RESUMO
Vascular endothelial cell growth factor (VEGF) binds to 2 related receptor tyrosine kinases, known as kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1). The KDR has been shown to mediate VEGF-stimulated endothelial cell mitogenesis, migration, and permeability. The Flt-1 receptor has been suggested to mediate VEGF-stimulated endothelial branching morphogenesis, a process whereby endothelial cells, in the presence of a 3D milieu composed of extracellular matrix components and a mixture of growth factors, undergo a morphological transition into a tubular network with many lumina. In the present study, we have used 2 independent endothelial cell tube formation models and highly selective VEGF mutants for the KDR and Flt-1 receptors. We demonstrate that KDR, not Flt-1, stimulation is responsible for the induction of endothelial tubulogenesis. In addition, we demonstrate a modulatory role for Flt-1 in VEGF-mediated tube formation. We also report that VEGF-driven endothelial tube formation is inhibited by selective inhibitors of mitogen-activated protein kinase activation and p38 protein kinase.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Vasos Sanguíneos/crescimento & desenvolvimento , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Morfogênese , Mutação , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We report that, contrary to common belief, polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein are functionally displayed on the phage surface. In a phagemid display system, carboxy-terminal fusion through optimized linker sequences resulted in display levels comparable to those achieved with conventional amino-terminal fusions. These findings are of considerable importance to phage display technology because they enable investigations not suited to amino-terminal display, including the study of protein-protein interactions requiring free carboxy-termini, functional cDNA cloning efforts, and the display of intracellular proteins.
Assuntos
Bacteriófago M13 , Proteínas de Ligação a DNA/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Fusão Gênica Artificial , Bacteriófago M13/genética , Sítios de Ligação , Proteínas do Capsídeo , Proteínas de Ligação a DNA/genética , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Dados de Sequência Molecular , Peptídeos/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteínas Virais de Fusão/genéticaRESUMO
PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.
Assuntos
Proteínas do Capsídeo , Capsídeo/química , Proteínas de Membrana/química , Proteínas do Tecido Nervoso/química , Biblioteca de Peptídeos , Sequência de Aminoácidos , Bacteriófago M13 , Sítios de Ligação , Sequência Conservada , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exerts a multitude of biological effects through its interaction with two receptor tyrosine kinases, fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 and kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Whereas it is commonly accepted that KDR is responsible for the proliferative activities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Based on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to Flt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage display strategy, two KDR-selective variants were discovered with three and four changes from wild-type, respectively. Both variants had approximately wild-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosphorylation and endothelial cell proliferation assays. These variants will serve as useful reagents in elucidating the roles of Flt-1 and KDR.
Assuntos
Fatores de Crescimento Endotelial/análise , Linfocinas/análise , Receptores Proteína Tirosina Quinases/análise , Receptores de Fatores de Crescimento/análise , Fatores de Crescimento Endotelial/metabolismo , Humanos , Linfocinas/metabolismo , Ligação Proteica , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Neuropilin-1 (NP-1) was first identified as a semaphorin receptor involved in neuron guidance. Subsequent studies demonstrated that NP-1 also binds an isoform of vascular endothelial growth factor (VEGF) as well as several VEGF homologs, suggesting that NP-1 may also function in angiogenesis. Here we report in vitro binding experiments that shed light on the interaction between VEGF165 and NP-1, as well as a previously unknown interaction between NP-1 and one of the VEGF receptor tyrosine kinases, VEGFR1 or Flt-1. BIAcore analysis demonstrated that, with the extracellular domain (ECD) of NP-1 immobilized at low density, VEGF165 bound with low affinity (K(d) = 2 microm) and fast kinetics. The interaction was dependent on the heparin-binding domain of VEGF165 and increased the affinity of VEGF165 for its signaling receptor