrate in this study such a locus on the long arm of chromosome 1 at band 1q32.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.     

Activation of bradykinin B2 receptors increases calcium entry and intracellular mobilization in C9 liver cells.

In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.     

Activation and infection of B cells by Epstein-Barr virus. Role of calcium mobilization and of protein kinase C translocation

Both the activation and the transformation of human B cells by EBV were inhibited by either the Ca2+ channel blocking agent verapamil or the combination of theophylline and dibutyryl cAMP: the day 4 and day 20 peaks of [3H]TdR incorporation were abolished; the EBNA marker was not expressed by day 10; lymphoblastoid cell lines did not arise. Short term incubation of B cells with EBV or verapamil showed that the effect of verapamil was reversible and took place early in the interaction between EBV and B cells. The effect of EBV on the early metabolic events of B cell response was thus examined in the presence and in the absence of the drugs. Compared to anti-mu stimulation, supernatant of the transforming B95-8 strain as well as that of the non-transforming P3HR1 strain induced a drug sensitive increase of the free cytosolic Ca2+ concentration. This increase was associated with a protein kinase C translocation from the cytosol to a membrane bound compartment. Moreover, B95-8 supernatant induced phosphatidyl inositol metabolism by human B cells but at least four times less than that induced by anti-mu antibody. These metabolic events induced by EBV were significantly inhibited by anti-CD21 antibodies whereas anti-mu induced metabolic events were not. The infection of EBV negative Ramos cell line was prevented by verapamil or by theophylline + dibutyryl cAMP. Verapamil did not modify the density of EBV receptors but negatively interfered with the penetration of the virus into B cells. Thus B cell activation through the EBV receptor and virus penetration share a common metabolic pathway which is also used for transduction of the signal delivered through the membrane Ig.     

Intracellular degradation of the complement C3b/C4b receptor in the absence of ligand

Human neutrophils (PMN) respond to various soluble stimuli by translocating intracellular complement C3b/C4b receptors (CR1) to the cell surface. Ligand-independent internalization of surface CR1 has been demonstrated previously, but the fate of total cellular CR1 during PMN stimulation has not been determined. In order to study the fate of CR1 during neutrophil activation, we have employed a unique approach for the quantitative analysis of intracellular antigens which allows simultaneous measurement of total cellular and surface membrane antigen pools. Stimulation of isolated PMN with N-formyl-Met-Leu-Phe or ionomycin resulted in a mean 7-fold increase in surface CR1 expression within 15 min. Total cellular CR1 decreased by as much as 45% within 15 min, with loss continuing for up to 1 h. Inclusion of NH4Cl during PMN stimulation inhibited the loss of total CR1 without affecting surface CR1 expression. Addition of phenylmethylsulfonyl fluoride inhibited loss of total CR1 and enhanced the stimulus-induced increases in surface CR1. These data suggest that intracellular degradation of CR1 occurs during stimulation of PMN and may involve proteolysis in an acidic intracellular compartment. Since our experiments were done with isolated PMN in the absence of serum and complement components, this degradation occurred in the absence of C3b, the ligand for CR1. To our knowledge, ligand-independent degradation of a cell surface receptor has not been previously detected.     

Polymorphism of the C3b/C4b receptor (CR1): characterization of a fourth allele

The receptor for C3b/C4b (C3bR or CR1) has an unusual polymorphism in which three codominant alleles determine variants with a large difference in Mr (160,000, 190,000, or 220,000). We found an individual who has, in addition to the common 190,000 Mr molecule, a C3bR whose Mr is 250,000. In this proband and in some members of his family, this novel heterozygous phenotype can be isolated from 125I surface-labeled cells by iC3 or iC4 affinity chromatography or by immunoprecipitation with the use of polyclonal or monoclonal anti-C3bR. Relative to the 190,000 Mr C3bR, E from individuals in this family have 20 to 30% of the total receptor counts in the 250,000 Mr C3bR. However, on C3bR-bearing leukocytes there is a much larger amount of the 250,000 Mr C3bR (approximately 60%) relative to the 190,000 Mr C3bR. Similar to the other three C3bR variants, the Mr is 5,000 greater on polymorphonuclear cells than on E, and treatment of this new C3bR with endoglycosidase F decreases its Mr by approximately 10,000. Therefore, because this variant is inherited and has structural and functional similarities to the other three C3bR, we conclude that this 250,000 Mr CR1 probably represents a fourth allele.     

Activation of protein kinase C alpha by lipid mixtures containing different proportions of diacylglycerols.

