Receptor activation by human C5a des Arg74 but not intact C5a is dependent on an interaction between Glu199 of the receptor and Lys68 of the ligand.

Despite the expression of only one type of receptor, there is great variation in the ability of different cell types to discriminate between C5a and its more stable metabolite, C5a des Arg74. The mechanism that underlies this phenomenon is not understood but presumably involves differences in the interaction with the C5a receptor. In this paper, we have analyzed the effects of a substitution mutation of the receptor (Glu199 --> Lys199) and the corresponding reciprocal mutants (Lys68 --> Glu68) of C5a, C5a des Arg74 and peptide analogues of the C-terminus of C5a on the ability of the C5a receptor to discriminate between ligands with and without Arg74. The use of these mutants indicates that the Lys68/Glu199 interaction is essential for activation of receptor by C5a des Arg74 but not for activation by intact C5a. The substitution of Asp for Arg74 of C5a [Lys68] produces a ligand with equal potency on both the wild-type and mutant receptors, suggesting that it is the C-terminal carboxyl group rather than the side chain of Arg74 that controls the responsiveness of the receptor to Lys68. In contrast, the mutation of Lys68 to Glu(68) has little effect on the ability of either C5a or C5a des Arg(74) to displace [(125)I]C5a from the receptors, indicating that binding of ligand and receptor activation are distinct but interdependent events. C5a and the truncated ligand, C5a des Arg74, appear to have different modes of interaction with the receptor and the ability of the human C5a receptor to discriminate between these ligands is at least partly dependent on an interaction with the receptor residue, Glu199.     

Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.     

Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes

We have developed a reverse-phase HPLC method to purify 125I-labeled products resulting from the chloramine-T-based iodination of glucagon and have used the products [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon) to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. The extent of binding of the three labeled glucagons to cell receptors differed at steady state (8.5, 11.9, and 12.6% of the three peptides, respectively, becoming cell-associated), but each of the labeled glucagons approached steady state binding at the same rate. Further, unlabeled glucagon competed for the binding of each of the labeled peptides in parallel under steady state conditions, and each of the peptides showed potent activity in inhibiting [14C]fructose incorporation into glycogen. Gel filtration of the acetic acid-extracted, cell-associated products of radiolabeled glucagon binding revealed 10-20% of the material as a shoulder on the descending limb of the peak of hormone for each of the three labeled peptides. Trypsin digestion of the lower molecular weight peptide derived from [(125I)iodoTyr13]glucagon resulted in a fragment containing residues 13 to 17 as the only detectable radiolabeled product. On the other hand, trypsin digestion of the analogous peptide derived from [(125I)iodoTyr10]glucagon revealed, in addition to the radiolabeled fragment containing residues 1 to 12, a major fragment identified by radiosequence analysis to contain residues 4 to 12 and a minor fragment identified to contain residues 7 to 12. We conclude that (a) notwithstanding apparent differences in affinities exhibited by [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon for binding to canine hepatocytes, the interactions of all three peptides with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH2-terminal region of the hormone has been modified by limited proteolytic cleavage.     

The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.     

A direct in vivo comparison of the inflammatory properties of human C5a and C5a des Arg in human skin

C5a is an 11,000-Da complement-derived inflammatory glycoprotein that has been shown to mediate inflammatory reactions in vitro as well as in vivo in human skin. The C5a degradation product, C5a des Arg, is rapidly formed after exposure of C5a to serum carboxypeptidase N and may represent the relevant C5-derived inflammatory peptide in vivo. To examine the biologic activity of human C5a des Arg in vivo and to compare it with that seen with human C5a, we purified and characterized homogeneous preparations of human C5a and C5a des Arg and injected them intradermally into seven normal volunteers. C5a des Arg exhibited biochemical and biologic properties in vitro that were different from those of C5a. When injected into human skin, C5a des Arg was less potent than C5a, in respect to both minimal dose eliciting wheal and flare reactions and maximal wheal and flare elicited at a given dose, but C5a des Arg still elicited cutaneous wheal and flare reactions at physiologically relevant concentrations. Histologically, C5a des Arg skin test sites showed dense polymorphonuclear neutrophil-rich infiltrates associated with leukocytoclasis, dermal mast cell degranulation, and endothelial cell swelling. These were virtually indistinguishable from reactions elicited by C5a and occurred with concentrations attainable in vivo. Cutaneous wheal and flare reactions elicited by either C5a or C5a des Arg were partially inhibited by H1 antihistamines but were unaffected by selected nonsteroidal anti-inflammatory agents.     

