Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

The alpha, beta, and gamma subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the alpha chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for alpha and beta. However, these putative homologies were not apparent when tryptic maps of the biosynthetically ([3H]leucine) labeled subunits were analyzed.     

Ascorbate induced cross-linking of oxyhemoglobin subunits

Ascorbic acid during oxidation in vitro can generate H2O2 which induces non-disulphide covalent cross-linking of coincubated oxyhemoglobin. The cross-linking phenomenon mediated by H2O2 takes place possibly without the involvement of hydroxyl radicals as evident from the failure of radical scavengers like mannitol and dimethyl sulphoxide as well as metal-chelator, to inhibit the process. This pro-oxidant effect of ascorbic acid may have physiological significance in red blood cells in vivo.     

Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114

Above its critical micelle concentration, Triton X-114 in solution forms two phases at room temperature: a lower phase containing supramicellar aggregates and an upper phase largely depleted of detergent. This property of the detergent is potentially useful for separating under mild conditions proteins that bind detergent from those that do not (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). We studied the distribution of the receptor for immunoglobulin E (IgE) and its subunits in the two phases. IgE and IgE complexed either with intact receptors or with the alpha chains of the receptor alone are principally partitioned into the upper phase, whereas the unliganded receptor as well as the isolated alpha, and especially the beta and gamma chains of the receptor, preferentially partition into the lower detergent phase. Chromatography of IgE and of the subunits of the receptor on a hydrophobic support showed that the beta and gamma chains have a considerably greater hydrophobic surface than the alpha chains or IgE. These results indicate that the distribution of a protein in the two phases of phase-separated Triton X-114 is not an all-or-none effect based upon whether it binds detergent or not. Rather, it reflects the overall balance between the hydrophobic and hydrophilic properties of the protein's surface.     

Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking

The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipids stabilize the interaction between the alpha and beta subunits of the solubilized receptor for immunoglobulin E

The cell-surface component (alpha) which binds monomeric immunoglobulin E with high affinity is associated with a second polypeptide (beta) in the plasma membrane. The latter component tends to dissociate during purification of the alpha chain from detergent extracts of cells, even at neutral pHs and physiological ionic strengths. We now report that the interaction of alpha and beta can be stabilized by maintaining an appropriate phospholipid to detergent ratio. Under such conditions, other discrete components reproducibly copurify with the alpha and beta chains. These results suggest that the subunits of this membrane protein--or the interaction of it with other constituents in the cell--may be stabilized in ways not observed with ordinary soluble proteins.     

UV cross-linking/immunoprecipitation assay: glucocorticoid receptor-adrenergic receptor gene sequence interaction

The rat beta 1-adrenergic receptor (beta 1-AR) gene contains glucocorticoid response element (GRE) half-sites at positions -2767 and -945. In electrophoretic mobility shift assay (EMSA) experiments, neither beta 1-AR GRE half-site recognized glucocorticoid receptors (GRs) obtained from baculovirus high-level expression systems or from mammalian cells. We have developed a sensitive UV cross-linking/immunoprecipitation assay, using a 524-bp fragment containing the prototypical GRE obtained from the rat tyrosine aminotransferase promoter sequence and using antibodies recognizing mammalian GR. Using this assay, we provide evidence that rat beta 1-AR gene sequences recognize mammalian GRs expressed in mouse 3T3 cells and that the site of GR interaction does not appear to specifically contain the beta 1-AR GRE half-sites. This represents one of the first reports demonstrating the utility of a UV cross-linking/immunoprecipitation assay in the detection of mammalian GR interaction with beta 1-AR sequences, is consistent with the lack of specific DNA-GR protein complexes observed in EMSA experiments using oligonucleotide probes containing the beta 1-AR GRE half-sites, and provides evidence that mammalian GR interaction occurs at complex rate beta 1-AR gene sequences.     

