Showing papers on "Protease-activated receptor 2 published in 1990"

Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor.

Abstract: Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, we obtained a homologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells--i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. We therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the alpha and beta subunits of the GM-CSF receptor, respectively.

Coiled-coil fibrous domains mediate ligand binding by macrophage scavenger receptor type II

Abstract: THE macrophage scavenger receptor, which has been implicated in the pathogenesis of atherosclerosis1,2, has an unusually broad binding specificity1. Ligands include modified low-density lipoprotein and some polyanions (for example, poly(I) but not poly(C)). The scavenger receptor type I (ref. 3) has three principal extracellular domains that could participate in ligand binding: two fibrous coiled-coil domains (α-helical coiled-coil domain IV and collagen-like domain V), and the 110-amino-acid cysteine-rich C-terminal domain VI. We have cloned complementary DNAs encoding a second scavenger receptor which we have termed type II. This receptor is identical to the type I receptor, except that the cysteine-rich domain is replaced by a six-residue C terminus. Despite this truncation, the type II receptor mediates endocytosis of chemically modified low-density lipoprotein with high affinity and specificity, similar to that of the type I receptor. Therefore one or both of the extracellular fibrous domains are responsible for the unusual ligand-binding specificity of the receptor.

The 5HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors.

Abstract: Serotonin exerts its diverse physiological effects by interacting with multiple distinct receptor subtypes. We have isolated a rat brain 5HT2 serotonin receptor cDNA by virtue of its homology with the 5HT1c receptor. The 5HT2 receptor is a member of the family of receptors that are linked to guanine nucleotide-binding proteins and are predicted to span the lipid bilayer seven times. Overall sequence identity between the 5HT2 and 5HT1c receptors is 49%, but identity within the transmembrane domains is 80%. Expression of both the 5HT2 and 5HT1c receptors in transfected mouse fibroblasts activates phospholipase C signaling pathways and promotes cellular transformation. However, RNA blotting shows that these two receptor subtypes are differentially expressed in the central nervous system. In this manner, structurally and functionally homologous receptor subtypes may elicit distinct physiologic actions.

Three different mRNAs encoding human granulocyte colony-stimulating factor receptor.

Abstract: Three cDNAs for the human granulocyte colony-stimulating factor (G-CSF) receptor were isolated from the cDNA libraries of human U937 leukemia cells and placenta by using a murine G-CSF receptor cDNA as the probe. The human G-CSF receptor containing 813 amino acids had a marked homology (62.5%) with its murine counterpart and consisted of extracellular, transmembrane, and cytoplasmic domains. The WSXWS motif found in members of the newly identified growth factor receptor family was also present in the extracellular domain of the human G-CSF receptor. Expression of the cloned cDNA in monkey COS cells gave rise to a protein that could specifically bind G-CSF with a high affinity (Kd, 550 pM). Two other classes of the human G-CSF receptor were also identified, one of which had a deletion of the transmembrane domain and seemed to encode a secreted, soluble receptor. The third class of the G-CSF receptor contained a 27-amino acid insertion in the cytoplasmic domain and was highly expressed in placenta.

Cloning, expression, and pharmacological characterization of a human alpha 2B-adrenergic receptor.

Abstract: An alpha 2-adrenergic receptor subtype has been isolated from a human genomic spleen library using the human 5-hydroxytryptamine1A receptor gene (also known as G-21) as a probe. This adrenergic receptor gene encodes a protein of 450 amino acids and does not contain any consensus sequences for N-linked glycosylation in its amino terminus or extracellular loops. This receptor is also distinguished by the presence of 12 consecutive glutamic acid residues in the region of its third intracellular loop. The deduced amino acid sequence shows greatest homology to previously cloned human alpha 2-adrenergic receptors and has structural similarities to other guanine nucleotide-binding protein-coupled receptors. The DNA encoding the human alpha 2 receptor was stably transfected into mouse fibroblast Ltk- cells and radioligand binding studies were performed using the alpha 2 antagonist [3H]rauwolscine. [3H]Rauwolscine bound with high affinity (Kd = 0.33 nM) and in a saturable manner (Bmax = 1.4 pmol/mg of protein). Pharmacological characterization of this receptor indicated a rank order of potency of yohimbine greater than prazosin greater than oxymetazoline. Additionally, 100 microM 5'-guanylylimidodiphosphate, produced a rightward shift in the epinephrine competition curve, with resultant increases in both the Ki value and Hill coefficient, suggestive of a functional interaction of the cloned receptor with native guanine nucleotide-binding protein(s) of Ltk- membranes. The data presented here are consistent with previous biochemical and pharmacological studies on alpha 2 receptors and are supportive of the designation of this receptor as an alpha 2B subtype.

