Stoichiometry of interaction between interferon gamma and its receptor.

The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein. The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering. Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein.     

Expression of recombinant extracellular domain of the type II transforming growth factor-beta receptor: utilization in a modified enzyme-linked immunoabsorbent assay to screen TGF-beta agonists and antagonists

TGF-beta is a ubiquitous protein that exhibits a broad spectrum of biological activity. The prokaryotic expression and purification of the extracellular domain of the type II TGF-beta receptor (T beta R-II-ED), without the need for fusion protein cleavage and refolding, is described. The recombinant T beta R-II-ED fusion protein bound commercially available TGF-beta 1 and displayed an affinity of 11.1 nM. In a modified ELISA, receptor binding to TGF-beta1 was inhibited by TGF-beta 3. The technique lends itself to high-throughput screening of combinatorial libraries for the identification of TGF-beta agonists and antagonists and this, in turn, may have important therapeutic implications.     

The growth hormone binding protein as a paradigm of the erythropoietin superfamily of receptors.

The growth hormone (GH) receptor belongs to a novel receptor family which shares significant amino-acid sequence homology and includes prolactin receptors, erythropoietin receptors and several cytokines' receptors. GH and three other members of this family of receptors have been shown to have circulating soluble forms. The present review summarizes our knowledge on receptor related binding proteins, discusses their possible biological effects and suggests their use in novel assays for their ligands. The GH-binding protein (GH-BP) was the first to have been described and is used as a model for the concept. A series of indirect pieces of evidence suggest that the measurement of circulating GH-BP may enable an evaluation of the GH-receptor. When covalently bound to GH, GH-BP has been shown to slow the clearance of GH. On the other hand GH-BP competes with the GH-receptor for GH binding and, thus, diminishes the biological effect of GH. We suggest a biological role for GH-BP as follows: an increase in the availability of GH results not only in the upregulation of the GH-receptor but also in increased turnover of this receptor, its internalization and recycling. This is followed by a concomitant increase in GH-BP which, in turn, mitigates the effect of GH by competing with the receptor on GH binding. The extracellular domain of the GH-receptor is homologous, to a large extent, with the sequence of several receptors for hormones and cytokines, which have recently been cloned.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of predicted extracellular domains of human growth hormone secretagogue receptor 1a

The Class A family of guanine nucleotide-binding protein (G protein)-coupled receptors that includes receptors for motilin, ghrelin, and growth hormone secretagogue (GHS) has substantial potential importance as drug targets. Understanding of the molecular basis of hormone binding and receptor activation should provide insights helpful in the development of such drugs. We previously reported that Cys residues and the perimembranous residues in the extracellular loops and amino-terminal tail of the motilin receptor are critical for peptide ligand, motilin, binding and biological activity. In the current work, we focused on the predicted extracellular domains of the human GHS receptor 1a, and identified functionally important residues by using sequential deletions ranging from one to twelve amino acid residues and site-directed replacement mutagenesis approach. Each construct was transiently expressed in COS cells, and characterized for ghrelin- and growth hormone releasing peptide (GHRP)-6-stimulated intracellular calcium responses and ghrelin radioligand binding. Cys residues in positions 116 and 198 in the first and second extracellular loops and the perimembranous Glu¹⁸⁷ residue in the second extracellular loop were critical for ghrelin and GHRP-6 biological activity. These results suggest that Cys residues in the extracellular domains in this family of Class A G protein-coupled receptor is likely involved in the highly conserved and functionally important disulfide bond, and that the perimembranous residues contribute peptide ligand binding and signaling.     

Sulfation of tyrosine 174 in the human C3a receptor is essential for binding of C3a anaphylatoxin

The complement anaphylatoxin C3a and its cellular seven-transmembrane segment receptor, C3aR, are implicated in a variety of pathological inflammatory processes. C3aR is a G-protein-coupled receptor with an exceptionally large second extracellular loop of 172 amino acids. Previously reported deletion studies have shown that at least part of this region plays a critical role in binding C3a. Our data now demonstrate that five tyrosines in the second extracellular loop of the C3aR are posttranslationally modified by the addition of sulfate. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 184, 188, 317, and/or 318 does not affect ligand binding or signal transduction. However, when tyrosine 174 is mutated to phenylalanine, binding of native C3a is completely blocked. This variant efficiently mobilizes calcium in response to synthetic C3a agonist peptides, but not to native C3a. This finding is consistent with a two-site model of ligand association typical of many peptide ligand-receptor interactions and identifies sulfotyrosine 174 as the critical C3a docking site. Tyrosine sulfation in the amino-terminal extracellular domain has been shown to be important in several other seven-transmembrane segment receptors. Our data now demonstrate that tyrosine sulfate in other extracellular domains can function for ligand interactions as well.     

