Two PDGF-B chain residues, arginine 27 and isoleucine 30, mediate receptor binding and activation.

PDGF may be involved in the pathogenesis of a variety of disorders including atherosclerosis and certain types of cancer. There is currently little understanding of the molecular structure of PDGF and of the critical amino acid residues involved in receptor binding and cell activation. Two such PDGF-B chain residues, arginine 27 and isoleucine 30, have been identified by a site-directed mutagenesis programme. Substitutions in these positions can lead to PDGF mutants defective in both receptor affinity and cell activation as judged by displacement of [125I]PDGF-BB, mitogenic assay and inositol lipid turnover. Circular dichroism and fluorescence spectroscopy show that such mutations do not disrupt the structure of PDGF.     

Kinetic and biophysical analysis of the m2 muscarinic receptor

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.     

Receptor binding of PDGF-AA and PDGF-BB,and the modulation of PDGF receptors by TGF-β, in human periodontal ligament cells

The growth factors PDGF-AA and PDGF-BB have previously been shown to be potent mitogens for human periodontal ligament (hPDL) cells in vitro. Additionally, the mitogenic response to PDGF-AA has been shown to be specifically inhibited by TGF-β. The purpose of the present investigation was to examine the binding of PDGF-AA and PDGF-BB, and the modulation of PDGF binding by TGF-β, in hPDL cells. Scatchard analysis identified an average of 32,000 PDGF-AA high-affinity binding sites per cell with a dissociation constant (K_d) of 0.66 nM and an average of 36,000 PDGF-BB binding sites per cell with a dissociation constant (k_d) of 0.44 nM. After treatment with TGF-β, the receptor number for PDGF-AA was found to specifically decrease by approximately 50%, with no change in binding affinity. This reduced number of binding sites was shown to correlate with both a decrease in levels of receptor tyrosine phosphorylation and a decreased number of α receptor subunits. Northern blot analysis identified the TGF-β-mediated decrease in PDGF α receptor subunit mRNA levels. PDGF-BB showed little change in the number of binding sites or in the binding affinity with TGF-β treatment, and the data were consistent with an increase in the number of β receptor subunits. These results demonstrate nearly equivalent numbers of receptors for both PDGF-AA and PDGF-BB in hPDL cells. Also, modulation of PDGF binding, by TFG-β, was shown to result in a reduced number of α receptor subunits with an increase in the number of β receptor subunits. © 1995 Wiley-Liss, Inc.     

Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.     

Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation

Platelet-derived growth factor (PDGF) was found to induce dimerization of purified B-type PDGF receptors, as analyzed by sodium dodecyl sulfate gel electrophoresis after covalent cross-linking using disuccinimidyl suberate. PDGF-BB was 20-fold more effective than PDGF-AB; PDGF-AA was without effect. The dimerization was dose-dependent and was maximal at 0.5-2 micrograms/ml PDGF-BB; at higher concentrations dimerization was less abundant. This indicates that dimerization occurred when one PDGF-BB molecule bound two receptor molecules. The dimerization correlated to activation of the tyrosine kinase of the receptor, determined as autophosphorylation, but was not dependent on phosphorylation reactions because it occurred also in the absence of ATP. Furthermore, dimerization of the receptor correlated with the ability to phosphorylate phosphofructokinase, an exogenous substrate. The complex of ligand and receptor dimer was stable; it resisted electrophoresis under nondenaturing conditions, as well as gel chromatography. The present data indicate that intermolecular mechanisms are involved in signal transduction from the external ligand binding domain to the internal effector domains of the B-type PDGF receptor.     

Expression of biologically active human platelet-derived growth factor (PDGF) B-receptor in Xenopus laevis oocytes

Platelet-derived growth factor (PDGF) consists of three different isoforms, PDGF-AA, PDGF-AB and PDGF-BB, which bind to at least two types of receptors: the B-receptor, to which only PDGF-BB binds, and the A/B receptor, to which all three isoforms bind. Microinjection of synthetic mRNA in Xenopus laevis oocytes was used to obtain cell-surface expression of the human PDGF B-receptor. The production of receptor molecules of correct size (190 kd) was demonstrated by specific immunoprecipitation; the binding properties of the membrane- associated PDGF B-receptor were investigated with highly purified recombinant [125I] labeled human PDGF-BB and -AA. Unlike Swiss mouse 3T3 cells, which possess both B- and A/B-receptors and, therefore, bind both isoforms with high affinity, the mRNA-injected oocytes bound only the BB isoform. Mock-injected oocytes showed no specific binding.     