Binding of insulin-like growth factor II (IGF-II) by human cation-independent mannose 6-phosphate receptor/IGF-II receptor expressed in receptor-deficient mouse L cells.

Abstract: Mouse L cells deficient in expression of the murine cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor (CI-MPR/IGF-IIR) were stably transfected with a plasmid containing the cDNA for the human receptor. Transfected cells expressed high levels of the human receptor which functioned in the transport of lysosomal enzymes and was capable of binding 125I-IGF-II, both at the cell surface and intracellularly. Cell surface binding of 125I-IGF-II by the receptor could be inhibited by pretreatment of cells with antibodies to the receptor or by coincubation with the lysosomal enzyme, beta-glucuronidase. Expression of the receptor conferred on transfected cells the ability to internalize and degrade 125I-IGF-II. Cells transfected with the parental vector and those expressing the human CI-MRP/IGF-IIR were found to express an atypical binding site for IGF-II that was distinct from the CI-MPR/IGF-IIR and the type I IGF-receptor. The availability of two cell lines, one of which overexpresses the human CI-MPR/IGF-IIR and one deficient in expression of the murine receptor, may help in the analysis of the role of the receptor in mediating the biological effects of IGF-II. They should also be useful in examining the significance of binding of ligands, such as transforming growth factor-beta 1 precursor and proliferin to this receptor.

Insulin binding to its receptor induces a conformational change in the receptor C-terminus.

Abstract: Antibodies against peptides corresponding to sequences in the C-terminus of the insulin receptor beta-subunit were used to approach the putative role of this receptor domain in signal generation. Two sequences were chosen and correspond to peptide C1, comprising amino acids 1309-1326, and peptide C2, comprising amino acids 1294-1317. The two antibodies produced distinct immunoprecipitation patterns as a function of the insulin receptor form and recognized changes in the insulin receptor molecule induced by ligand binding and autophosphorylation. Both antipeptides, anti-C1 and anti-C2, showed an important decrease in their recognition capacity for the receptor occupied by insulin when compared to the empty receptor. Further, anti-C1 had a lower affinity for the phosphorylated receptor compared to the unphosphorylated receptor and failed to recognize a fraction of the phosphoreceptor population. In contrast, anti-C2 had similar affinities for the phosphorylated and unphosphorylated receptors but was unable to interact with part of the unphosphorylated receptors. Finally, using immunoblotting of the receptor to analyze the denatured molecules, we showed that the phosphorylation-induced changes detected by anti-C1 are retained, suggesting that they are likely not of a conformational nature. In contrast, the insulin-induced changes in the receptor molecule disappear with receptor denaturation which points to their reversible nature. We conclude from these data that (i) antipeptides against the receptor C-terminal sequence are able to distinguish between phosphorylated and unphosphorylated receptor forms and (ii) binding of insulin to its receptor leads to a reversible, phosphorylation-independent, and possibly conformational change at the level of the receptor C-terminal domain.

Glucocorticoid receptor binding to calf thymus DNA. 2. Role of a DNA-binding activity factor in receptor heterogeneity and a multistep mechanism of receptor activation.

Abstract: In the preceding paper [Cavanaugh, A H, & Simons, S S, Jr (1990) Biochemistry (preceding paper in this issue)], we characterized an apparently identical factor in the cytosol and the nuclear extract of HTC cells that is required for the DNA binding of approximately 43% of the activated receptor-glucocorticoid complexes In the present study, both those activated complexes that are influenced by this factor and the role of this factor in the process of activation are examined We find that sodium arsenite inhibits only the DNA binding of those complexes that require factor Conversely, methyl methane-thiolsulfonate inhibits the DNA binding of only those complexes that are independent of factor These results provide direct chemical evidence for two populations of activated complexes Double-reciprocal plots revealed that the increase in DNA binding with endogenous factor occurred by recruiting new complexes for DNA binding as opposed to increasing the binding affinity of existing complexes These results further suggest that factor associates only with the receptor-steroid complex and does not additionally interact with DNA A saturable association of factor with complexes was indicated since the amount of available factor in cytosolic solutions decreased after activation of the complexes Sodium molybdate is known to inhibit the activation of HTC cell receptor-steroid complexes When factor was added to complexes that had been subjected to activating conditions in the presence of the inhibitor sodium molybdate, no increased DNA binding was observed(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of the human IFN-gamma receptor expression in Raji and IM9 lymphoblastoid cells versus THP-1 monocytic cells by IFN-gamma and phorbol myristate acetate.

