Molecular dissection of the human alpha2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling

alpha(2)-Macroglobulin (alpha(2)M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in alpha(2)M are responsible for its various functions, we hypothesized that the overall effects of alpha(2)M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). FP6 rapidly and robustly activated Akt and ERK/MAP kinase in Schwann cells and PC12 cells. This response was blocked by LRP-1 gene silencing or by co-incubation with the LRP-1 antagonist, receptor-associated protein. The activity of FP6 also was blocked by mutating Lys(1370) and Lys(1374), which precludes LRP-1 binding. FP3 blocked activation of Akt and ERK/MAP kinase in response to nerve growth factor-beta (NGF-beta) but not FP6. In PC12 cells, FP6 promoted neurite outgrowth and expression of growth-associated protein-43, whereas FP3 antagonized the same responses when NGF-beta was added. The ability of FP6 to trigger LRP-1-dependent cell signaling in PC12 cells was reproduced by the 18-kDa RBD, isolated from plasma-purified alpha(2)M by proteolysis and chromatography. We propose that the effects of intact alpha(2)M on cell physiology reflect the degree of penetration of activities associated with different domains, such as FP3 and FP6, which may be regulated asynchronously by conformational change and by other regulatory proteins in the cellular microenvironment.     

Growth factor-binding sequence in human alpha2-macroglobulin targets the receptor-binding site in transforming growth factor-beta

alpha(2)-Macroglobulin (alpha(2)M) binds transforming growth factor-beta1 (TGF-beta1) and TGF-beta2, forcing these growth factors into a state of latency. The mechanism by which this occurs remains unclear. In this paper, we demonstrate that peptides, derived from the structure of human alpha(2)M (amino acids 714-729), bind directly to TGF-beta1 and block the binding of TGF-beta1 to the type I and II TGF-beta receptors. The alpha(2)M-derived peptides are notable for hydrophobic tripeptide sequences (WIW or VVV) and acidic residues (Glu(714) and Asp(719) in the mature alpha(2)M subunit), which may function analogously to the structural elements that mediate TGF-beta-binding in the type II receptor. Mutating Glu(714) and Asp(719) in the alpha(2)M-peptide-GST fusion protein, FP3, which contains the putative growth factor-binding site, significantly decreased the binding affinity of FP3 for TGF-beta1. The alpha(2)M-derived peptides, which bind TGF-beta1, inhibited the interaction of TGF-beta1 with its receptors in fetal bovine heart endothelial cells. The same peptides also inhibited the activity of TGF-beta1 in endothelial cell proliferation assays. These results demonstrate that alpha(2)M-derived peptides target the receptor-binding sequence in TGF-beta.     

Identical or overlapping sequences in the primary structure of human alpha(2)-macroglobulin are responsible for the binding of nerve growth factor-beta, platelet-derived growth factor-BB, and transforming growth factor-beta

alpha(2)-Macroglobulin (alpha(2)M) functions as a proteinase inhibitor and as a carrier of diverse growth factors. In this study, we localized binding sites for platelet-derived growth factor-BB (PDGF-BB) and nerve growth factor-beta (NGF-beta) to a linear sequence in the 180-kDa human alpha(2)M subunit which includes amino acids 591-774. A glutathione S-transferase fusion protein containing amino acids 591-774 (FP3) bound PDGF-BB and NGF-beta in ligand blotting assays whereas five other fusion proteins, which collectively include amino acids 99-590 and 775-1451 did not. The K(D) values for PDGF-BB and NGF-beta binding to immobilized FP3 were 300 +/- 40 and 180 +/- 30 nM, respectively; these values were comparable with those determined using methylamine-modified alpha(2)M, suggesting that higher-order alpha(2)M structure is not necessary for PDGF-BB and NGF-beta binding. PDGF-BB and NGF-beta blocked the binding of transforming growth factor-beta1 (TGF-beta1) to FP3. Furthermore, murinoglobulin, which is the only known member of the alpha-macroglobulin family that does not bind TGF-beta, also failed to bind PDGF-BB and NGF-beta. These results support the hypothesis that either a single linear sequence in human alpha(2)M or overlapping sequences are responsible for the binding of TGF-beta, PDGF-BB, and NGF-beta, even though there is minimal sequence identity between these three growth factors. FP3 blocked the binding of PDGF-BB to a purified chimeric protein, in which the extracellular domain of the PDGF beta receptor was fused to the IgG(1) Fc domain, and to PDGF receptors on NIH 3T3 cells. Thus, FP3 may inhibit the activity of PDGF-BB.     