VEGFR2 or kinase insert domain-containing receptor. The affinity of VEGF165 for the NP-1 ECD was greatly enhanced either by increasing the density of immobilized NP-1 (K(d) = 113 nm) or by the addition of heparin (K(d) = 25 nm). We attribute these affinity enhancements to avidity effects mediated by the bivalent VEGF165 homodimer or multivalent heparin. We also show that the NP-1 ECD binds with high affinity (K(d) = 1.8 nm) to domains 3 and 4 of Flt-1 and that this interaction inhibits the binding of NP-1 to VEGF165. Based on these results, we propose that NP-1 acts as a coreceptor for various ligands and that these functions are dependent on the density of NP-1 on the cell membrane. Furthermore, Flt-1 may function as a negative regulator of angiogenesis by competing for NP-1.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Heparina/fisiologia , Humanos , Camundongos , Neovascularização Fisiológica/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuropilina-1 , Ligação Proteica , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Fatores de Crescimento Endotelial/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Linfocinas/imunologia , Alanina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Antígenos/química , Antígenos/imunologia , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Cristalografia por Raios X , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/química , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/farmacologia , Linfocinas/antagonistas & inibidores , Linfocinas/química , Linfocinas/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Biblioteca de Peptídeos , Conformação Proteica , Termodinâmica , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Formation of a complex between Apo2L (also called TRAIL) and its signaling receptors, DR4 and DR5, triggers apoptosis by inducing the oligomerization of intracellular death domains. We report the crystal structure of the complex between Apo2L and the ectodomain of DR5. The structure shows three elongated receptors snuggled into long crevices between pairs of monomers of the homotrimeric ligand. The interface is divided into two distinct patches, one near the bottom of the complex close to the receptor cell surface and one near the top. Both patches contain residues that are critical for high-affinity binding. A comparison to the structure of the lymphotoxin-receptor complex suggests general principles of binding and specificity for ligand recognition in the TNF receptor superfamily.
Assuntos
Apoptose , Glicoproteínas de Membrana/química , Receptores do Fator de Necrose Tumoral/química , Fator de Necrose Tumoral alfa/química , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Linhagem Celular , Cristalografia por Raios X , Escherichia coli , Linfotoxina-alfa/química , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/química , Alinhamento de Sequência , Ligante Indutor de Apoptose Relacionado a TNFRESUMO
The human growth hormone (hGH)-receptor interaction was used to study the relationship between hormone-receptor affinity and bioactivity. hGH has two nonequivalent sites, called site 1 and site 2, that bind two molecules of receptor in a sequential fashion. We produced both site 1 and site 2 high-affinity hGH variants either by combining alanine mutants previously found to improve affinity at site 1 or by random mutagenesis of residues in site 2 followed by phage display and receptor binding selections. The two high-affinity variants, as well as one which combined them, were used in cell proliferation assays with FDC-P1 cells expressing the hGH receptor. Interestingly, none of these variants produced a change in the EC50 for cell proliferation or the levels of JAK2 tyrosine kinase phosphorylation. Next we studied the effect of a reduction in site 1 affinity on cell proliferation. A systematic series of hGH mutants were produced in which affinity for site 1 was reduced from 5- to 500-fold. Surprisingly, the EC50 for cell proliferation was unaffected until affinity was reduced about 30-fold from wild-type hGH. Thus, native hGH-receptor affinity is much higher than it needs to be for maximal JAK2 phosphorylation or cell proliferation. These studies begin to define basic functional tolerances for receptor activation that need to be considered in the design of hGH mimics.
Assuntos
Hormônio do Crescimento Humano/metabolismo , Proteínas Proto-Oncogênicas , Receptores da Somatotropina/metabolismo , Animais , Bacteriófagos/genética , Sítios de Ligação/genética , Divisão Celular/genética , Linhagem Celular , Sequência Consenso , Vetores Genéticos/síntese química , Hormônio do Crescimento Humano/genética , Humanos , Janus Quinase 2 , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.