Abstract: Expression of the human (hu) IFN-gamma-R has been studied in Raji and IM9 cells (two B lymphoblastoid cell lines) and in THP-1 cells (a monocytic cell line) with respect to IFN-gamma binding sites, receptor protein and mRNA levels. Although, in these three cell lines, the hu-IFN-gamma-R mRNA was expressed to the same extent, the high affinity receptor was expressed differently both in cell surface receptor binding and amount of receptor protein. Various ligands are able to modulate the expression of their own receptor. We investigated the modulation of the hu-IFN-gamma-R by its ligand. Hu-IFN-gamma induced a rapid and dose-dependent decrease of its cell surface receptor number without alteration of receptor affinity, amounts of receptor protein or hu-IFN-gamma-R mRNA accumulation and stability. Thus, in Raji, IM9, and THP-1 cells, the hu-IFN-gamma had no effect on its receptor gene expression and the cell surface decrease was simply due to ligand blocking and receptor internalization rather than true down-regulation. The second messenger in the hu-IFN-gamma signal transduction pathway is not well characterized, but activation of protein kinase C has been reported in some cases. Therefore, the modulation of the hu-IFN-gamma-R expression by PMA, a potent activator of protein kinase C and a modulator of other receptor expression, has been investigated. In Raji and IM9 cells, PMA had no or few effects on the cell surface receptor number and no detectable effect on the receptor protein or on mRNA levels. In contrast, in THP-1 cells, PMA treatment induced a time and dose-dependent five- to sixfold increase of the cell surface receptors due to a rapid and persistent increase of the hu-IFN-gamma-R gene expression in THP-1 cells was specifically inhibited or reversed by hu-IFN-gamma treatment. The modulation of the hu-IFN-gamma-R expression by PMA in THP-1 cells and by hu-IFN-gamma in PMA-treated THP-1 cells seems associated with their effect on monocyte-macrophage differentiation and/or macrophage activation.

Modulation of mannose receptor activity by proteolysis.

Abstract: Macrophages express a receptor on the cell surface that functions to clear glycoproteins from the extracellular milieu. The activity of this receptor is sensitive to treatment with trypsin. In inflammatory situations, macrophages are activated and exposed to increased levels of extracellular proteases. Under these conditions, mannose receptor activity on the macrophages is diminished. We therefore decided to study the effects of trypsin treatment on the structure and activity of cell-associated and purified receptor that might contribute to the activation-associated receptor down-regulation. Trypsin treatment (1 microgram/ml for 3 h) resulted in the production of a 140 kDa, trypsin-resistant fragment from both intact cells and isolated receptor. This fragment was no longer able to bind ligand. The remaining 35 kDa fragment apparently is further degraded into smaller fragments, since no evidence of this domain was found on Coomassie Blue-stained gels. The 140 kDa fragment retained immunoreactivity and contained at least a portion of the iodinated tyrosine residues following surface labelling with Na125I. Neither calcium nor ligand protected the receptor from proteolysis. In addition, prior treatment with oxidants did not increase the susceptibility of the receptor to trypsin digestion. We conclude from these results that the macrophage mannose receptor is clipped by the serine protease trypsin at the cell surface, resulting in the release and further degradation of the binding domain, and the production of a membrane-associated 140 kDa fragment. This trypsin-mediated down-regulation of receptor activity might be important in controlling glycoprotein clearance during inflammation.

Second messenger modulation of epidermal growth factor receptor function does not occur at the level of receptor dimerization.

