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U Gether

Publications and source records attributed to U Gether.

49 records · Page 3Linked to original sources
PubMed1993

Chimeric NK1 (substance P)/NK3 (neurokinin B) receptors. Identification of domains determining the binding specificity of tachykinin agonists.

The NK1 (substance P) and NK3 (neurokinin B) receptors are G protein-coupled receptors sharing approximately 70% identity within the membrane-spanning domains. However, they each have a distinct pharmacological profile in respect of peptide binding. To identify epitopes that determine their selectivity for natural and synthetic tachykinin peptides, we constructed a series of chimeric NK1/NK3 receptors in which carboxyl-terminal segments of increasing length in the NK1 receptor were exchanged with the corresponding segments from the NK3 receptor. The general, structural integrity of the chimeric constructs was confirmed by the amphibian tachykinin peptide, eledoisin, which was recognized equally well by both of the wild-type receptors and bound with a similar or even higher affinity to all the chimeric receptors. Competition binding studies showed that the affinity of the two natural ligands, substance P and neurokinin B, changed gradually through the series of chimeric receptors indicating that several binding epitopes throughout the receptor structure are involved in the selective recognition of these peptides. However, whereas the single, largest change in binding affinity for substance P occurred when segments in the amino-terminal end of the receptor were exchanged, this occurred for neurokinin B in the carboxyl-terminal end of the receptor. The affinity of the NK1-selective ligand, [Sar9,Met(O2)11]SP, changed even more gradually through the series of chimeric receptors than that of substance P. In contrast, the NK3-selective hexapeptide, senktide, was recognized only when transmembrane segment III and IV from the NK3 receptor were incorporated into the chimeric constructs. These data suggest that several receptor domains contribute to the binding specificity of tachykinin agonists but in varying degrees for each peptide. It is concluded that the tachykinin peptides, in partially different ways, interact with multiple epitopes scattered throughout the receptor structure, but conceivably these epitopes are closely located in space around a hypothetical receptor center.

Amino Acid Sequence↗
PubMed1993

Different binding epitopes on the NK1 receptor for substance P and non-peptide antagonist.

Non-peptide ligands for peptide receptors have been discovered in several systems through file screening programs, but the mechanism of action for these candidate drugs is obscure as they do not chemically resemble the native peptides. The compound CP 96345 is a high-affinity, non-peptide antagonist of the substance P (NK1) receptor, which is important in pain perception and neurogenic inflammation. Here we identify epitopes on the NK1 receptor responsible for the specific binding of CP 96345 by systematic exchange of corresponding segments between the NK1 receptor and the homologous NK3 (neurokinin B) receptor, which does not bind the non-peptide ligand. Non-conserved residues, in two epitopes around the top of transmembrane segment V and in one epitope at the top of transmembrane segment VI, are essential for the specific action of CP 96345 on the NK1 receptor, but are surprisingly not important for the binding of the natural peptide ligand, substance P. Susceptibility to the non-peptide antagonists can be conveyed to the previously unresponsive NK3 receptor by mutational transfer of this discontinuous epitope from the NK1 receptor.

Amino Acid Sequence↗
PubMed1993

Efficient amidation of C-peptide deleted NPY precursors by non-endocrine cells is affected by the presence of Lys-Arg at the C-terminus.

Post-translational processing of peptide precursors producing amidated, biologically active peptides generally occurs in specially differentiated endocrine or neural cells. However, we have previously shown that a C-peptide-deleted precursor of neuropeptide Y (NPY1-39) in which the precursor terminates in the sequence Gly-Lys-Arg was partially amidated by the non-endocrine cell line, CHO. In the present study we show that two other non-endocrine cell lines, NIH 3T3 and BHK, also possess amidating activities and that the NPY1-39 precursor was completely converted to NPY1-36 amide by the NIH 3T3 cell line. The role of the two basic residues (Lys-Arg) in the C-terminus was studied by transfection of a construct encoding a NPY precursor terminating with glycine alone. Both the CHO and NIH 3T3 cell lines, transfected with this construct, secreted a significantly smaller fraction of NPY reactive material as amidated NPY compared to the fraction of amidated NPY secreted by the cells transfected with the NPY1-39 precursor. It is concluded that the capacity to perform C-terminal amidation appears to be a universal feature of eukaryotic cells and that the carboxypeptidase E-like enzyme influences the amidation process, beyond its known ability to remove the C-terminal basic residues.

3T3 Cells↗
PubMed1992

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.

Animals↗
PubMed1991

Comparison of peptidyl-glycine alpha-amidation activity in medullary thyroid carcinoma cells, pheochromocytomas, and serum.

With an assay based on the radioimmunological detection of the formation of the C-terminal amide function on a neuropeptide Y-like substrate, amidation enzyme activity with apparent Mr of 56,000 and 38,000 was found in pheochromocytoma extracts. The larger molecular form of amidating enzyme was also expressed and secreted from medullary thyroid carcinoma cells in a dexamethasone-suppressible way. Serum contained high levels of amidating enzyme activity with no difference between normal subjects and patients with pheochromocytomas. However, the majority of the amidating activity in serum was of much larger size, Mr between 80 and 105,000, compared to that released from the endocrine cells. No major difference was found between the molecular forms of amidation enzyme from tissues and from serum either in respect of enzyme kinetics or in respect of requirements for the cofactors copper and ascorbate. The major serum forms of enzyme were relatively independent of exogenous copper; however, they could still be quenched by cobber chelating agents. It is concluded that the molecular weight forms of the amidating enzyme circulating in serum are much larger than the soluble enzyme stored and secreted from most endocrine tissues.