Assuntos
Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Dimerização , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/química , Fatores de Crescimento Endotelial/metabolismo , Humanos , Imunoglobulina G/metabolismo , Linfocinas/antagonistas & inibidores , Linfocinas/química , Linfocinas/metabolismo , Ligação Proteica , Receptores de Fatores de Crescimento do Endotélio Vascular , Especificidade por Substrato , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Peptides that inhibit binding of vascular endothelial growth factor (VEGF) to its receptors, KDR and Flt-1, have been produced using phage display. Libraries of short disulfide-constrained peptides yielded three distinct classes of peptides that bind to the receptor-binding domain of VEGF with micromolar affinities. The highest affinity peptide was also shown to antagonize VEGF-induced proliferation of primary human umbilical vascular endothelial cells. The peptides bind to a region of VEGF known to contain the contact surface for Flt-1 and the functional determinants for KDR binding. This suggests that the receptor-binding region of VEGF is a binding "hot spot" that is readily targeted by selected peptides and supports earlier assertions that phage-derived peptides frequently target protein-protein interaction sites. Such peptides may lead to the development of pharmacologically useful VEGF antagonists.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Peptídeos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Crescimento Endotelial/química , Endotélio Vascular/citologia , Inibidores do Crescimento/farmacologia , Humanos , Linfocinas/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Distribuição Aleatória , Receptores Proteína Tirosina Quinases/química , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor (VEGF) is a homodimeric hormone that induces proliferation of endothelial cells through binding to the kinase domain receptor and the Fms-like tyrosine kinase receptor (Flt-1), the extracellular portions of which consist of seven immunoglobulin domains. We show that the second and third domains of Flt-1 are necessary and sufficient for binding VEGF with near-native affinity, and that domain 2 alone binds only 60-fold less tightly than wild-type. The crystal structure of the complex between VEGF and the second domain of Flt-1 shows domain 2 in a predominantly hydrophobic interaction with the "poles" of the VEGF dimer. Based on this structure and on mutational data, we present a model of VEGF bound to the first four domains of Flt-1.
Assuntos
Fatores de Crescimento Endotelial/química , Fatores de Crescimento Endotelial/genética , Linfocinas/química , Linfocinas/genética , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/química , Células Cultivadas , Cristalografia , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Dados de Sequência Molecular , Mutagênese , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Homologia de Sequência de Aminoácidos , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Derivatives of human growth hormone (hGH) of increasing size were produced by reaction with the N-hydroxysuccinimide ester of polyethylene glycol-5000 (PEG5000), a 5-kDa reagent that selectively conjugates to primary amines. By adjusting the reaction conditions and purification procedure, it was possible to isolate hGH derivatives containing up to seven PEG moieties that altered the Stokes radius and thereby the effective molecular masses of the unmodified hormone from 22 to 300 kDa. Fortunately, the most reactive amines were ones that did not lie in either of the two sites important for receptor binding. Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor and increased the EC50 in a cell-based assay up to 1500-fold. Most of the reduction in affinity was the result of slowing the association rate for the receptor. The clearance rate of hGH in rats was inversely proportional to effective molecular weight and closely fit a filtration model. We have tested the potency of these analogs by injecting them daily or every 6 days into hypophysectomized rats and determining the effects on body and organ growth. The efficacy of these analogs was optimal for hGH conjugated with 5 eq of PEG5000, and the potency was increased by about 10-fold compared with unmodified hGH. Such PEG-hGH derivatives show promise as long-acting alternatives to daily injections of hGH. More generally these studies show that improving hormone clearance properties, even at the expense of reducing receptor binding affinity, can lead to dramatic increases in hormone efficacy.