Lipid activation of protein kinase C alpha (PKC alpha) was studied using a model mixture containing POPC/POPS (molar ratio 4:1) and different proportions of either DPG or POG. The lipid mixtures containing DPG were physically characterized by using different physical techniques, and a phase diagram was constructed by keeping a constant POPC/POPS molar ratio of 4:1 and changing the concentration of 1,2-DPG. The phase diagram displayed three regions delimited by two compounds: compound 1 (CO(1)) with 35 mol % of 1,2-DPG and compound 2 (CO(2)) with 65 mol % of 1,2-DPG. PKC alpha activity was assayed at increasing concentrations of 1,2-DPG, maximum activity being reached at 30 mol % 1,2-DPG, which decreased at higher concentrations. Maximum activity occurred, then, at concentrations of 1,2-DPG which corresponded to the transition from region 1 to region 2 of the phase diagram. It was interesting that this protein was maximally bound to the membrane at all DPG concentrations. Similar results were observed when the enzyme was activated by POG, when a maximum was reached at about 10 mol %. This remained practically constant up to 50 mol %, about which it decreased, the binding level remaining maximal and constant at all POG concentrations. The fact that in the assay conditions used maximal binding was already reached even in the absence of diacylglycerol was attributed to the interaction of the C2 domain with the POPS present in the membrane through the Ca(2+) ions also present. To confirm this, the isolated C2 domain was used, and it was also found to be maximally bound at all DPG concentrations and even in its absence. Since the intriguing interaction patterns observed seemed to be due then to the C1 domain, the PKC alpha mutant D246/248N was used. This mutant has a decreased Ca(2+)-binding capacity through the C2 domain and was not activated nor bound to membranes by increasing concentrations of DPG. However, POG was able to activate the mutant, which showed a similar dependence on POG concentration with respect to activity and binding to membranes. These data underline the importance of unsaturation in one of the fatty acyl chains of the diacylglycerol.     

Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.     

A Revised Mechanism for the Activation of Complement C3 to C3b: A MOLECULAR EXPLANATION OF A DISEASE-ASSOCIATED POLYMORPHISM*

The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg¹⁰². In 50 mm NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg¹⁰²–Glu¹⁰³² salt bridge. In physiological 137 mm NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg¹⁰²–Glu¹⁰³² salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg¹⁰²-Glu¹⁰³² salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg¹⁰²) and disease-linked C3F (Gly¹⁰²) allotypes of C3b were experimentally explained for the first time.     

Characterization of the 5-HT2b receptor in evaluation of aequorin detection of calcium mobilization for miniaturized GPCR high-throughput screening

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein-coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 microL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550) and kinetic flash luminometry (CyBi Lumax). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC(50) in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains.     

Activation of calcium mobilization and calcium influx by alpha 1-adrenergic receptors in a smooth muscle cell line

Alpha 1-adrenergic receptor (alpha 1R) mediated increases in the cytosolic levels of free Ca+2 and the inositol phosphates were measured in a smooth muscle cell line, DDT1. Norepinephrine (NE) stimulated a rapid increase in cytosolic Ca+2 by two distinct components: 1) release of Ca+2 from intracellular sites (mobilization), and 2) influx of extracellular Ca+2. The mobilization component was not affected by removal of extracellular Ca+2 or addition of La+3 or Co+2 to the buffer. The influx component was abolished by EGTA, La+3, or Co+2, but was not affected by the voltage-operated Ca+2 channel blockers diltiazem or nifedipine. Depolarization of DDT1 cells with 100 mM KCl or with gramicidin did not induce Ca+2 influx. NE also increased inositol trisphosphate to 78% over basal levels within 1 minute. These results suggest that alpha 1R on DDT1 cells are coupled to both the mobilization of intracellular Ca+2 and to receptor-operated Ca+2 channels in the plasma membrane, and that polyphosphoinositide hydrolysis may play a role in these phenomena.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils

As measured by fluorescence microscopy and radioligand binding, C3b/C4b receptors (CR1) became attached to the detergent-insoluble cytoskeleton of human neutrophils when receptors were cross-linked by affinity-purified polyclonal F(ab')2 anti-CR1, dimeric C3b, or Fab monoclonal anti-CR1 followed by F(ab')2 goat anti-mouse F(ab')2. CR1 on neutrophils bearing monovalent anti-CR1 was not attached to the cytoskeleton. In contrast, cross-linked CR1 on erythrocytes and cross-linked MHC Class I on neutrophils were not cytoskeleton associated. A possible role for filamentous actin (F-actin) in the binding of cross-linked CR1 to neutrophil cytoskeleton was suggested by three observations. When neutrophils were differentially extracted with either Low Salt-detergent buffer or High Salt-detergent buffer, stained with FITC-phalloidin, and examined by fluorescent flow cytometry, the residual cytoskeletons generated with the former buffer were shown to contain polymerized F-actin, whereas cytoskeletons generated with the latter buffer were found to be depleted of F-actin. In parallel experiments, High Salt-detergent buffer was also found to release cross-linked CR1 from neutrophils. Second, depolymerization of F-actin by DNAse I released half of the cytoskeletal-associated cross-linked CR1. Third, immunoadsorbed neutrophil CR1, but not MHC Class I or erythrocyte CR1, specifically bound soluble 125I-actin. In addition, Fc receptor and CR3, other phagocytic membrane proteins of neutrophils, specifically bound 125I-actin. These data demonstrate that CR1 cross-linked on neutrophils becomes associated with detergent-insoluble cytoskeleton and that this interaction is mediated either directly or indirectly by actin.     

