Synthesis and biological characterization of sqbazide, a novel biotinylated photoaffinity probe for the study of the human platelet thromboxane A2 receptor

SQBAzide, a biotinylated, azido derivative of the TXA2 receptor antagonist, SQ31,491, was synthesized and characterized. The compound specifically inhibited human platelet aggregation mediated by TXA2 receptor activation and irreversibly labeled platelet TXA2 receptors upon exposure to ultraviolet light. This probe should prove to be of significant value for the study of the receptor-ligand binding domain.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2 recognition

The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.     

Structural and functional characterization of a novel type of ligand-independent RXR-USP receptor

Retinoid X receptor (RXR) and Ultraspiracle (USP) play a central role as ubiquitous heterodimerization partners of many nuclear receptors. While it has long been accepted that a wide range of ligands can activate vertebrate/mollusc RXRs, the existence and necessity of specific endogenous ligands activating RXR-USP in vivo is still matter of intense debate. Here we report the existence of a novel type of RXR-USP with a ligand-independent functional conformation. Our studies involved Tribolium USP (TcUSP) as representative of most arthropod RXR-USPs, with high sequence homology to vertebrate/mollusc RXRs. The crystal structure of the ligand-binding domain of TcUSP was solved in the context of the functional heterodimer with the ecdysone receptor (EcR). While EcR exhibits a canonical ligand-bound conformation, USP adopts an original apo structure. Our functional data demonstrate that TcUSP is a constitutively silent partner of EcR, and that none of the RXR ligands can bind and activate TcUSP. These findings together with a phylogenetic analysis suggest that RXR-USPs have undergone remarkable functional shifts during evolution and give insight into receptor-ligand binding evolution and dynamics.     

Expression, purification, and characterization of the active immunoglobulin-like domain of human granulocyte-colony-stimulating factor receptor in Escherichia coli

We succeeded in the expression, purification, and refolding of the immunoglobulin-like (Ig) domain of human granulocyte-colony-stimulating factor (G-CSF) receptor with amino-terminal His-tag in Escherichia coli. The refolded Ig domain bound to a G-CSF affinity column and could be eluted with free G-CSF as a receptor-ligand complex, demonstrating that the Ig domain has the information necessary for binding its ligand, G-CSF. The eluted His-Ig/G-CSF complex could be separated from excess G-CSF by Ni-NTA column chromatography. The yield of this active recombinant His-Ig protein is about 0.72 mg per liter of culture. Its small size and the ease of production make this receptor fragment a useful reagent for the structural analysis of its complex with G-CSF.     

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties

Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N-terminal extracellular moiety of the receptor and (iii) to set up a fluorescence-based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N-terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino-acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy-pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor-ligand complexes, relative donor (EGFP)-acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino-terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence-based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.     

The glycan domain of thrombopoietin enhances its secretion

Thrombopoietin (TPO) is the major regulator of megakaryocyte development and platelet production. The hormone is structurally characterized by an amino-terminal receptor binding domain (amino acid residues 1-152) predicted to encode a left-handed four-helix bundle structure, and a carboxyl-terminal domain (residues 153-335) that is remarkable for its abundant carbohydrate modification and a lack of homology to other proteins. To investigate the functional role of the carboxyl-terminal glycan domain, we generated truncated forms of murine TPO (TPO1-238, TPO1-174, and TPO1-152) and glycomuteins in which the predicted asparagine (N)-linked sites of glycosylation were sequentially mutated to glutamine (Q), and assayed their secretion and function by comparing them to the native sequence (TPO1-335). Following transient transfection of the corresponding cDNA expression vectors into mammalian cell lines, the secretory efficiencies of the proteins were compared with those of the native hormone. Transfection efficiencies were monitored by cotransfection and reporter gene assay, and TPO secretion was assessed by functional and immunologic assays. We found that full-length TPO was 5-29-fold more efficiently secreted than any of the truncated forms of the hormone in fibroblast and hepatocyte cell lines. Elimination of carboxyl-terminal sites of N-linked glycosylation had a minor impact on secretion of the protein. We conclude that the carboxyl-terminal domain of TPO serves the important role of enhancing secretion of the protein, and in this manner functions as a prosequence.     

Misfolding of vWF to Pathologically Disordered Conformations Impacts the Severity of von Willebrand Disease

The primary hemostatic von Willebrand factor (vWF) functions to sequester platelets from rheological blood flow and mediates their adhesion to damaged subendothelium at sites of vascular injury. We have surveyed the effect of 16 disease-causing mutations identified in patients diagnosed with the bleeding diathesis disorder, von Willebrand disease (vWD), on the structure and rheology of vWF A1 domain adhesiveness to the platelet GPIbα receptor. These mutations have a dynamic phenotypical range of bleeding from lack of platelet adhesion to severe thrombocytopenia. Using new rheological tools in combination with classical thermodynamic, biophysical, and spectroscopic metrics, we establish a high propensity of the A1 domain to misfold to pathological molten globule conformations that differentially alter the strength of platelet adhesion under shear flow. Rheodynamic analysis establishes a quantitative rank order between shear-rate-dependent platelet-translocation pause times that linearly correlate with clinically reported measures of patient platelet counts and the severity of thrombocytopenia. These results suggest that specific secondary structure elements remaining in these pathological conformations of the A1 domain regulate GPIbα binding and the strength of vWF-platelet interactions, which affects the vWD functional phenotype and the severity of thrombocytopenia.     