Assuntos
Hormônio do Crescimento/síntese química , Polietilenoglicóis/química , Animais , Sítios de Ligação , Divisão Celular , Ensaio de Imunoadsorção Enzimática , Excipientes , Hormônio do Crescimento/farmacologia , Meia-Vida , Humanos , Hipofisectomia , Cinética , Peso Molecular , Conformação Proteica , Ratos , Receptores da Somatotropina/genética , Timidina/metabolismo , Transfecção , Aumento de Peso/efeitos dos fármacosRESUMO
We investigated the mechanism of action of the human prolactin (hPRL) receptor on four different breast cancer cell lines, T-47D, MCF-7, BT-474, and SK-BR3, that express elevated levels of the receptor compared with normal cells. Cells treated with human growth hormone (hGH), which binds and activates the hPRL receptor, exhibited bell-shaped dose-response growth curves consistent with the sequential dimerization mechanism proposed for the hPRL receptor (Fuh, G., Colosi, P., Wood, W.I., and Wells, J.A. (1993) J. Biol. Chem. 268, 5376-5381). Growth stimulation was enhanced by Zn2+, which preferentially increases the affinity of hGH for the hPRL receptor. Furthermore, receptor-selective variants of hGH that bind the hPRL receptor but not the hGH receptor were agonistic, providing additional support that specific binding to the hPRL receptor can stimulate these breast cancer cells to grow. On this basis we produced variants of hGH and human placental lactogen (hPL) that were potential antagonists because they bind but do not dimerize the hPRL receptor. The hPL-based antagonist was less potent than the hGH-based antagonist toward the growth of MCF-7 cells, consistent with the lower affinity of hPL for hPRL receptor than for hGH. However, the hPL-based antagonist was more potent than the hGH antagonist for BT-474 cells. Antibodies to the hPRL receptor inhibited growth of FDC-P1 cells transfected with the hPRL receptor; these also inhibited MCF-7 cells and T47D cells but not BT-474 cells. A unique feature of BT-474 cells was found when screening its cDNA revealed the presence of a novel alternative splice of the hPRL receptor that codes for the soluble extracellular domain; this may explain these differential inhibitory effects. These studies provide further molecular insight into the potential role of the hPRL receptor in breast cancer and demonstrate that hPRL receptor antagonists can inhibit the growth of breast cancer cells.
Assuntos
Neoplasias da Mama/patologia , Receptores da Prolactina/antagonistas & inibidores , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , DNA Complementar , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Células Tumorais CultivadasRESUMO
The mechanism of action of two forms of the prolactin (PRL) receptor was studied using analogs of human growth hormone (hGH). At low concentrations (approximately 1 pM), hGH binds and stimulates proliferation of Nb2 cells containing the 391-residue PRL receptor as well as murine lymphoid FDC-P1 cells transfected with the 591-residue hPRL receptor. However, at high concentrations (approximately 70 microM) hGH inhibits proliferation of both these cell lines. Such a "bell-shaped" hormone response curve was observed for hGH stimulation of the hGH receptor (Fuh, G., Cunningham, B.C., Fukunaga, R., Nagata, S., Goeddel, D. V., and Wells, J.A. (1992) Science 256, 1677-1680) and is consistent with the sequential formation of an active hormone-(receptor)2 complex in which hGH binds through a first site (Site 1) to a first receptor and then through a second site (Site 2) to a second receptor. By analogy to hGH activation of the hGH receptor, we find that hGH variants that are mutated in Site 1 or Site 2 are greatly reduced as agonists. Similarly, only Site 2 mutants are potent antagonists of either hGH or hPRL stimulated cell proliferation. These and other data support the notion that hGH and hPRL activate the PRL receptor by sequential dimerization and provide a rational basis for the design of potent antagonists to the prolactin receptor.