Abstract: Epidermal growth factor (EGF)-induced receptor dimerization may provide a mechanism for activation of the receptor protein tyrosine kinase and for initiation of post-receptor signalling pathways. We have examined whether second messengers and agents that modulate EGF receptor function act at the level of receptor dimerization. Both the Ca2+ ionophore ionomycin and the tumour promotor tetradecanoylphorbol acetate (TPA), added shortly before EGF, inhibit EGF receptor protein tyrosine kinase activity in intact cells. In permeabilized cells, elevation of Ca2+ similarly inhibits EGF receptor function. The inhibitory effect of Ca2+, unlike that of TPA, appears not to be dependent on protein kinase C activity. Neither ionomycin nor phorbol ester affects EGF-induced receptor dimerization, as shown by cross-linking and immunoblotting techniques, although the phosphotyrosine content of both monomeric and dimeric receptors is strongly decreased. Furthermore, we show that EGF receptor dimerization is not affected by increases in cyclic AMP or intracellular pH, nor by changes in transmembrane potential, medium osmolarity or the glycosylation state of the receptor. These result suggest that modulation of EGF receptor function occurs at a step other than receptor dimerization.

[35] Receptor identification

Abstract: Publisher Summary This chapter focuses on the methods of receptor identification for receptors localized on the cell surface. Receptors play a key role in regulating cellular processes by enabling the cell to interact with the outside environment. A receptor is a molecular entity on the target tissue that, when occupied by an agonist, initiates a biological response.Purification of a protein is an essential step if one has to determine its amino acid sequence by traditional methods. Receptor proteins are, however, large, poorly soluble in aqueous media, and partially buried in the lipid environment of the cell membrane. Hence, the first step in purifying a receptor protein involves solubilization of the membrane receptor as described in the chapter , solubilization of receptors. When multiple sources of tissues are available for receptor purification, their specific binding capacities may be used as a guide to choosing the tissue most likely to provide the best yield of receptor.

Colony-Stimulating Factor 1 Receptor

Abstract: Colony stimulating factors (CSFs) regulate the development of granulocytes and macrophages from hemopoietic precursor cells (Stanley and Jubinsky, 1984; Metcalf, 1986; Clark and Kamen, 1987). One of these growth factors, CSF-1, is specifically required for the in vitro survival, proliferation, and differentiation of mononuclear phagocytes (precursor cell-monoblast->promonocyte->monocyte-macrophage). The response to CSF-1 is pleiotropic and varies with mononuclear phagocyte cell type (Stanley et al., 1983). The actions of CSF-1 are mediated via a specific receptor that is selectively expressed on all mononuclear phagocytes and their precursors (Guilbert and Stanley, 1980; Byrne et al., 1981) and tropho-blastic cells (Rettenmier et al., 1986). The CSF-1 receptor has been shown immunologically and functionally identical to the c-fms proto-oncogene product (Sherr et al., 1985; Sacca et a1.,1986; Roussel et al., 1987) that belongs, with the platelet-derived growth factor receptor, to a subclass of tyrosine receptor kinases (Coussens et al., 1986; Yarden and Ullrich, 1988). Chemical crosslinking studies (Morgan and Stanley, 1984) indicated that the cell surface CSF-1 receptor is a macromolecule of approximately 165 kDa and is not disulfide bonded to any other macromolecule in the macrophage membrane. In this chapter we describe a two-step method that was initially developed in 1983 for the purification of the CSF-1 receptor from a CSF-1 independent, receptor bearing cell line.

Protease resistance of the beta subunit of the hamster fibronectin receptor. Evidence for differential cleavage of membrane-bound and soluble receptor.

Abstract: The structural stability of the hamster fibronectin receptor has been studied using limited proteolytic digestion and anti-(fibronectin receptor) monoclonal antibodies of known specificity. Treatment of the solubilized intact receptor or of the dissociated alpha and beta chains with any one of several proteases generated large protease-resistant fragments (92-110 kDa). Western blot analysis of tryptic digests using subunit-specific monoclonal antibodies revealed the large trypsin-generated fragment to be of beta-subunit origin. No products from the alpha subunit were detected. The protease-resistant fragment is lost upon exposure to reducing conditions; thus, the highly disulfide-bonded region of the beta subunit is important in the maintenance of the tertiary structure of the entire subunit. In contrast to solubilized fibronectin receptor, membrane-bound receptor is much more stable to proteolysis, and tryptic cleavage results in two large immunoreactive fragments of approximately 100 kDa and 95 kDa. This suggests a difference in the conformation and/or oligomeric organization of the membrane-bound receptor as compared with the solubilized heterodimeric receptor.