Characterization of HasB, a Serratia marcescens TonB-like protein specifically involved in the haemophore-dependent haem acquisition system

In Gram-negative bacteria, the TonB-ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC-ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC-dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.     

Evaluation of the role of electrostatic residues in human epidermal growth factor by site-directed mutagenesis and chemical modification.

Four residues in the carboxy-terminal domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site-directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor-ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge-conservative replacement of glutamate 40 with aspartate resulted in a decrease in receptor binding affinity to 30% relative to wild-type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitution of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side-chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274-2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor-ligand association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of porcine brain alpha 2-adrenergic receptors by Na+,H+ and inhibitors of Na+/H+ exchange

Previous reports from this laboratory have demonstrated that alpha 2-adrenergic receptors accelerate Na+/H+ exchange in NG108-15 neuroblastoma X glioma cells and evoke platelet secretion via a pathway involving Na+/H+ exchange. The present studies were designed to examine whether agents that interact with Na+/H+ antiporters also might influence alpha 2-adrenergic receptor-ligand interactions. We observed that Na+ decreases receptor affinity for the agonists epinephrine, norepinephrine, and UK14304 and slightly increases receptor affinity for the antagonists yohimbine and idazoxan in digitonin-solubilized preparations from porcine brain cortex. Increases in [H+] also decrease receptor affinity for agonists and cause either a slight increase or no change in receptor affinity for antagonists. Amiloride analogs accelerate the rate of [3H] yohimbine dissociation from digitonin-solubilized receptors with a relative effectiveness that parallels their ability to block Na+/H+ exchange in other systems. Interestingly, these modulatory effects of Na+,H+ and 5-amino-substituted analogs of amiloride are retained in homogeneous preparations of the alpha 2-adrenergic receptor, suggesting that the allosteric-binding sites for these agents are on the receptor-binding protein itself.     

Mechanical modulation of receptor-ligand interactions at cell-cell interfaces

Cell surface receptors have been extensively studied because they initiate and regulate signal transduction cascades leading to a variety of functional cellular outcomes. An important class of immune receptors (e.g., T-cell antigen receptors) whose ligands are anchored to the surfaces of other cells remain poorly understood. The mechanism by which ligand binding initiates receptor phosphorylation, a process termed “receptor triggering”, remains controversial. Recently, direct measurements of the (two-dimensional) receptor-ligand complex lifetimes at cell-cell interface were found to be smaller than (three-dimensional) lifetimes in solution but the underlying mechanism is unknown. At the cell-cell interface, the receptor-ligand complex spans a short intermembrane distance (15 nm) compared to long surface molecules (LSMs) whose ectodomains span >40 nm and these LSMs include phosphatases (e.g., CD45) that dephosphorylate the receptor. It has been proposed that size-based segregation of LSMs from a receptor-ligand complex is a mechanism of receptor triggering but it is unclear whether the mechanochemistry supports such small-scale segregation. Here we present a nanometer-scale mathematical model that couples membrane elasticity with the compressional stiffness and lateral mobility of LSMs. We find robust supradiffusive segregation of LSMs from a single receptor-ligand complex. The model predicts that LSM redistribution will result in a time-dependent tension on the complex leading to a decreased two-dimensional lifetime. Interestingly, the model predicts a nonlinear relationship between the three- and two-dimensional lifetimes, which can enhance the ability of receptors to discriminate between similar ligands.     

Misfolding of vWF to Pathologically Disordered Conformations Impacts the Severity of von Willebrand Disease

The primary hemostatic von Willebrand factor (vWF) functions to sequester platelets from rheological blood flow and mediates their adhesion to damaged subendothelium at sites of vascular injury. We have surveyed the effect of 16 disease-causing mutations identified in patients diagnosed with the bleeding diathesis disorder, von Willebrand disease (vWD), on the structure and rheology of vWF A1 domain adhesiveness to the platelet GPIbα receptor. These mutations have a dynamic phenotypical range of bleeding from lack of platelet adhesion to severe thrombocytopenia. Using new rheological tools in combination with classical thermodynamic, biophysical, and spectroscopic metrics, we establish a high propensity of the A1 domain to misfold to pathological molten globule conformations that differentially alter the strength of platelet adhesion under shear flow. Rheodynamic analysis establishes a quantitative rank order between shear-rate-dependent platelet-translocation pause times that linearly correlate with clinically reported measures of patient platelet counts and the severity of thrombocytopenia. These results suggest that specific secondary structure elements remaining in these pathological conformations of the A1 domain regulate GPIbα binding and the strength of vWF-platelet interactions, which affects the vWD functional phenotype and the severity of thrombocytopenia.     