Covalent cross-linking of prostaglandin E receptor from bovine adrenal medulla with a pertussis toxin-insensitive guanine nucleotide-binding protein

Prostaglandin E2 (PGE2) specifically bound to 100,000 X g pellet prepared from bovine adrenal medulla, and [3H]PGE2-bound proteins were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The dissociation of bound [3H]PGE2 from the proteins was enhanced by GTP. [3H]PGE2-specifically bound proteins were adsorbed onto a wheat germ agglutinin column and GTP treatment decreased the amount of [3H]PGE2 retained on the column. When [3H]PGE2-bound proteins were cross-linked in the membrane by dithiobis(succinimidyl propionate) and solubilized, bound [3H]PGE2 was no longer dissociated by GTP treatment, suggesting that cross-linking produced a stable and high-affinity complex of PGE receptor with a GTP-binding protein. Covalent cross-linking of the complex was attested by adsorption of dithiobis(succinimidyl propionate)-treated [3H]PGE2-bound proteins to GTP-Sepharose, and co-elution of [35S]guanosine 5'-O-(3-thiotriphosphate) binding activity and immunoreactivities of alpha o and beta subunits of a GTP-binding protein. The cross-linked [3H]PGE2-bound complex was eluted as an apparently single radioactive peak at the position of Mr = 200,000 by gel filtration. These results have demonstrated that PGE receptor is a glycoprotein with an approximate Mr of 110,000, assuming that the Mr of the GTP-binding protein is 90,000. PGE2 neither activated nor inhibited adenylate cyclase activity, and pertussis toxin (islet-activating protein) did not affect PGE2 binding and its GTP sensitivity. These results suggest that the PGE receptor may be functionally associated with a pertussis toxin-insensitive GTP-binding protein and is not coupled to the adenylate cyclase system in bovine adrenal medulla.     

Covalent cross-linking and degradation of proteins caused by ozone

Molecular products of ozonolysis of tyrosine in proteins have been investigated by means of spectroscopy. The processes of covalent crosslinking and destruction of proteins have been studied. Possible mechanism of crosslinking induced by ozone is discussed.     

Covalent cross-linking of secreted bovine thyroglobulin by transglutaminase.

Extracellular storage of thyroglobulin (TG) is a prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of TG within the follicle lumen is achieved by compactation and by the formation of covalent cross-links between TG molecules. In bovine thyroids, approximately 75% of the cross-links are other than disulfide bonds (J. Cell Biol. 180, 1071-1081). We have now shown that polymeric TG contains a large number of N(epsilon)(gamma-glutamyl)lysine cross-links and that only traces of these can be found in the soluble form of TG. Because such isopeptide bridges are generated usually by the action of a transglutaminase, it is reasonable to propose that the covalent polymerization of TG in the globules is under the control of this enzyme. Soluble TG was shown to be a substrate for transglutaminase in vitro; moreover, the presence of transglutaminase was demonstrated by immunofluorescence and by immunoblotting in freshly isolated bovine thyroid globules. With immunoelectron microscopy, transglutaminase was detected in the cytoplasm of thyrocytes, but not in compartments of the secretory pathway. Only one messenger RNA for transglutaminase was found by Northern blotting. Sequencing of the cloned gene failed to reveal a secretory signal, which supports the notion that the thyroid transglutaminase is the cytosolic type. Apparently, the enzyme reaches the lumen of the follicle by an as yet unknown pathway to catalyze the covalent cross-linking of thyroid globules in this extracellular compartment.     

Dissociation of the receptor for immunoglobulin E in mild detergents

We previously showed that, in the absence of phospholipids, exposure of the tetrameric receptor for immunoglobulin E to mild detergents dissociates the intact beta chain and two gamma chains from the alpha chains. Having developed a practical method for assaying the dissociation, we have now explored a variety of different detergents, detergent concentrations, temperatures, times, salts, pHs, and other factors that influence the detergent-induced dissociation. Our findings should be useful for optimizing the stability of the receptor and for future studies on recombination of the subunits. The data suggest the following: (1) The critical perturbant is micellar detergent. (2) Unlike solubilization of membranes, where a molar ratio of micellar detergent:lipid of 2 is adequate, dissociation of the receptor is incomplete even at molar ratios of micellar detergent:receptor of greater than 10(5) and may be limited by a reversible component. (3) Detergents that are best for solubilizing membranes are also best for dissociating the receptors. (4) The latter observation and other data implicate bound lipid as stabilizing the receptor. Our findings may be applicable to the study of interactions between membrane proteins in general.     

Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by alpha-neurotoxin cross-linking

[3H]Methyl-alpha-neurotoxin prereacted with dithiobis(succinimidyl propionate) (DTSP) can be covalently linked to each of the subunits of the nicotinic acetylcholine receptor in membranes from the electric tissue of Torpedo californica. Pronounced changes in the cross-linking pattern are observed upon prior incubation with receptor specific ligands and upon reduction and/or alkylation of the receptor. d-Tubocurarine has been shown to bind to two different sites in receptor-rich membranes. These sites are present in equal numbers but have different affinities [Neubig, R. R., & Cohen, J. B. (1979) Biochemistry 18, 5464-5475; Sine, S., & Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699]. Using d-tubocurarine inhibition of [3H]-methyl-alpha-neurotoxin binding, we demonstrate two inhibitory constants for d-tubocurarine of 67 +/- 21 nM and 4.9 +/- 1.7 microM in unreduced membranes. We utilize the large difference in Ki's to preferentially block toxin cross-linking at the high affinity site for d-tubocurarine. Low concentrations of this competitive antagonist selectively block the cross-linking of toxin to the beta and gamma subunits of the receptor, suggesting that these subunits are located close to the toxin binding site which is also the high-affinity binding site for d-tubocurarine. Reduction of disulfide bonds alters the affinity of the receptor for alpha-neurotoxin. Alterations are also seen in the cross-linking pattern of DTSP-activated [3H]methyl-alpha-neurotoxin to reduced and alkylated membranes in the presence of tubocurarine. The constants for d-tubocurarine inhibition of [3H]methyl-alpha-neurotoxin binding to reduced and alkylated membranes are 172 +/- 52 nM and 2.4 +/- 0.4 microM. The effects of bromoacetylcholine, carbamoylcholine, gallamine, and procaine on the cross-linking pattern are also examined. Our observations are consistent with an arrangement of the subunits in the membrane of alpha beta alpha gamma delta.     

A previously unrecognized subunit of the receptor for immunoglobulin E

Our laboratory previously found that under conditions that stabilized the interaction between the alpha and beta subunits of the receptor for immunoglobulin E, two new components were recovered having apparent molecular weights of 45 000 and 20 000, respectively. In this paper, we characterize the 20-kDa material. We demonstrate that it consists of a disulfide-linked dimer of 10-kDa polypeptides and that these have all the characteristics expected for subunits of the receptor. We propose that they be termed gamma chains and that the receptor consists of four chains: one alpha, one beta, and two gamma chains. The gamma chains share many of the labeling properties of the beta chain and, like the latter, are likely to be embedded in the plasma membrane and exposed on the internal but not the external surface of the bilayer.     

Affinity improvement of the high-affinity immunoglobulin E receptor by phage display

The immunoglobulin E (IgE)-binding site of its high-affinity receptor is localized in the second immunoglobulin-like domain (D2) of the alpha-subunit (Fc epsilon RI alpha). In this study, the randomized pentapeptides were introduced between Glu(132) and Ile(138) of Fc epsilon RI alpha D2 and displayed on a filamentous phage. After eight rounds of panning, a phage clone having a mutation of Asp(135)Tyr(136)Met(137) in Fc epsilon RI alpha D2 was obtained. The binding affinity of the mutant phages to immobilized IgE was approximately 500 times higher than that of the wild type. The mutant phages competitively inhibited the binding of IgE to the soluble receptor at a 50% inhibition (IC(50)) value of 116 pM. The mutant Fc epsilon RI alpha D2, which had been expressed as a fusion protein with glutathione S-transferase in Escherichia coli, also showed higher IgE-binding capacity than the wild type. The mutant Fc epsilon RI alpha D2 is expected to manifest its improved IgE-binding affinity together with any fusion partner.     

Molecular structure of human lymphocyte receptor for immunoglobulin E

We have isolated and sequenced a cDNA clone encoding the human lymphocyte receptor for IgE (Fc epsilon R). The deduced protein sequence reveals that Fc epsilon R consists of 321 amino acids, without any signal sequence, and is oriented with its N-terminus on the cytoplasmic side and its C-terminus on the outside of the cell. This molecule shows striking sequence homology with chicken asialoglycoprotein receptor (hepatic lectin), suggesting a possible role for Fc epsilon R in endocytosis. Fc epsilon R mRNA is expressed in B cells, B cell lines, and macrophage cell lines. It is not expressed in T cells or T cell lines, with the exception of an HTLV-transformed T cell line. mRNAs expressed in a macrophage line and in the latter T cell line differ in size from mRNA expressed in B cells. Human BSF-1 (or IL-4) induces the expression of Fc epsilon R mRNA in B cells, but not in T cells.     

Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.     

Covalent cross-linking of proteins without chemical reagents

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.