Biomedical subjects

B C Cunningham

Publications and source records attributed to B C Cunningham.

At least 19 recordsLinked to original sources

PubMed1999

Growth hormone binding affinity for its receptor surpasses the requirements for cellular activity.

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.

Animals↗

PubMed1998

Novel peptides selected to bind vascular endothelial growth factor target the receptor-binding site.

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.

Amino Acid Sequence↗

PubMed1998

Crystal structure of the complex between VEGF and a receptor-blocking peptide.

Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and, therefore, a prime therapeutic target for the development of antagonists for the treatment of cancer. As a first step toward this goal, phage display was used to generate peptides that bind to the receptor-binding domain (residues 8-109) of VEGF and compete with receptor [Fairbrother, W. J., Christinger, H. W., Cochran, A. G., Fuh, G., Keenan, C. J., Quan, C., Shriver, S. K., Tom, J. Y. K., Wells, J. A., and Cunningham, B. C. (1999) Biochemistry 38, 17754-17764]. The crystal structure of VEGF in complex with one of these peptides was solved and refined to a resolution of 1.9 A. The 20-mer peptide is unstructured in solution and adopts a largely extended conformation when bound to VEGF. Residues 3-8 form a beta-strand which pairs with strand beta6 of VEGF via six hydrogen bonds. The C-terminal four residues of the peptide point away from the growth factor, consistent with NMR data indicating that these residues are flexible in the complex in solution. In contrast, shortening the N-terminus of the peptide leads to decreased binding affinities. Truncation studies show that the peptide can be reduced to 14 residues with only moderate effect on binding affinity. However, because of the extended conformation and the scarcity of specific side-chain interactions with VEGF, the peptide is not a promising lead for small-molecule development. The interface between the peptide and VEGF contains a subset of the residues recognized by a neutralizing Fab fragment and overlaps partially with the binding site for the Flt-1 receptor. The location of the peptide-binding site and the hydrophilic character of the interactions with VEGF resemble more the binding mode of the Fab fragment than that of the receptor.

Amino Acid Sequence↗

PubMed1998

VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 A resolution and mutational analysis of the interface.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a highly specific angiogenic growth factor; anti-angiogenic treatment through inhibition of receptor activation by VEGF might have important therapeutic applications in diseases such as diabetic retinopathy and cancer. A neutralizing anti-VEGF antibody shown to suppress tumor growth in an in vivo murine model has been used as the basis for production of a humanized version. RESULTS: We present the crystal structure of the complex between VEGF and the Fab fragment of this humanized antibody, as well as a comprehensive alanine-scanning analysis of the contact residues on both sides of the interface. Although the VEGF residues critical for antibody binding are distinct from those important for high-affinity receptor binding, they occupy a common region on VEGF, demonstrating that the neutralizing effect of antibody binding results from steric blocking of VEGF-receptor interactions. Of the residues buried in the VEGF-Fab interface, only a small number are critical for high-affinity binding; the essential VEGF residues interact with those of the Fab fragment, generating a remarkable functional complementarity at the interface. CONCLUSIONS: Our findings suggest that the character of antigen-antibody interfaces is similar to that of other protein-protein interfaces, such as ligand-receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptide chain, whereas those essential for binding to the receptor are distributed over four different segments and span across the dimer interface.

Alanine↗

PubMed1997

Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site.

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor beta2 (TGF-beta). We have determined its crystal structure at a resolution of 2.5 A, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-beta. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional "hot spots" composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-beta. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

Binding Sites↗

PubMed1997

Minimized proteins.

The design of bioactive small molecules for interaction at larger protein-protein interfaces remains a challenge. Recent progress towards minimizing proteins into significantly smaller polypeptides has been achieved via both rational design processes and selection from vast combinatorial libraries. Such 'mini-proteins' represent a potential intermediate step toward the development of drugs targeted to protein-protein interfaces.

Amino Acid Sequence↗

PubMed1996

Crystallization of the receptor binding domain of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the production of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P2(1), with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, beta = 90.0 degrees, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF.

Binding Sites↗

PubMed1996

Recombinant human growth hormone (GH)-binding protein enhances the growth-promoting activity of human GH in the rat.

To address the role of the GH-binding protein (GHBP) in GH physiology, two forms of recombinant human GHBP (rhGHBP) were given alone or in combination with rhGH to hypophysectomized rats or GH-deficient dwarf rats. Hypophysectomized rats were given daily sc injections of excipient, hGH, rhGHBP, or rhGH plus rhGHBP (produced in Escherichia coli) for 7 days. Injections of rhGH induced dose-related body weight gain and bone growth that were increased by the coadministration of rhGHBP with rhGH; rhGHBP alone had no effect. Serum insulin-like growth factor increased 24 h later when rhGH was given together with rhGHBP (P < 0.01), but not when rhGH was given alone. E. coli-derived rhGHBP also enhanced the bioactivity of coadministered rhGH in the GH-deficient dwarf rat. In contrast, the glycosylated rhGHBP, made in human A293 cells, inhibited the growth-promoting activity of coadministered rhGH. The opposite effects of these two forms of rhGHBP could be explained by clearance studies that showed radiolabeled rhGH bound to A293 cell-derived rhGHBP to be cleared more rapidly from the blood than free rhGH. Natural rabbit GHBP and E. coli-derived rhGHBP both prolonged the presence of rhGH in blood. It is proposed that by slowing the clearance of GH, GHBP increased the bioactivity of GH. In summary, codelivery of rhGHBP and rhGH caused a dose-dependent enhancement of the activity of rhGH in two rat models of GH deficiency. This suggests that endogenous circulating GHBP may increase the activity of blood-borne GH in a similar manner.