Probing for integrin alpha v beta3 binding of RGD peptides using fluorescence polarization

Integrin alpha(v)beta(3) is an adhesion molecule involved in tumor invasion, angiogenesis, and metastasis. There is substantial interest in developing novel agents that bind to integrin alpha(v)beta(3). Here we report the synthesis and characterization of a fluorescent integrin alpha(v)beta(3) probe and its use in a nonradioactive, simple, sensitive fluorescence polarization (FP) assay to quantify binding to integrin alpha(v)beta(3). For assay validation, the FP assay was compared to a cell adhesion assay. In the two assays, probe binding to integrin alpha(v)beta(3) showed a similar dependence on probe concentration. The FP assay was successfully applied to measure the binding affinity to integrin alpha(v)beta(3) of several cyclic peptides containing the Arg-Gly-Asp (RGD) motif. The FP assay we describe here may be appropriate for high-throughput screening for integrin alpha(v)beta(3)-binding ligands used for anti-integrin therapy or noninvasive imaging of integrin expression.     

Characterization of human beta-interferon-binding sites on human cells

Radioiodinated recombinant human beta-interferon (rHuIFN beta Ser), with almost full (greater than 90%) biological activity, was used to study the binding of human beta-interferon to Daudi cells. Specific binding was not observed with less biologically active (less than or equal to 10%) radioiodinated interferon. The bound radioiodinated interferon was shown to compete with human beta-interferon (HuIFN beta), rHuIFN beta Ser, human alpha-interferon (HuIFN alpha) and with human gamma-interferon (HuIFN gamma). Scatchard plot analyses suggest the presence of about 10,000 binding sites for HuIFN beta/Daudi cell. About 6,600 of these sites can be blocked by HuIFN alpha and 3,700 sites can be blocked by HuIFN gamma. The apparent Kd for HuIFN beta is 2.7 nM. The apparent Kd values for HuIFN alpha and HuIFN gamma are 3.7 and 1.1 nM, respectively. It was possible to demonstrate the cross-linking of HuIFN beta to two macromolecular components of Mr = 128,000 and 103,000. We propose the existence of at least two binding sites for HuIFN beta in Daudi cells, one site recognizing both HuIFN beta and HuIFN gamma, the other site recognizing both HuIFN beta and HuIFN alpha. Each site is capable of recognizing only HuIFN gamma or HuIFN alpha.     

Engineering and use of (32)P-labeled human recombinant interleukin-11 for receptor binding studies.

Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FP Delta IL-11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with (32)P. A high radiological specific activity (250,000 c.p.m x ng(-1) of protein) was obtained with the retention of full biological activity of the protein. The binding of (32)P-labeled FP Delta IL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors alpha and beta chains (IL-11R alpha and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea.

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly puridied nuclei exhibited specific binding with 125I-labeled human choriogonadotropin, [3H]prostaglandin E1 and [3H]prostaglandin F2 alpha. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E1) and 8.9% (prostaglandin F2 alpha) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F2 alpha binding site and 5'-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21--26% (prostaglandins) of original specific binding despite virtual disappearance of 5'-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound 125I-labeled human choriogonadotropin and [3H]prostaglandin F2 alpha to the same extent or significantly more ([3H]prostaglandin E1, P less than 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Haem degradation in human haemoglobin in vitro. Separation of the contribution of the alpha- and beta-subunits.

As part of an investigation into whether alpha 1-foetoprotein (alpha 1-FP) plays the same transport role in foetal serum as albumin does in the adult, the binding properties of both proteins were compared with respect to the binding of a series of compounds known to be bound by albumin's specific drug-binding sites. The binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid by rat alpha 1-FP and serum albumin was studied by equilibrium dialysis at 4 degrees C. Rat alpha 1-FP was shown to have neither albumin's high-affinity site II (diazepam as marker) nor its site III (digitoxin and cholic acid as markers). High-affinity binding by alpha 1-FP was found for the specific markers (warfarin, phenylbutazone, azapropazone) of albumin's drug-binding site I. However, instead of albumin's one high-affinity site/molecule, a mean value of 0.5 site/molecule was obtained with rat alpha 1-FP. Charcoal treatment at neutral pH of rat serum albumin did not affect its measured binding properties, but treatment of the alpha 1-FP led to an increased affinity for warfarin, phenylbutazone and azapropazone without a change in the measured number of sites, indicating competition for binding at this site by (an) endogenous ligand(s). These results are discussed in terms of the structures of the two proteins and with respect to the physiological implications of the differences found.     

Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites

Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.     

Bimatoprost (Lumigan((R))) is an agonist at the cloned human ocular FP prostaglandin receptor: real-time FLIPR-based intracellular Ca(2+) mobilization studies

Bimatoprost is the ethyl amide derivative of 17-phenyl-trinor prostaglandin F(2alpha). Here, we show that bimatoprost (K(i)=9250+/-846nM) and bimatoprost free acid (17-phenyl-trinor prostaglandin F(2alpha); K(i)=59+/-6nM) bind to the FP receptor and displace [(3)H]-travoprost acid, a selective FP agonist. Bimatoprost (EC(50)=3070+/-1330nM), Lumigan((R)) (bimatoprost 0.03% ophthalmic solution; EC(50)=1150+/-93nM) and bimatoprost acid (EC(50)=15+/-3nM) mobilized intracellular Ca(2+) ([Ca(2+)](i)) in <5s in HEK-293 cells expressing the cloned human ciliary body FP receptor on a fluorometric imaging plate reader (FLIPR). Furthermore, agonist effects of bimatoprost and bimatoprost acid were blocked by AL-8810 (11beta-fluoro-15-epi-15-indanyl prostaglandin F(2alpha); K(i)=0.7-2.1 MicroM), an FP receptor-selective antagonist. Therefore, the prodrug bimatoprost and its hydrolytic product, bimatoprost free acid, bind to and activate the human ocular FP prostaglandin receptor to mobilize [Ca(2+)](i), thus behaving as FP receptor agonists.     

Secretion of alpha 1-antitrypsin by an established human hepatoma cell line and by human/mouse hybrids

The human hepatoma cell line SK-HEP-1 has been shown by radioimmunoelectrophoresis to synthesize and secrete a protein which coprecipitates with human alpha 1-antitrypsin. This protein was indistinguishable from serum alpha 1-antitrypsin in terms of electrophoretic mobility, apparent subunit molecular weight (47,000), and binding to concanavalin-A. The protein identified as alpha 1-antitrypsin (alpha 1 AT) was secreted by seven clones derived from SK-HEP-1 and by twelve out of eighteen hybrid clones derived from the fusion of SK-HEP-1 with mouse RAG cells. There was no correlation between the expression of alpha 1 AT and that of human enzymes assigned to sixteen different autosomes. There was an imperfect correlation between the expression of alpha 1 AT and of the two chromosome 9 marker enzymes AK1 and AK3 (two discordant clones).     

Homology model of human interferon-alpha 8 and its receptor complex.

Human interferon-alpha 8 (HuIFN alpha 8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic super-family that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFN alpha 8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which HGH receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFN alpha 8 with the receptor subunits were built by superpositioning the conserved C alpha backbone of the HuIFN alpha 8 and receptor subunit models with HGH and its receptor complex. The HuIFN alpha 8 model was constructed from the C alpha coordinates of murine interferon-beta crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFN alpha 8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFN alpha 8 correspond to functionally important residues determined previously for human IFN alpha 1, IFN alpha 2, and IFN alpha 4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFN alpha 8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-alpha subtypes and IFN-beta) and their respective receptor components.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

The LG1-3 tandem of laminin alpha5 harbors the binding sites of Lutheran/basal cell adhesion molecule and alpha3beta1/alpha6beta1 integrins

The laminin-type globular (LG) domains of laminin alpha chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, alpha-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin alpha5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin alpha5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified alpha chains, such that all or part of alpha5LG was replaced with analogous segments of human laminin alpha1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing alpha5 domains LN through LG3 fused to human laminin alpha1LG4-5. However, Lu/B-CAM did not bind other recombinant laminins containing alpha5LG3 unless alpha5LG1-2 was also present. A recombinant alpha5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the alpha5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin alpha5. Our results also suggest that the binding site for Lu/B-CAM on laminin alpha5 may overlap with that of integrins alpha3beta1 and alpha6beta1.     

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.