Differential effects of PDGF and PDGF-BB on vascular smooth muscle cells

Several biological effects of recombinant PDGF-BB and PDGF obtained from human platelets were examined with vascular smooth muscle cells. Although PDGF and PDGF-BB were equally potent mitogens for these cells, 5 fold higher levels of PDGF were required to displace 125I-PDGF-BB binding than PDGF-BB itself. Higher concentrations of PDGF relative to PDGF-BB were also required to stimulate the phosphorylation of a 163K protein in membrane preparations. PDGF-BB, but not PDGF, treatment of intact cells resulted in the phosphorylation on tyrosine residues of 168, 53, 48, and 45K proteins. The data suggest that PDGF and PDGF-BB stimulate smooth muscle cell mitogenesis by different mechanisms.     

Chimeric alpha- and beta-platelet-derived growth factor (PDGF) receptors define three immunoglobulin-like domains of the alpha-PDGF receptor that determine PDGF-AA binding specificity

Binding of platelet-derived growth factor (PDGF) to its cell surface receptors stimulates a variety of biochemical and biological responses. Two receptor gene products (designated alpha and beta) have been identified, and the different binding affinities of various PDGF isoforms for these receptors are prime determinants of the spectrum of responses observed. The beta receptor binds PDGF-BB, but not PDGF-AA, while the alpha receptor binds PDGF-AA and PDGF-BB. We utilized these different ligand binding specificities to investigate the PDGF-AA binding site in the human alpha-PDGF receptor by constructing chimeric molecules between the human alpha- and beta-PDGF receptors. Our results demonstrate that amino acids 1-340 of the alpha-PDGF receptor comprise the region that confers PDGF-AA binding specificity. This region corresponds to immunoglobulin-like sub-domains 1, 2, and 3 of its external domain.     

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.     

The relation of conformation and association of insulin to receptor binding; x-ray and circular-dichroism studies on bovine and hystricomorph insulins.

Crystal and solution structure studies on insulins of different sequences and of widely different receptor binding affinities are reported. Bovine insulin, studied as a control, has a circular dichroism spectrum which is dependent both on protein concentration and zinc concentration. The spectrum appears to be related to the level of association of the insulin molecules. This implies that when using circular dichroism to compare solution structures of insulin derivatives or species variants one must make the comparison at equivalent levels of association and not merely at the same concentration. Changes in circular dichroism are related to the known crystal structure of zinc insulin hexamers. The chinchilla insulin spectrum shows a reduced zinc dependence in low-salt conditions which correlates with the inability to form crystals in similar conditions. This is attributed to an amino acid substitution at position B4. Crystals are obtained in high-salt conditions and X-ray diffraction patterns show these to be isomorphous with bovine 4Zn insulin crystals. Guinea pig insulin failed to crystallise under conditions which are normally conducive to the formation of crystals of zinc insulin hexamers and the circular dichroism showed no zinc dependence. This is consistent with a monomeric structure. The significance of the association behaviour of chinchilla and guinea pig insulins may be in the storage of the hormone in vivo. Whereas the monomeric form of chinchilla insulin has a structure closely related to bovine insulin, the circular dichroism indicates a gross structural difference for guinea pig insulin. This may be similar to that in des-A21, des-B30-insulin, as both lack the Arg-B22--Asn-A21 carboxylate ion pair. The similarity of structure of chinchilla and bovine insulins is reflected in their receptor binding whereas the low receptor binding of guinea pig insulin probably results from the changes in its conformation rather than an alteration in residues of a receptor binding region.     

Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone

Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.     

Crystal structure of the alpha1beta1 integrin I-domain: insights into integrin I-domain function.

The alpha1beta1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a 200 amino acid inserted 'I'-domain contained in the extracellular part of the integrin alpha chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.     

The Full-Length Mu-Opioid Receptor: A Conformational Study by Circular Dichroism in Trifluoroethanol and Membrane-Mimetic Environments

The secondary structure content of the recombinant human mu-opioid receptor (HuMOR) solubilized in trifluoroethanol (TFE) and in detergent micelles was investigated by circular dichroism. In both conditions, this G protein-coupled receptor adopts a characteristic alpha-helical structure, with minima at 208 and 222 nm as observed in the circular dichroism spectra. After deconvolution of spectra, the alpha-helix contents were estimated to be in the range of 50% in TFE and in sodium dodecyl sulfate at pH 6. These values are in accordance with the predicted secondary structure content determined for the mu-opioid receptor. A pH-dependent effect was observed on the secondary structure of the receptor solubilized in detergents, which demonstrates the essential role of ionic and hydrophobic interactions on the secondary structure. Circular dichroism spectra of EGFP-HuMOR, a fusion protein between the enhanced green fluorescent protein (EGFP) and the mu-opioid receptor, and EGFP solubilized in TFE were also analyzed as part of this study.     