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites (Ile(116), Arg(175,) Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-d-cyclohexylalanine-cyclohexylalanine-d-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 (peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-d-cyclohexylalanine-Trp-Arg] (peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.     

Discrimination of galanin receptor subtypes in RINm5F cells by structurally different galanin radioligands

Galanin (GAL) is a biologically active peptide that is involved in a variety of physiological functions. The purpose of this study was to evaluate whether porcine and rat galanin radioligands could be used as probes to discriminate GAL receptors (GALR) subtypes using a cell line, RINm5F, that express multiple GALR subtypes. Data from parallel equilibrium binding experiments using the same RINm5F membrane homogenates reveal that [125I]pGAL labels 20% more GALRs with a 2-fold lower affinity than those values identified when using [125I]rGAL. Competition studies using various GAL peptides showed different rank order of potencies depending on the radioligand used. Preincubation of RINm5F membranes with GppNHp, a non-hydrolizable GTP analog, prior to radioligand labeling suggests that a portion of GALRs is precoupled to G proteins. In addition, receptors labeled by [125I]rGAL appear more sensitive to GppNHp-induced uncoupling of G proteins than those labeled by [125I]pGAL. In conclusion, our data suggest that pGAL and rGAL radioligands define different pharmacological profiles of GALRs, and hence, these ligands can be used as pharmacological tools to discriminate GALR subtypes. Additionally, our data suggests that GALRs exist in a precoupled state with their respective G-proteins prior to interaction with the agonist.     

Complement C5a Receptor Assay for High Throughput Screening

Abstract

The complement C5a receptor on U937 cells, a human histiocytic lymphoma cell line, stimulated with dibutyryl-cAMP have been stabilized for at least 3 months at a diluce, ready to use concentration. [¹²⁵I]-Bolton Hunter labeled C5a, (recombinant, human) has been prepared by reverse phase HPLC to 2200 Ci/mmol. Using a filtration binding assay the Kd from receptor saturation analysis is 10–40 pM and there are 50,000–100,000 receptor sites per cell. These reagents have permitted the development of a reliable, reproducible and convenient drug screening assay, in kit format, for compounds acting at the C5a receptor.     

Human C5a and C5a des Arg exhibit chemotactic activity for fibroblasts

C5a and C5a des Arg are chemotactic factors for inflammatory cells but it is not known whether these agents are chemoattractants for fibroblasts. Accordingly, C5a, purified from zymosan-activated human, and C5a des Arg, prepared by incubating C5a with immobilized porcine carboxypeptidase B, were studied for fibroblast chemotactic activity. We observed that both C5a and C5a des Arg stimulated human skin fibroblasts and fetal bovine ligament fibroblasts to migrate in a concentration-dependent fashion, and that the migratory responses were similar in magnitude to the responses achieved with optimal concentrations of two known fibroblast chemoattractants, platelet-derived growth factor and human fibrinopeptide B. The peak responses to C5a and C5a des Arg occurred at approximately 10(-9)M. With ligament fibroblasts, there was a greater response to C5a des Arg than to C5a, but with human fibroblasts there was no difference. Cochemotaxin, which enhances the chemotactic activity of C5a des Arg for neutrophils, had no effect on C5a des Arg fibroblast chemotactic activity but appeared to increase the fibroblast chemotactic activity of C5a. These results indicate that the effects of C5a and C5a des Arg in vivo may extend to the recruitment of mesenchymal cells. Moreover, the findings represent another example of an activity retained by C5a after removal of its carboxyl terminal arginine.     