Independent assembly and secretion of a dimeric adhesive domain of von Willebrand factor containing the glycoprotein Ib-binding site

von Willebrand factor (vWF) is a multimeric glycoprotein that supports platelet adhesion on thrombogenic surfaces as part of the normal hemostatic response to vascular injury. We have employed a domain-specific expression strategy to analyze the biosynthetic processing steps and minimum structural requirements for assembly of the platelet receptor glycoprotein Ib-binding domain of vWF. A chimeric cDNA that codes for the vWF signal peptide and a segment of vWF internal primary sequence, residues 441-730, directs the secretion of a functional vWF fragment from mammalian cells. The recombinant molecule intrinsically assembles through intermolecular disulfide bond formation into a dimeric adhesive domain without contributions from other regions of vWF, including propeptide, previously indicated as essential for vWF multimer assembly. Prevention of N-linked glycosylation on the recombinant domain does not impair dimer formation or the ability to support platelet aggregation. These results identify a minimum structural element for vWF subunit assembly and provide new insights into the processing steps to produce vWF multimers and adhesive domains.     

Mobility of GPIIb-IIIa receptors within membranes of surface- and suspension-activated platelets does not depend on assembly and contraction of cytoplasmic actin

Investigations using fibrinogen coupled to colloidal gold (Fgn/Au) have shown that glycoprotein IIb-IIIa (GPIIb-IIIa) receptors are mobile and undergo centripetal reorganization on spread platelets following surface activation. The assembly of cytoplasmic actin and its constriction into an inner filamentous zone have been proposed as the mechanism driving the Fgn/Au receptor complexes across the plasma membrane. The present study has used cytochalasin B (CB), an agent known to inhibit actin assembly and cause breakdown of newly formed actin filaments to test that hypothesis. At a concentration which inhibited pseudopod formation and spreading, CB did not block movement of Fgn/Au receptor complexes toward platelet centers or into the open canalicular system (OCS). Channels of the OCS filled with Fgn/Au receptor complexes were evident in over 90% of the CB-treated, surface-activated platelets. The findings do not support the concept that assembly of cytoplasmic actin and its contraction move receptor-ligand complexes on the platelet plasma membrane.     

Increase in GP IIb-IIIa and P2Y12 receptors in activated platelets as the possible indicator of de novo protein synthesis

Although platelets lack nuclei, they are capable of de novo protein synthesis. We speculate that key platelet receptors are involved in the regulation of this process, and the changes in their number indicate the de novo protein synthesis in platelets. The object of our study was native platelets obtained from healthy donors. Using flow cytometry and Western blot, we determined the number of GP IIb-IIIa receptors (fibrinogen receptor) and P2Y12 receptors (ADP receptor) on the surface of platelets upon their activation with ADP and collagen. To verify the approaches and techniques used, we studied IL-1β protein, which was previously shown to be synthesized de novo in activated platelets. GP IIb-IIIa receptor numbers correlate with the number of P2Y12 receptors on the cell surface (R = 0.45, p = 0.03). It was demonstrated that the platelet receptor numbers are higher on the surface of the cells with high functional activity. According to the data obtained by Western blot, upon the cell activation with ADP, the number of GP IIb-IIIa and P2Y12 receptors increases, which may serve as evidence of these proteins being synthesized in the activated platelets. It was observed that the level of P2Y12 and IL-1β was lower in the samples where GP IIb-IIIa receptor was blocked by the selective inhibitor, i.e., the Fab fragment of the antibodies that specifically recognizes the GP IIb-IIIa complex. This suggests the important role of GP IIb-IIIa receptor in the regulation of protein synthesis.     

A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen

The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.     

Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function.

The human platelet thrombin receptor is activated when thrombin cleaves its receptor's amino-terminal extension to reveal a new amino terminus that functions as a tethered peptide ligand. Exactly how this "agonist peptide domain" remains cryptic within the uncleaved receptor and becomes functional after receptor cleavage is unknown. In this report we define the structural features of the thrombin receptor's agonist peptide domain important for receptor activation. Studies with mutant thrombin receptors have suggested that agonist peptide domain residues 2-6 contained determinants critical for receptor activation, and the synthetic peptide SFLLR-NH2 representing the 1st 5 amino-terminal residues of the agonist peptide domain was sufficient to specify agonist activity. Acetylating or removing the agonist peptide's amino-terminal ammonium group greatly attenuated agonist activity. Agonist peptide residue Phe2 was vital for agonist function; residues Leu4 and Arg5 individually played less important roles. These structure-function relationships held for both platelet activation and activation of the cloned receptor expressed in transfected mammalian cells. Our studies suggest that structures at the extreme amino terminus of the thrombin receptor's agonist peptide domain, in particular the free ammonium group of Ser1 and the phenyl ring of Phe2, are critical for receptor activation and that the agonist function of this domain is expressed when receptor proteolysis unmasks such determinants. In addition to revealing details of the thrombin receptor's proteolytic triggering mechanism, these studies open avenues to the development of drugs targeting the thrombin receptor and to further definition for the role of the thrombin receptor in cellular regulation.