The solution structure of BMPR-IA reveals a local disorder-to-order transition upon BMP-2 binding

The structure of the extracellular domain of BMP receptor IA was determined in solution by NMR spectroscopy and compared to its structure when bound to its ligand BMP-2. While most parts of the secondary structure are highly conserved between the bound and unbound forms, large conformational rearrangements can be observed in the beta4beta5 loop of BMPR-IA, which is in contact with BMP-2 and harbors the main binding determinants for the BMPR-IA-BMP-2 interaction. In its unbound form, helix alpha1 in BMPR-IA, which is in the center of the binding epitope for BMP-2, is missing. Since BMP-2 also shows conformational changes in the type I receptor epitope upon binding to BMPR-IA, both binding partners pass through an induced fit mechanism to adapt their binding interfaces to a given interaction surface. The inherent flexibility of both partners possibly explains the promiscuous ligand-receptor interaction observed in the BMP protein superfamily.     

Dynamic localization and persistent stimulation of factor-dependent cells by a stem cell factor / cellulose binding domain fusion protein

The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.     

Flt3配体的同源二聚体构建及其生物学特性

Flt3配体 (FL)是一种早期造血生长因子 ,与相应受体结合后 ,驱动受体二聚化 ,从而启动细胞内信号转导。体内 ,可溶性的FL主要以单体形式存在。将 2个相同的编码可溶性FL的基因片段 ,通过连接序列以首尾相连的方式构建成FL FL基因 ,并在毕氏酵母 (Pichiapastoris)中表达。SDS PAGE试验表明 ,FL FL融合蛋白 (rFL FL)分子量约为 4 3kD ,与预期相符 ,并为Western印迹试验所证实。骨髓祖细胞体外集落形成试验提示 ,rFL FL具有更强的生物学活性 ,约是单体FL(rhFL)的 10倍之多。另外 ,FL FL融合蛋白还能促进和延长饥饿的Raji细胞存活 ,与rhFL相比 ,也表现出较强的生物学功能。     

A common model for cytokine receptor activation: combined scissor-like rotation and self-rotation of receptor dimer induced by class I cytokine

The precise mechanism by which the binding of a class I cytokine to the extracellular domain of its corresponding receptor transmits a signal through the cell membrane remains unclear. Receptor activation involves a cytokine-receptor complex with a 1∶2 stoichiometry. Previously we used our transient-complex theory to calculate the rate constant of the initial cytokine-receptor binding to form a 1∶1 complex. Here we computed the binding pathway leading to the 1∶2 activation complex. Three cytokine systems (growth hormone, erythropoietin, and prolactin) were studied, and the focus was on the binding of the extracellular domain of the second receptor molecule after forming the 1∶1 complex. According to the transient-complex theory, translational and rotation diffusion of the binding entities bring them together to form a transient complex, which has near-native relative separation and orientation but not the short-range specific native interactions. Subsequently conformational rearrangement leads to the formation of the native complex. We found that the changes in relative orientations between the two receptor molecules from the transient complex to the 1∶2 native complex are similar for the three cytokine-receptor systems. We thus propose a common model for receptor activation by class I cytokines, involving combined scissor-like rotation and self-rotation of the two receptor molecules. Both types of rotations seem essential: the scissor-like rotation separates the intracellular domains of the two receptor molecules to make room for the associated Janus kinase molecules, while the self-rotation allows them to orient properly for transphosphorylation. This activation model explains a host of experimental observations. The transient-complex based approach presented here may provide a strategy for designing antagonists and prove useful for elucidating activation mechanisms of other receptors.     