Comparative affinities of adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) for [125I] AM and [125I] CGRP specific binding sites in porcine tissues.

We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM > or = human AM > or = human AM(22-52) > h alpha CGRP > or = h alpha CGRP(8-37) > sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20-300 fold more potent than hAM (22-52). When the same experiments were performed using [125I] h alpha CGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > h alpha CGRP > h alpha CGRP(8-37) in most of the tissues except in spleen and liver where h alpha CGRP was the most potent ligand. In lung, h alpha CGRP was almost as potent as rAM and hAM in displacing [125I] h alpha CGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.     

Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN)

Elucidation of the interactions between C5a and granulocytes is central to understanding the role of C5a in inflammation. In this study, interactions between C5a and PMN have been studied at two levels. Binding of human C5a to intact human cells has been characterized by using the radiolabeled ligand 125I-C5a. Binding is shown to be reversible, saturable, and to reach equilibrium in 60 to 90 min at 0 degrees C. Results show high affinity C5a binding sites with Kd = 2 X 10(-9) M and a range of 50,000 to 113,000 binding sites per PMN. These values for C5a receptors are comparable with the number of fMLP and LTB4 receptors on PMN. Binding of C5a to PMN fails to reach equilibrium at 37 degrees C because there is an irreversible loss of available surface receptors caused by an active internalization of the ligand-receptor complex. Interactions between C5a and human PMN were characterized further by cross-linking experiments, with the use of ethylene glycol bis succinimidylsuccinate (EGS). Cross-linking of 125I-C5a to intact PMN followed by subcellular fractionation revealed a single radioactive band present only in the plasma membrane fraction and visualized by autoradiography. Similar experiments resulted in a covalent linkage between 125I-C5a and a component in the isolated plasma membrane of PMN. The covalent complex containing C5a and a putative receptor has been visualized by autoradiography as a single 60,000 Mr complex on SDS-PAGE. The complex is not present when experiments are performed in the presence of excess unlabeled C5a or in the absence of EGS. Therefore, the putative receptor for C5a on human neutrophils is estimated to be approximately 48,000 Mr, assuming contribution of 12,000 to 13,000 daltons by the ligand 125I-C5a.     

A derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors

We examined the mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of [3H]-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 microM FMLP for 10 min at 4 degrees C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using [125I]-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. PMN did internalize [125I]-WGA-D, however, when stimulated with FMLP. Internalization of WGA-D by FMLP-stimulated PMN was rapid, dependent on the concentration of FMLP, and specific. Internalization of [125I]-WGA-D by PMN did not occur when highly purified human C5a, instead of FMLP, was used as a stimulus. Subcellular fractionation studies demonstrated that [125I]-WGA-D and [3H]-FMLP were co-internalized by PMN, and segregated to a compartment co-migrating with Golgi markers. Western blot analysis, using PMN plasma membranes, demonstrated that WGA-D bound to a single membrane glycoprotein that migrated with an apparent m.w. of 62,000. The data indicate that WGA-D, perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking the re-expression (or recycling) of a population of receptors required for continuous migration.     