Assuntos
Hormônio do Crescimento/farmacologia , Receptores da Prolactina/antagonistas & inibidores , Animais , Sítios de Ligação , Hormônio do Crescimento/análogos & derivados , Humanos , Fígado/metabolismo , Camundongos , Prolactina/metabolismo , Ratos , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/metabolismo , Células Tumorais CultivadasRESUMO
High-resolution mutational and structural analyses of purified components have revealed a great deal about the molecular basis for growth hormone action. The structural and functional aspects of the interactions between hGH and its receptors have been largely elaborated. From these studies it has been possible to engineer homologues of hGH to bind to the hGH receptor and act as potential antagonists. Receptor-selective and high-affinity analogs have also been constructed based on a combination of alanine scanning and monovalent phage display. From this molecular work much has been revealed about the biology of hGH (Fig.9). Our data suggest that hGH is stored in the pituitary as a (Zn2+,hGH)2 complex. On release from somatotropic vesicles it dissociates into a monomeric form and reveals its primary receptor binding site (site 1). Free hGH can bind to the hGHbp in serum to form monomeric or dimeric complexes that slow the clearance of hGH (Moore et al., 1989). However, because the affinity for the full-length receptor is greater, hGH can bind to it preferentially. Furthermore, the constitutive levels of the hGHbp (approximately 0.5 to 1 nM) (Baumann et al., 1986; Herrington et al., 1986) are considerably below the levels of hGH after pulsatile release (approximately 2 to 5 nM) (Thompson et al., 1972). Our data indicate that hGH binds to the hGH receptor on cell membranes through site 1 and subsequently forms dimers through site 2. We believe a similar process may occur for hGH to activate the hPRL receptor, except that Zn2+ is required for site 1 association. Such receptor dimers are then activated and capable of interacting with other cellular components that may mediate the hGH "signal." Recently, based upon this proposed mechanism, we produced potent antagonists to the hGH receptor (Fuh et al., 1992) and hPRL receptor (G. Fuh, P. Colosi, W. Wood, and J. Wells, unpublished results). These antagonists bind tightly to site 1 but are blocked in their ability to bind site 2 and dimerize the receptor. We believe these methods and discoveries will be relevant to the study of signaling by other hematopoietic hormones and receptors as well as other hormones and receptors.
Assuntos
Hormônio do Crescimento/metabolismo , Receptores da Somatotropina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Hormônio do Crescimento/química , Hormônio do Crescimento/genética , Humanos , Dados de Sequência Molecular , Mutagênese , Lactogênio Placentário/metabolismo , Prolactina/metabolismo , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/químicaRESUMO
A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially--a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.
Assuntos
Receptores da Somatotropina/fisiologia , Anticorpos Monoclonais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hormônio do Crescimento/análise , Hormônio do Crescimento/fisiologia , Humanos , Modelos Moleculares , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Recombinant human growth hormone (HuGH) and human prolactin (HuPRL), but not GH of bovine or porcine origin, prime human neutrophils for enhanced superoxide anion (O2-) secretion. Since HuGH, but not GH of other species, effectively binds to the HuPRL receptor (HuPRL-R), we used a group of HuGH variants created by site-directed mutagenesis to identify the receptor on human neutrophils responsible for HuGH priming. A monoclonal antibody (MAb) directed against the HuPRL-R completely abrogated O2- secretion by neutrophils incubated with either HuGH or HuPRL, whereas a MAb to the HuGH-R had no effect. The HuGH variant K172A/F176A, which has reduced affinity for both the HuGH-binding protein (BP) and the HuPRL-BP, was unable to prime human neutrophils. This indicates that priming is initiated by a ligand-receptor interaction, the affinity of which is near that defined for receptors for PRL and GH. Another HuGH variant, K168A/E174A, which has relatively low affinity for the HuPRL-BP but slightly increased affinity for the HuGH-BP, had much reduced ability to prime neutrophils. In contrast, HuGH variant E56D/R64M, which has a similar affinity as wild-type HuGH for the HuPRL-BP but a lower affinity for the HuGH-BP, primed neutrophils as effectively as the wild-type HuGH. Finally, binding of HuGH to the HuPRL-BP but not to the HuGH-BP has been shown to be zinc dependent, and priming of neutrophils by HuGH was also responsive to zinc. Collectively, these data directly couple the binding of HuGH to the HuPRL-R with one aspect of functional activation of human target cells.