Inhibition of platelet-derived growth factor-mediated signal transduction by transforming ras. Suppression of receptor autophosphorylation.

The function of ras protein and its relationship to growth-factor mediated signal transduction remain unclear. The demonstration that the expression of transforming ras (v-ras) can block the stimulation of growth-related gene expression and cell division mediated by the platelet-derived growth factor (PDGF) may provide a model for the functional interation of ras with growth factor receptors. In the current studies, we have demonstrated that this blockade by v-ras of PDGF-BB signal transduction occurs very early in signal transduction, at the level of PDGF receptor autophosphorylation. Although the expression of PDGF receptor as detected by Western blot with anti-PDGF receptor antibody was not diminished in v-ras-transformed murine Balb/c 3T3 fibroblasts, the autophosphorylation of PDGF receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-ras-mediated PDGF receptor autophosphorylation inhibition was also demonstrated in normal rat kidney fibroblasts. Further, factor(s) present in v-ras-expressing fibroblasts found in the membrane fractions of these cells can dominantly inhibit the autophosphorylation of the PDGF receptor obtained from normal fibroblasts. These findings suggest a role for ras in one of the earliest steps of the signal transduction pathway.     

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer.

Human platelet-derived growth factor (PDGF) occurs as three isoforms which are made up of disulfide-bonded A and B chains. The isoforms bind with different affinities to two different but structurally related cell surface receptors. The A type receptor binds all three isoforms (PDGF-AA, PDGF-AB, PDGF-BB) with high affinity, whereas the B type receptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity but does not appear to bind PDGF-AA. We have utilized the differential effects of the three isoforms on actin reorganization and membrane ruffling in human foreskin fibroblasts to probe the idea that ligand-induced receptor dimerization is associated with receptor activation. Actin reorganization was found to be induced only by PDGF-AB and PDGF-BB and is therefore likely to be mediated by the B type receptor. Simultaneous addition of PDGF-AA, or downregulation of the A type receptor blocked the effect of PDGF-AB but not that of PDGF-BB. This is compatible with a model by which PDGF-AB binds to and dimerizes one A and one B type receptor; PDGF-AB therefore requires A type receptors in order to be functionally active at physiological concentrations. In cells with down-regulated A type receptors, high concentrations of PDGF-AB inhibited the effect of PDGF-BB on actin reorganization. We believe that this is due to a monovalent binding of PDGF-AB to the B type receptors which prevents PDGF-BB from dimerizing the receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topology of receptor binding domains of mouse IFN-gamma.

IFN-gamma is an essential immunoregulatory lymphokine for a variety of immunologic functions including upregulation of MHC Ag. The elucidation of the structure, particularly the receptor binding domains, should further enhance our understanding of its mechanism of action, and provide a rational basis for modulation of its activity by alteration of its structure. A predicted model of murine IFN-gamma structure has been constructed based on data derived from our synthetic peptide studies, circular dichroism spectra, and predictive algorithms for secondary structure, surface accessibility, and tertiary structure, as well as predicted structural similarities to IL-2. Direct synthetic peptide competition studies using five overlapping peptides that encompassed the entire IFN-gamma sequence of 133 amino acids showed that only the N-terminus of IFN-gamma, IFN-gamma(1-39), blocked binding to receptor, suggesting that the N-terminus is directly involved in receptor binding. Rabbit antibodies to the N-terminal (IFN-gamma(1-39)) and C-terminal (IFN-gamma(95-133)) peptides neutralized IFN-gamma activity, whereas antibodies to the three peptides that spanned the internal region, sequence 36-107, were without effect. Thus, the antibody data support the involvement of the N-terminus as a receptor binding domain and also suggest that the C-terminus of the molecule is also a binding domain. Predictive algorithms assign six alpha-helices and five turns to the molecule and circular dichroism spectra of both intact human and murine IFN-gamma and synthetic peptides of murine IFN-gamma showed that the molecule is mainly alpha-helical in structure. Drawing mainly on the four alpha-helix bundle model, a common motif in globular proteins such as IFN-gamma and IL-2 (whose crystalline structure is known), we constructed a simple model of the tertiary structure of IFN-gamma that fits well with our synthetic peptide and circular dichroism data. The model consists of the four-alpha-helical bundle along with N- and C-terminal helices that are predicted to be closely associated and together form the receptor binding domains. The model presented should contribute to further understanding the molecular basis of IFN-gamma action and allow us to begin modulating the function of IFN-gamma through the design of agonists and antagonists.