Structural insights into the mode of action of a pure antiestrogen.

BACKGROUND: Estrogens exert their effects on target tissues by binding to a nuclear transcription factor termed the estrogen receptor (ER). Previous structural studies have demonstrated that each class of ER ligand (agonist, partial agonist, and SERM antagonist) induces distinctive orientations in the receptor's carboxy-terminal transactivation helix. The conformation of this portion of the receptor determines whether ER can recruit and interact with the components of the transcriptional machinery, thereby facilitating target gene expression. RESULTS: We have determined the structure of rat ERbeta ligand binding domain (LBD) in complex with the pure antiestrogen ICI 164,384 at 2.3 A resolution. The binding of this compound to the receptor completely abolishes the association between the transactivation helix (H12) and the rest of the LBD. The structure reveals that the terminal portion of ICI's bulky side chain substituent protrudes from the hormone binding pocket, binds along the coactivator recruitment site, and physically prevents H12 from adopting either its characteristic agonist or AF2 antagonist orientation. CONCLUSIONS: The binding mode adopted by the pure antiestrogen is similar to that seen for other ER antagonists. However, the size and resultant positioning of the ligand's side chain substituent produces a receptor conformation that is distinct from that adopted in the presence of other classes of ER ligands. The novel observation that binding of ICI results in the complete destabilization of H12 provides some indications as to a possible mechanism for pure receptor antagonism.

Evidence of functional gastric inhibitory polypeptide (GIP) receptors in human insulinoma. Binding of synthetic human GIP 1-31 and activation of adenylate cyclase.

Specific gastric inhibitory polypeptide (GIP) receptors were characterized in human benign insulinoma plasma membranes employing [mono-[125I]iodo-Tyr10]-GIP (125I-GIP) as the radioligand. GIP 1-42 inhibited 125I-GIP binding with an IC50 value of 10(-9) M. Scatchard analysis showed two classes of binding sites: a high-affinity site (Kd = 2.23 x 10(-10) M; Bmax = 24 fmol/mg protein) and a low-affinity site (Kd = 8.39 x 10(-9) M; Bmax = 118 fmol/mg protein). A synthetic replicate of human GIP 1-31 inhibited 125I-GIP binding with an IC50 value of 10(-8) M. The GIP binding sites of human insulinoma were coupled to adenylate cyclase stimulation. GIP 1-31 regulated the adenylate cyclase activity to the same extent as GIP 1-42. The concentrations of GIP required for maximal activity ranged from 10(-9) to 10(-8) M for either GIP 1-42 or GIP 1-31. The existence of functional GIP receptors in human insulinoma substantiates our recent reports demonstrating the presence of GIP binding sites in transplantable hamster insulinoma and indicates that GIP could exert a direct control of the beta-cell function in humans through a purely endocrine pathway.

Evidence for and characterization of specific high affinity binding sites for the gastric inhibitory polypeptide in pancreatic beta-cells.

High affinity binding sites have been found in membrane preparations from hamster beta-cell tumors by using radiolabeled gastric inhibitory polypeptide (125I-GIP). HPLC of 125I-GIP resulted in two major peaks (A III and B III), with identical specific binding. It was verified that peaks A III and B III stimulate insulin release from the isolated perfused rat pancreas to an extent at least equal to that obtained with unlabeled GIP at 10(-9) M. Natural GIP competitively inhibited the binding of 125I-GIP in the range of 10(-10) -10(-6) M and half-maximal inhibition was observed at 1.9 +/- 0.19 X 10(-9) M GIP. The number of high affinity sites was 219 +/- 8 fmol/mg protein and the dissociation constant was 2.05 +/- 0.1 X 10(-9) M. None of 10 regulatory peptides tested exhibited any effect on the 125I-GIP binding at concentrations in the range of 10(-6) -10(-4) M. Consequently, saturable, high affinity and specific binding sites for the GIP have been found and characterized in the plasma membranes of beta-cells. This model can be of use in studying the interaction of GIP with its preponderant target tissue.