Chemotactic peptides modulate adherence of human polymorphonuclear leukocytes to monolayers of cultured endothelial cells

We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.0 nM), and FMLP (1.0 nM) significantly reduced the percentage of PMN that adhered to endothelial monolayers. Adherence also was reduced by C5a des Arg that was generated by incubating (37 degrees C, 30 min) fresh human serum with either zymosan or purified C5a. High concentrations of C5a (greater than 1.0 nM) and FMLP (greater than 50 nM) that diminished PMN chemotaxis significantly enhanced the percentage of PMN that adhered tightly to endothelial cells (adherent cells resisted a dislodgment force of 1200 X G). Tight adherence of PMN to endothelial cells also was increased by high concentrations of C5a that were added to human serum in which carboxypeptidase N activity was destroyed by heating (56 degrees C, 30 min), and by C5a that was generated by incubating (37 degrees C, 30 min) fresh human serum with zymosan in the presence of the carboxypeptidase N inhibitor, epsilon-aminocaproic acid. High concentrations of C5a des Arg (up to 80 nM) neither enhanced adherence of PMN to endothelial cells nor decreased PMN migration. Thus, a reciprocal relation exists between PMN migration and PMN adherence to endothelial cells in response to chemotactic factors. At concentrations that are chemotactic for human PMN, C5-derived peptides and FMLP reduce the adherence of PMN to endothelial monolayers. Only at concentrations that decrease PMN migration do C5a and FMLP augment PMN adherence.     

The chemoattractant des-Arg74-C5a regulates the expression of its own receptor on a monocyte-like cell line.

The interaction of the chemoattractant des-Arg74-C5a (C5a des Arg) with its receptor on a human monocyte-like cell line, U-937, was examined. The data obtained suggest that C5a des Arg receptor expression is regulated by the extracellular concentration of C5a des Arg itself.     

Identification of a chemotactic epitope in human transforming growth factor-β1 spanning amino acid residues 368–374

TGF-b?1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-b?1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-b?1 that contained residues 368–374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-b?1 monomer were able to compete with [¹²⁵I]hrTGF-b?1 for binding to TGF-b? cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed. © 1995 Wiley-Liss, Inc.     

Comparative Affinities of Adrenomedullin (AM) and Calcitonin Gene-Related Peptide (CGRP) for [125I] AM and [125I] CGRP Specific Binding Sites in Porcine Tissues

Abstract

We have investigated the binding characteristics of rat [¹²⁵I] adrenomedullin (AM) and human [¹²⁵I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [¹²⁵I] rat AM and [¹²⁵I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [¹²⁵I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [¹²⁵I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [¹²⁵I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [¹²⁵I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

The product of a novel growth factor-activated gene, fic, is a biologically active "C-C"-type cytokine.

We have characterized a new member of the superfamily of proinflammatory peptides encoded by a growth factor-inducible gene, fic, previously isolated by differential screening of a cDNA library of mRNA from serum-stimulated NIH 3T3 cells. Immunoprecipitation analyses showed that the protein was rapidly induced following serum stimulation and secreted unglycosylated into the medium. The fic protein, FIC, shows highest sequence homology (57%) to human and rabbit monocyte chemoattractant protein 1 (MCP-1), an established monocyte activator. To determine the biological activity of FIC and to compare it with that of mouse MCP-1 (muMCP-1), both proteins were expressed in the baculovirus system. FIC and muMCP-1 were purified to near homogeneity by a two-step chromatography protocol. Both proteins elicited changes in intracellular calcium concentration in human monocytes. The effect was dependent on external Ca2+ and was inhibited by pretreatment of cells with pertussis toxin. FIC did not desensitize human monocytes to the three related cytokines muMCP-1, human MCP-1 (huMCP-1), and huMCP-2. However, pretreatment with muMCP-1 or huMCP-1, but not with huMCP-2, desensitized human monocytes to FIC. Specific binding of [125I]FIC was found in human monocytes, mouse monocytic cultured cells, and human endothelial cells but not in lymphocytes, neutrophils, or primary mouse fibroblasts. Scatchard analysis of the binding of [125I]FIC to human monocytes showed the presence of two classes of receptors, with apparent KdS of 1.2 and 7.7 nM and receptor numbers per cell of 2,400 and 6,300, respectively. FIC, muMCP-1, and huMCP-1 competed to the same extent for the binding of [125I]FIC to human monocytes, contrary to huMCP-2, which competed very ineffectively, if at all.