Assuntos
Hormônio do Crescimento/farmacologia , Neutrófilos/metabolismo , Receptores da Prolactina/fisiologia , Superóxidos/metabolismo , Adulto , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Divisão Celular , Células Cultivadas , Hormônio do Crescimento/metabolismo , Humanos , Linfoma/patologia , Suínos , Zinco/farmacologiaRESUMO
Human growth hormone (hGH) elicits a diverse set of biological activities including lactation that derives from binding to the prolactin (PRL) receptor. The binding affinity of hGH for the extracellular binding domain of the hPRL receptor (hPRLbp) was increased about 8000-fold by addition of 50 micromolar ZnCl2. Zinc was not required for binding of hGH to the hGH binding protein (hGHbp) or for binding of hPRL to the hPRLbp. Other divalent metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Co2+) at physiological concentrations did not support such strong binding. Scatchard analysis indicated a stoichiometry of one Zn2+ per hGH.hPRLbp complex. Mutational analysis showed that a cluster of three residues (His18, His21, and Glu174) in hGH and His188 from the hPRLbp (conserved in all PRL receptors but not GH receptors) are probable Zn2+ ligands. This polypeptide hormone.receptor "zinc sandwich" provides a molecular mechanism to explain why nonprimate GHs are not lactogenic and offers a molecular link between zinc deficiency and its association with altered functions of hGH.
Assuntos
Cloretos/farmacologia , Hormônio do Crescimento/metabolismo , Receptores da Prolactina/metabolismo , Compostos de Zinco , Zinco/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Plasmídeos , Conformação Proteica , Receptores da Prolactina/efeitos dos fármacos , Receptores da Prolactina/genética , Mapeamento por Restrição , Zinco/metabolismoRESUMO
A gene fragment encoding the extracellular domain of the human growth hormone (hGH) receptor from liver was cloned into a plasmid under control of the Escherichia coli alkaline phosphatase promoter and the heat-stable enterotoxin (StII) signal peptide sequence. Strains of E. coli expressing properly folded hGH binding protein were identified by blotting colonies with 125I-hGH. The E. coli strain capable of highest expression (KS330) secreted 10 to 20 mg/liter of culture of properly processed and folded hGH receptor fragment into the periplasmic space. The protein was purified to near homogeneity in 70 to 80% yield (in tens of milligram amounts) using ammonium sulfate precipitation, hGH affinity chromatography, and gel filtration. The unglycosylated extracellular domain of the hGH receptor has virtually identical binding properties compared to its natural glycosylated counterpart isolated from human serum, suggesting glycosylation is not important for binding of hGH. The extracellular binding domain codes for 7 cysteines, and we show that six of them form three disulfide bonds. Peptide mapping studies show these disulfides are paired sequentially to produce short loops (10-15 residues long) as follows: Cys38-Cys48, Cys83-Cys94, and Cys108-Cys122. Cys241 is unpaired, and mutagenic analysis shows that the extreme carboxyl end of the receptor fragment (including Cys241) is not essential for folding or binding of the protein to hGH. High level expression of this receptor binding domain and its homologs in E. coli will greatly facilitate their detailed biophysical and structural analysis.
Assuntos
Cisteína , Escherichia coli/genética , Receptores da Somatotropina/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dissulfetos/análise , Genes , Cinética , Fígado/metabolismo , Dados de Sequência Molecular , Plasmídeos , Conformação Proteica , Receptores da Somatotropina/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Microvascular endothelial cells isolated from abdominal skin of diabetic and nondiabetic adults were maintained in culture by serial passage. Both cell types showed typical endothelial cell morphology, expressed factor VII-associated antigen, contained Weibel-Palade bodies, and produced an extensive subendothelial extracellular matrix containing type IV (basement membrane) procollagen. Biosynthetic studies using radioactive amino acids indicated that under the conditions of cell culture the matrix proteins newly synthesized by MEC of both cell types were similar in type and amount. Both cell types produced type IV procollagen, laminin, and fibronectin, which were deposited in the matrix. Electron microscopy showed that the matrices of both cell types had a similar multilayered, discontinuous, filamentous ultrastructure. Immunoperoxidase staining showed type IV collagen to be distributed similarly, in a fibrillar meshwork, in both matrices. The extractability of individual matrix macromolecules from both matrices was identical; 4 M urea or guanidine-HCl partially removed fibronectin and thrombospondin, but reducing agent was required to solubilize type IV procollagen. The results suggest that diabetic microangiopathy is not due to an inherent defect in the endothelium, and that this in vitro system may be useful for examining environmental factors possibly involved in its development.