Nucleotide sequence analysis of cDNAs encoding a bovine galanin precursor protein in the adrenal medulla and chemical isolation of bovine gut galanin.

A description is given of the primary structure of a bovine adrenal medullary precursor protein (123 amino acids), containing a nonrepetitive galanin sequence, different in four amino acid positions from pig galanin, structural information deduced from the nucleotide sequence analysis of two cDNA clones. Pairs of lysine and arginine residues separate the galanin sequence N-terminally from the initiating methionine and a 29 amino acid leader peptide and C-terminally from a 59 amino acid peptide. The deduced amino acid composition was confirmed by the isolation and determination of the amino acid composition for bovine intestinal galanin. Northern blot analysis revealed the presence of an apparent single galanin mRNA species, of approx. 850-900 bases in size, which was present in a population of chromaffin cells scattered throughout the bovine adrenal medulla.

A C-terminal fragment of hemoglobin beta-chains in extracts of porcine upper intestine.

A 32-residue peptide has been isolated from extracts of porcine upper intestine. Amino acid sequence determination showed that the peptide is a fragment of hemoglobin, corresponding to the C-terminal part of the beta-chain. The region in the beta-chain which precedes the isolated fragment is hydrophobic (8 Leu/Val/Ile) and has no charges at 10 preceding positions. It therefore, to some extent, resembles the structure of 'signal sequences' which may suggest a specific cleavage site in hemoglobin beta-chains.

A proform of secretin with high secretin-like bioactivity.

A variant form of the heptacosapeptide amide secretin, with C-terminal -Val-Gly-Lys-Arg instead of valine amide, has been isolated from porcine upper intestinal tissue. Unexpectedly, this triacontapeptide exhibited a substantially higher bioactivity than the heptacosapeptide amide.

The primary structure of two polypeptide chains from preparations of homeostatic thymus hormone (HTH alpha and HTH beta) entire-chain identities to two histones.

The primary structures of the 2 polypeptide chains (HTH alpha and HTH beta) of the homeostatic thymus hormone (HTH) were determined. The entire structures were found to be identical to those of histones H2A and H2B, respectively, without evidence for sub-types, proteolytic processings, or other peptide fragments. The results show that suggestions for new extranuclear and hormone-like histone functions apply to HTH preparations with intact protein chains of the H2 histones.

Characterization of a polypeptide from human seminal plasma with inhibin (inhibition of FSH secretion)-like activity.

A polypeptide in preparations of 'large' form (Mr approximately 14000) material with inhibin-like activity (inhibiting FSH secretion) has been isolated from human seminal plasma. Its amino acid composition, cleavage pattern with CNBr, N-terminal sequence, and properties on reverse-phase high-performance liquid chromatography establish this inhibin-like preparation to be homogeneous. The polypeptide contains close to 130 residues, has a free N-terminal serine residue, a methionine residue in position 19, and a dibasic structure (Arg-Lys) in positions 16-17.

The preparation of biotinyl-epsilon-aminocaproylated forms of the vasoactive intestinal polypeptide (VIP) as probes for the VIP receptor.

Vasoactive intestinal polypeptide (VIP) was biotinyl-epsilon-aminocaproylated using sulfosuccinimidyl-6-(biotinamido) hexanoate thereby producing a series of products that were separated by high performance liquid chromatography (HPLC). Seven VIP-derivatives were isolated and the number and location of biotinyl-epsilon-aminocaproylation was determined by a combination of enzymatic degradation and plasma desorption mass spectrometry (PDMS). Receptor binding experiments with the VIP biotinyl-epsilon-aminocaproylated derivatives revealed IC50 values for the monobiotinyl-epsilon-aminocaproylated peptides that were 1.3-3.2 times higher than for natural VIP. All isolated biotinyl-epsilon-aminocaproylated derivatives possess VIP-like bioactivity as shown by an assay measuring pancreatic juice secretion in cat, VIP biotinyl-epsilon-aminocaproylated in position lysine being almost equipotent with natural VIP.