Fibroblast growth factor-binding protein is a novel partner for perlecan protein core

Perlecan, a widespread heparan sulfate proteoglycan, functions as a bioactive reservoir for growth factors by stabilizing them against misfolding or proteolysis. These factors, chiefly members of the fibroblast growth factor (FGF) gene family, are coupled to the N-terminal heparan sulfate chains, which augment high affinity binding and receptor activation. However, rather little is known about biological partners of the protein core. The major goal of this study was to identify novel proteins that interact with the protein core of perlecan. Using the yeast two-hybrid system and domain III of perlecan as bait, we screened approximately 0.5 10(6) cDNA clones from a keratinocyte library and identified a strongly interactive clone. This cDNA corresponded to FGF-binding protein (FGF-BP), a secreted protein previously shown to bind acidic and basic FGF and to modulate their activities. Using a panel of deletion mutants, FGF-BP binding was localized to the second EGF repeat of domain III, a region very close to the binding site for FGF7. FGF-BP could be coimmunoprecipitated with an antibody against perlecan and bound in solution to recombinant domain III-alkaline phosphatase fusion protein. Immunohistochemical analyses revealed colocalization of FGF-BP and perlecan in the pericellular stroma of various squamous cell carcinomas suggesting a potential in vivo interaction. Thus, FGF-BP should be considered a novel biological ligand for perlecan, an interaction that could influence cancer growth and tissue remodeling.     

Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence

A putative growth hormone receptor from detergent-solubilized rabbit liver membranes and the growth hormone binding protein from rabbit serum have been purified 59,000- and 400,000-fold, respectively, primarily by affinity chromatography. Both purified proteins exhibit high affinity binding for human growth hormone; K alpha = 9-30 x 10(9) M-1 for the liver receptor and K alpha = 6 x 10(9) M-1 for the binding protein. The apparent molecular weight of the liver receptor is 130,000 by reduced sodium dodecyl sulfate gel electrophoresis, while that of the binding protein is 51,000. Both contain N-linked carbohydrate. The amino-terminal sequences of the liver growth hormone receptor and the serum binding protein were found to be the same, indicating that the binding protein corresponds to the extracellular domain of the liver receptor. Ubiquitin was found covalently linked to the liver receptor but not to the serum binding protein. The amino acid sequences of several peptides from the liver receptor were also determined after tryptic and V8 protease digestion.     

Identification of a region critical for proteolysis of the human growth hormone receptor.

Release of soluble growth hormone binding protein (GHBP) corresponding to the extracellular domain of the GH receptor (GHR) occurs via distinct mechanisms depending on species. In human, proteolysis of full length GHR results in liberation of GHBP into the extracellular medium. A putative protease responsive for GHR cleavage has been identified, however, the residues involved are still unknown. In this study, using the mutational approach to the extracellular domain of the human GHR, we demonstrated that deletion of three residues located close to the transmembrane domain abolishes constitutive GHBP shedding without change in cellular GH binding. Deletion also significantly decreased the phorbol 12-myristate 13-acetate (PMA)-induced release of GHBP and the accumulation of membrane-anchored remnant proteins. Taken together, these results suggest that integrity of the juxtamembrane region of GHR is necessary for its biochemical cleavage and that a common mechanism is involved in constitutive and PMA-induced shedding.     

Crystallization and preliminary X-ray analysis of a 1:1 complex between a designed monomeric interferon-gamma and its soluble receptor.

A variant of human interferon-gamma (IFN-gamma) has been created in which the two chains of the homodimeric cytokine were linked N- to C-terminus by an eight residue polypeptide linker. The sequence of this linker was derived from a loop in bira bifunctional protein, and was determined from a structural database search. This "single-chain" variant was used to create an IFN-gamma molecule that binds only a single copy of the alpha-chain receptor, rather than the 2 alpha-chain receptor: 1 IFN-gamma binding stoichiometry observed for the native hormone. Crystals have been grown of a 1:1 complex between this single-chain molecule and the extracellular domain of its alpha-chain receptor. These crystals diffract beyond 2.0 A, significantly better than the 2.9 A observed for the native 2:1 complex. Density calculations suggest these crystals contain two complexes in the asymmetric unit; a self-rotation function confirms this conclusion.     