BANK regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP(3) receptor.

B-cell activation mediated through the antigen receptor is dependent on activation of protein tyrosine kinases (PTKs) such as Lyn and Syk and subsequent phosphorylation of various signaling proteins. Here we report on the identification and characterization of the B-cell scaffold protein with ankyrin repeats (BANK), a novel substrate of tyrosine kinases. BANK is expressed in B cells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR) stimulation, which is mediated predominantly by Syk. Overexpres sion of BANK in B cells leads to enhancement of BCR-induced calcium mobilization. We found that both Lyn and inositol 1,4,5-trisphosphate receptor (IP(3)R) associate with the distinct regions of BANK and that BANK promotes Lyn-mediated tyrosine phosphorylation of IP(3)R. Given that IP(3)R channel activity is up-regulated by its tyrosine phosphorylation, BANK appears to be a novel scaffold protein regulating BCR-induced calcium mobilization by connecting PTKs to IP(3)R. Because BANK expression is confined to functional BCR-expressing B cells, BANK-mediated calcium mobilization may be specific to foreign antigen-induced immune responses rather than to signaling required for B-cell development.     

The muscarinic receptor of chick embryo cells: correlation between ligand binding and calcium mobilization

In this report we characterize muscarinic cholinergic receptor on embryonic cells. We established dose-response curves by fluorometric measurement of Ca2+ mobilization in cell suspensions of whole chick embryos stage 23/24. Ca2+ mobilization was quantitated by standardization of chlorotetracycline (CTC) fluorescence changes after stimulation with muscarinic agonists. We determined ED50 values for the agonists acetylcholine and carbachol as 3.4 X 10(-6) and 2.7 X 10(-5) M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with inhibition constants (Kl) of 5.0 X 10(-6) and 1.4 X 10(-6) M, respectively. Bethanechol, which induced only 23% of the maximal effect obtained by acetylcholine, was a partial agonist with an ED50 of 4.8 X 10(-4) M. Its antagonistic component is expressed by an inhibition constant of 1.9 X 10(-4) M. In parallel, binding studies were performed in a competition assay with [3H]-quinuclidinylbenzilate. For the agonists acetylcholine and carbachol, binding parameters were best fitted by a "two binding-sites model." Comparison with dose-response curves indicated that Ca2+ mobilization was triggered via the high-affinity binding site. The inhibition constants of antagonists derived from the shift of dose-response curves corresponded to the fitted KD values of the binding studies when a "one binding-site model" was applied. Combination of dose-response and binding data showed close proportionality between receptor occupancy and calcium mobilization. No spare receptors were present.     

Translation of the human C3b/C4b receptor mRNA in a cell-free system and by Xenopus oocytes

The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190,000, 220,000, 250,000, and 280,000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220,000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CR1 and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CR1 monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.     

The relationship between alpha 1-adrenergic receptor occupation and the mobilization of intracellular calcium

We have simultaneously quantitated alpha 1-adrenergic receptor occupation and agonist-elicited Ca2+ mobilization monitored as unidirectional 45Ca2+ efflux from intact BC3H-1 muscle cells in order to examine the relationship between the number of surface receptors occupied and the functional response. [3H]Prazosin has been used to measure receptor number as well as the binding kinetics with surface receptors, and the observed equilibrium and kinetic constants are in close accord with values obtained previously in cellular homogenates. Since alpha 1-agonist-elicited 45Ca2+ efflux can be monitored over intervals of 3 min or less and prazosin dissociation from its receptor has a t 1/2 of 44 min, prazosin can be employed to produce a pseudoirreversible inactivation of receptors. A comparison of the remaining receptors and residual response reveals an inverse linear relationship between receptors inactivated by prazosin and 45Ca2+ efflux. A similar result is obtained following fractional receptor inactivation with the irreversible alkylating agent phenoxybenzamine. Parameters of receptor occupation and response also correlate well for the agonist phenylephrine and for the competitive antagonist phentolamine. The unitary relationship between sites available for occupation and response indicates that the alpha 1 receptor does not function as an oligomer where fewer bound antagonist molecules are required to block the receptor than sites of agonist occupation necessary for activation. Moreover, substantial evidence has accrued in intact smooth muscle for a receptor reserve or nonlinear coupling between alpha 1 receptor occupation and contraction in smooth muscle. Our findings demonstrate that such behavior does not exist for alpha 1 receptor-elicited mobilization of Ca2+ in the BC3H-1 muscle cell.