Chemical detection of natural peptides by specific structures. Isolation of chicken galanin by monitoring for its N-terminal dipeptide, and determination of the amino acid sequence.

We have isolated galanin from chicken intestine by monitoring for the N-terminal glycyltryptophan, which constitutes a conserved part characteristic of the peptide. This monitoring method complements that previously used for C-terminal amide detection and proves chemical monitoring of specific structures to be useful. The isolation allowed determination of the structure, which was found to be unidentical to any of the known galanins. However, N-terminal pentadecapeptide parts are identical, showing this segment to be of special importance. In addition to common substitutions at positions 16, 18, 23, 26 and 29, chicken galanin has phenylalanine at position 28, where all known mammalian galanins have leucine.

Simultaneous solubilization of high-affinity receptors for VIP and glucagon and of a low-affinity binding protein for VIP, shown to be identical to calmodulin.

Anion-exchange chromatography of solubilized pig liver cell membranes on DEAE-Sepharose gave a fraction with high affinity binding proteins for VIP and glucagon distinct from each other. Scatchard analysis indicated the presence of one binding site for VIP (Kd 1.5 +/- 0.6 nM and Bmax 1.3 +/- 0.4 pmol/mg). The order of potency for VIP-related peptides to inhibit [125I]VIP binding was: VIP > peptide histidine isoleucine amide (PHI) > rat growth hormone releasing factor (rGRF) > secretin. GTP-gamma-S inhibited [125I]VIP binding and reduced the affinity of VIP binding sites to 6.5 nM. In the same isolated fraction, [125I]glucagon binding was displaced by glucagon preferentially to oxyntomodulin, and GTP did not affect this [125I]glucagon binding. Scatchard analysis indicated the presence of one binding site for glucagon (Kd 0.08 +/- 0.03 nM and Bmax 0.31 +/- 0.01 pmol/mg). A low-affinity VIP binding protein (IC50 0.7 microM) was detected in a fraction eluting later and exhibited a peptide specificity: rGRF > VIP > VIP(10-28) > secretin > PHI. This rGRF-preferring protein (18 kDa) was purified and had a partial amino-acid sequence identical to that of calmodulin. Its [125I]VIP binding was competitively inhibited by VIP and calmidazolium in a manner similar to that for pig brain calmodulin. Thus we have co-solubilized VIP and glucagon high affinity receptors from pig liver cell membranes and separated them from VIP-binding calmodulin.

Isolation of a brain peptide identical to the intestinal PHI (peptide HI).

The isolation of a brain peptide identical to the intestinal peptide PHI (peptide HI) is described. The peptide was isolated from porcine brain extract using a chemical assay method based on its C-terminal isoleucine amide structure. The complete amino acid sequence of the peptide was found to be: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser-Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence is identical to the intestinal peptide thus demonstrating PHI to be a brain-gut peptide. The role of PHI in the central nervous system as a neurotransmitter or neuromodulator is discussed.

Isolation and primary structure of human PHI (peptide HI).

The isolation of the human form of PHI (peptide HI) is described. The peptide was purified from human colonic extracts by using a chemical method for the detection of its C-terminal amidated structure. Human PHI consists of 27 amino acid residues and the complete amino acid sequence is: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Lys-Leu-Leu-Gly-Gln-Leu-Ser- Ala-Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Met-NH2. The differences between the structures of porcine and human PHI are at position 12 (Arg/Lys replacement) and at position 27 (Ile/Met).

Characterization of hemoglobin from the lizard Uromastix hardwickii.

Hemoglobin from the tropic lizard Uromastix hardwickii was isolated. Chain separations were studied, and the whole carboxymethylated globin was cleaved with trypsin. Peptides were pre-fractionated by exclusion chromatography and finally purified by reversed phase high-performance liquid chromatography. Amino acid sequence analysis permitted ordering of peptides in alpha- and beta-chains by homology with known structures in other hemoglobins. Results show large structural variations (about 50% homology between Uromastix and viper alpha-chains) and suggest chain heterogeneity with the presence of at least two types of both the alpha- and beta-chains in the preparations.