Animals↗

PubMed1995

Minimization of a polypeptide hormone.

A stepwise approach for reducing the size of a polypeptide hormone, atrial natriuretic peptide (ANP), from 28 residues to 15 while retaining high biopotency is described. Systematic structural and functional analysis identified a discontinuous functional epitope for receptor binding and activation, most of which was placed onto a smaller ring (Cys6 to Cys17) that was created by repositioning the ANP native disulfide bond (Cys7 to Cys23). High affinity was subsequently restored by optimizing the remaining noncritical residues by means of phage display. Residues that flanked the mini-ring structure were then deleted in stages, and affinity losses were rectified by additional phage-sorting experiments. Thus, structural and functional data on hormones, coupled with phage display methods, can be used to shrink the hormones to moieties more amendable to small-molecule design.

Amino Acid Sequence↗

PubMed1994

Solution conformation of an atrial natriuretic peptide variant selective for the type A receptor.

Two-dimensional NMR spectroscopy has been used to characterize the solution conformation of an atrial natriuretic peptide (ANP) variant which is selective for the human natriuretic peptide receptor A (NPR-A) relative to receptor C (NPR-C). The ANP mutant, containing six substitutions, has reduced flexibility in aqueous solution relative to wild-type ANP and allows the observation of sufficient NOE connectivities for structure determination by distance geometry and restrained molecular dynamics calculations. The solution conformation is reasonably well defined, having an average backbone atom rms deviation from the average coordinates of approximately 1.1 A for residues 7-27. The structure is consistent with available functional data and shows a spatial separation between known receptor binding determinants and residues found to be outside the hormone-receptor interface.

Amino Acid Sequence↗

PubMed1994

Production of an atrial natriuretic peptide variant that is specific for type A receptor.

Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein. These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution. This method also selected ANP variants with improved secretion expression in Escherichia coli. Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C. Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.

Amino Acid Sequence↗

PubMed1993

Comparison of a structural and a functional epitope.

A comprehensive analysis of the energetic importance of the 31 side-chains buried at the interface between human growth hormone (hGH) and the extracellular binding domain of its receptor (hGHbp) has been carried out to assess the roles of contact side-chains in modulating the affinity and kinetics of binding. Each side-chain in hGH was converted to alanine, and the kinetics and affinity were measured using a biosensor device. This detects binding of the mutated hormones to the immobilized hGHbp by changes induced in refractive index. The data generated on the biosensor match affinities obtained by radio-immune assay in solution. The study shows that only one-quarter of the side-chains buried at the interface can account for the majority of the binding energy. These residues cluster near the center of the structural epitope. The role of these side-chains is predominantly to slow dissociation because most of the effect of the alanine substitutions is to increase the off-rate, not to slow the on-rate. The hormone associates about 10,000 times slower than expected from random diffusion but 1000 times faster than may be expected if one imposes strict orientation restraints for a productive collision. Electrostatic interactions partly modulate association because mutations at Arg residues most affect association and together contribute a factor of about 20 to the on-rate. The data suggest that the hormone and receptor associate by diffusion and electrostatics to form an ensemble of weak collisional complexes. From these a bound complex is produced that is stabilized by only a small proportion of the contacts. We suggest that solvation energies and/or side-chains interactions within the free hormone or receptor may be so favorable that little energy is gained at most side-chains upon binding. The fact that the functional binding epitope is much smaller than the structural epitope suggests it may be possible to design smaller hormone mimics.

Amino Acid Sequence↗

PubMed1993

Evidence that the growth hormone receptor mediates differentiation and development of the mammary gland.

We have shown that nonlactogenic rat (r) GH is far more potent than rPRL in inducing rat mammary development. To determine the relative roles of GH and PRL in mammary development and their mechanisms of action, we have compared the abilities of a group of native and mutant GHs, PRLs, and placental lactogens (PLs) to induce mammary development, bind to GH receptors, and activate lactogenic receptors. Mammary development was assessed histologically by counting terminal end buds and alveolar structures in glands from sexually immature, hypophysectomized, castrated, estradiol-treated rats. Hormones were implanted, in Elvax pellets, into the lumbar mammary gland. Significant increases in terminal end buds (P < 0.03) over internal control values were obtained with rGH, recombinant human GH (rhGH), rbGH, and one of two mutant rhGHs. These four hormones were also found to bind to GH receptors with high affinity. In contrast, little development occurred with hPRL, rPRL, rhPL, ovine PRL, mutant forms of rhPRL and rhPL, and a mutant of rhGH altered to reduce binding to GH and PRL receptors. All of these substances are more than 50-fold reduced in binding to the GH receptor, yet can bind and activate lactogenic receptors. Thus, only those natural or mutant pituitary or placental hormones with high binding affinity to GH receptors induce mammary development, suggesting that GH receptors play a central role in this process.

Animals↗

PubMed1993

The molecular basis for growth hormone-receptor interactions.

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.

Amino Acid Sequence↗

PubMed1992

Rational design of potent antagonists to the human growth hormone receptor.

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.

Antibodies, Monoclonal↗

PubMed1992

Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis.

Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11-33), the loop between the first disulfide bond and helix 2 (residues 54-74), the beginning of helix 3 (residues 109-112), the carboxyl half of helix 4 (residues 167-182), and the final carboxyl terminus segment of the molecule (residues 184-191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54-74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.

Amino Acid Sequence↗

PubMed1992

Growth hormone augments superoxide anion secretion of human neutrophils by binding to the prolactin receptor.

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.

Adult↗

PubMed1991

Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule.

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.

Amino Acid Sequence↗