Investigation of Growth Hormone Releasing Hormone Receptor Structure and Activity Using Yeast Expression Technologies

Abstract

Growth hormone releasing hormone (GHRH) is the positive regulator of growth hormone synthesis and secretion in the anterior pituitary. The peptide confers activity by binding to a seven transmembrane domain G protein-coupled receptor. Signal transduction proceeds through subsequent Gas stimulation of adenylyl cyclase. To investigate ligand/receptor and receptor/G protein associations, the human GHRH receptor was expressed in a modified S. cerevisiae strain which allows for facile measurement of receptor activity by cell prototrophy mediated by a reporter gene coupled to the yeast pheromone response pathway. GHRH-dependent signal activation in this system required the substitution of yeast Gα protein with proteins containing C-terminal regions of Gαs. A D60G variant (analogous to the little mouse mutation) of the receptor failed to respond to agonist. In parallel studies, GHRH₂₉ and the N-terminal extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay. A specific interaction between these proteins was readily observed. The D60G mutation was engineered into the receptor fusion protein. This protein failed to interact with the ligand fusion, confirming the specificity of the association between unmodified proteins. These two yeast expression technologies should prove invaluable in additional structure/activity analyses of this ligand/receptor pair as well as other peptide ligands and receptors.     

Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling.

We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, <5% of labeled GH was internalized by cells expressing truncated GHR1-279. Using another truncated receptor, GHR1-317 fused to enhanced green fluorescent protein (EGFP), we have exploited fluorescence energy transfer to monitor the trafficking of ligand-receptor complexes. The data confirmed that internalization of this truncated receptor is very inefficient. It was possible to visualize the truncated GHR1-317-EGFP packaged in the endoplasmic reticulum, its rapid movement in membrane bound vesicles to the Golgi apparatus, and subsequent transport to the cell membrane. The GH antagonist, B2036, blocked Jak2-Stat5-mediated GHR signaling but was internalized with a similar time course to native GH. The results: 1) demonstrate the rapid internalization of GH when studied under physiological conditions; 2) confirm the hypothesis that internalization of cytoplasmic domain truncated human GHRs is very inefficient, which explains their dominant negative action; and 3) show that the antagonist action of B2036 is independent of receptor internalization.     

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.     

Mutational analysis of the ligand-binding domain of the prolactin receptor

The recent isolation and sequencing of the rat liver prolactin (PRL) receptor cDNA (clone F3) revealed that the receptor is a small molecular weight protein (nonglycosylated form, Mr 33,000; glycosylated form, Mr 42,000) comprised of 291 amino acids. A second form of the PRL receptor exists (591 amino acids) that contains a much longer cytoplasmic domain. In the present study, site-directed point mutations of the 5 conserved cysteine (Cys) residues and of the three potential N-linked glycosylation sites in the extracellular domain of the rat PRL receptor were constructed to assess their involvement in hormone binding. In addition, a truncation mutant (T delta 237) lacking 55 of 57 intracellular amino acids was constructed to determine the influence of the cytoplasmic domain on ligand-receptor interactions. Binding studies of transiently transfected COS-7 cells demonstrated that serine substitution of the first 4 Cys residues (Cys12, Cys22, Cys51, and Cys62) completely eliminated binding of 125I-ovine PRL and 125I-U5 and -U6, two monoclonal antibodies that bind the receptor molecule outside the PRL-binding domain. RNA blot analysis of the transfected cells showed that both the wild-type and mutant clones had similar levels of expression of receptor mRNA. Immunoblot analysis demonstrated that lack of PRL binding in these mutants was not due to incomplete processing of the protein, since the fully glycosylated Mr 42,000 form of the receptor was seen. Mutation of Cys184 had no effect on affinity or dimerization capacity of the receptor, suggesting the 5th cysteine is not directly involved in the binding domain. Carbohydrate groups of some receptors have been shown to be involved in ligand-receptor interactions as well as intracellular trafficking. This does not appear to be the case for the PRL receptor, since there was no corresponding decrease in affinity for PRL or cell surface receptor expression, following mutation of each of the 3 asparagine residues to aspartate. Interestingly, T delta 237 showed a 4-5-fold increase in affinity for PRL as well as a marked increase in the number of receptor sites. Whole cell binding assays also demonstrated that loss of the cytoplasmic domain lead to inefficient recycling of the receptor. These studies suggest that the first 4 conserved Cys residues are crucial for ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand- receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet- Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X- 100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu- Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G- protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet- Leu-Phe-associated G-protein.     

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.