Isolation and characterization of proSS1-32, a peptide derived from the N-terminal region of porcine preprosomatostatin.

A peptide derived from the N-terminal region of porcine prosomatostatin, proSS1-32, has been purified to homogeneity from extracts of porcine upper intestine. Amino acid analysis revealed that the peptide consists of 32 residues. The complete primary structure was determined as: A P S D P R L R Q F L Q K S L A A A A G K Q E L A K Y F L A E L. This sequence obviously comprises residues 1-32 of porcine prosomatostatin since it is identical to the corresponding sequence in human preprosomatostatin. The postulated cleavage site in porcine prosomatostatin is a Leu-Leu bond between residues 32 and 33, thus confirming previous studies of the processing of the somatostatin precursor in the rat and transgenic mouse.

Reptilian alcohol dehydrogenase. Heterogeneity relevant to class multiplicity of the mammalian enzyme.

Liver alcohol dehydrogenase of the ethanol-active type ('class I enzyme') from the lizard, Uromastix hardwickii, was purified and screened for relationships with other vertebrate forms of the enzyme. Two different acetylated N-termini (acetyl-Gly and acetyl-Ser) and further positional differences already in the N-terminal segments establish the presence of two types of protein chain. The multiplicity is different from that hitherto detected within vertebrate class I alcohol dehydrogenase isozymes but typical of that which would be expected for subunits of different classes. In particular, relationships to class II or to class II-related forms appear likely. This may indicate yet further vertebrate alcohol dehydrogenase multiplicity or discovery of a class II non-mammalian enzyme. The results give prospects of defining gene duplications corresponding to more than one alcohol dehydrogenase class split to at an early vertebrate stage.

Valosin: isolation and characterization of a novel peptide from porcine intestine.

The isolation and primary structure of a novel gastrointestinal peptide, designated valosin, is described. The peptide was purified from porcine upper gut extracts using an HPLC and N-terminal sequence screening strategy which depends on chromatographic and structural characteristics as isolation criterion. The amino acid sequence of this peptide consists of 25 amino acid residues:

A porcine gut polypeptide identical to the pancreatic hormone PP (pancreatic polypeptide).

A peptide hormone has been isolated from porcine intestine. Its primary structure was found to consist of 36 amino acid residues in a sequence identical to that of the porcine pancreatic polypeptide, previously not isolated from intestines or a tissue other than pancreas. The gut polypeptide significantly suppresses glucose-induced insulin secretion in vitro. Using an immunohistochemical technique, we also identified cells in the porcine gastrointestinal tract that were immunoreactive with pancreatic polypeptide. The immunoreactivity disappeared after absorption with the isolated gut polypeptide or synthetic human pancreatic polypeptide.

Structural insights into the mechanisms of agonism and antagonism in oestrogen receptor isoforms.

Here we summarise the results that have emerged from our structural studies on the oestrogen receptor (ER) ligand-binding domain. We have investigated the conformational effects of a variety of ligands on the structures of both ER isoforms. Each class of ligand (agonists, partial agonists and selective oestrogen receptor modulators) induces a unique conformation in the receptor's ligand-dependent transcriptional activation function. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity and the structural changes that underlie receptor agonism and antagonism.

Structural requirements for gastric inhibitory polypeptide (GIP) receptor binding and stimulation of insulin release.

The effect of bovine GIP 1-42 and several of its fragments in competing with the binding of 125I-GIP to beta-cell plasma membranes from transplantable hamster insulinoma, and in stimulating insulin release from the isolated perfused rat pancreas, was investigated. Our results, in association with the results of previous studies, indicate that the sequence 17-38 is necessary for receptor binding and biological activity of GIP. By contrast, the N-terminal portion of GIP can be removed without seriously impairing the activity of the molecule.