Adrenal Gland Neoplasms↗
PubMed1991

PreproVIP-derived peptides in tissue and plasma from patients with VIP-producing tumours.

To elucidate the biosynthetic processing of the precursor for vasoactive intestinal peptide (prepro-VIP) in tumours producing VIP we have used newly developed radioimmunoassays directed against the five functional domains of the VIP precursor molecule: preproVIP 22-79, peptide histidine methionine (PHM), preproVIP 111-122, VIP and preproVIP 156-170 in combination with HPLC to identify and quantify the peptides in tumour specimen and plasma from patients with the watery diarrhoea syndrome. Elevated quantities of all the five peptides were found in the 13 tumours (nine neurogenic tumours, one pheochromocytoma, three pancreatic carcinomas) examined. The preproVIP derived peptides were expressed in non-equimolar amounts and the relative proportion of the various peptides differed markedly from tumour to tumour. The pheochromocytoma was the only tumour type which contained large amounts of preproVIP 156-170 in comparison with the other peptides. A proportion of the VIP precursor which varied from 7% to 73% followed a pathway in which the dibasic conversion site after PHM was uncleaved as evidenced by the presence of PHV, a C-terminally extended form of PHM. It was also found that unlike normal tissue a fraction of the C-terminal VIP precursor peptide, preproVIP 156-170, was having its C-terminal lysine residue removed during processing. The findings indicate that various post-translational processing pathways of preproVIP exist. All the peptide sequences produced in the tumour tissue were secreted as evidenced by their presence in plasma in elevated concentrations. The plasma levels of preproVIP 22-79, preproVIP 111-122 and PHV exceeded those of the remaining preproVIP-derived peptides suggesting that determination of these peptides in patients with VIP-secreting tumours may be better markers than VIP.

Adult↗
PubMed1990

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase.

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗
PubMed1990

[Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist.

Two types of binding sites have previously been described for 36-amino acid neuropeptide Y (NPY), called Y1 and Y2 receptors. Y2 receptors can bind long C-terminal fragments of NPY-e.g., NPY-(13-36)-peptide. In contrast, Y1 receptors have until now only been characterized as NPY receptors that do not bind such fragments. In the present study an NPY analog is presented, [Leu31, Pro34]NPY, which in a series of human neuroblastoma cell lines and on rat PC-12 cells can displace radiolabeled NPY only from cells that express Y1 receptors and not from those expressing Y2 receptors. The radiolabeled analog, [125I-Tyr36] monoiodo-[Leu31, Pro34]NPY, also binds specifically only to cells with Y1 receptors. The binding of this analog to Y1 receptors on human neuroblastoma cells is associated with a transient increase in cytoplasmic free calcium concentrations similar to the response observed with NPY. [Leu31, Pro34]NPY is also active in vivo as it is even more potent than NPY in increasing blood pressure in anesthetized rats. It is concluded that [Leu31, Pro34]NPY is a specific Y1 receptor agonist and that the analog or variants of it can be useful in delineating the physiological importance of Y1 receptors.

Amino Acid Sequence↗
PubMed1988

Tyrosylation and purification of peptides for radioiodination.

A simple method for tyrosylation, purification and subsequent radioiodination of peptides which lack suitable acceptor groups for iodine substitution is presented. The reagent, tert.-butyloxycarbonyl-L-tyrosine N-hydroxysuccinimide ester, was used for conjugation to the amino groups of peptides. The derivatization was performed with relatively large amounts of reagents to ensure quantitative reactions. The derivatized peptides were purified by reversed-phase high-performance liquid chromatography or by gel filtration. Subsequent radioiodination was performed with sodium [125I]iodide and the sparingly soluble tetrachlorodiphenylglycouril as the oxidative agent to minimize possible oxidative damage to the peptides. The radiolabelled peptides were subsequently purified by isocratic high-performance liquid chromatography.

Amino Acid Sequence↗
PubMed1987

High-performance liquid chromatography of phenylthiocarbamyl derivatives of amino acids and side-chain derivatized amino acids.

Amino acid analysis by precolumn derivatization with phenyl isothiocyanate and subsequent characterization of the phenylthiocarbamyl derivatives by high-performance liquid chromatography on an inexpensive Supersphere C8 column is described. The phenylthiocarbamyl derivatives of common amino acids from protein hydrolysates are separated within 15 min. Suggestions for optimization of the system with respect to the separation of closely eluting derivatives are given. The characterization of side-chain-derivatized, e.g., phosphorylated and sulphated amino acids and of a series of biologically active amino acid analogues is described.

Amino Acids↗
PubMed1986

Cat pancreatic eicosapeptide and its biosynthetic intermediate. Conservation of a monobasic processing site.

Pancreatic eicosapeptide is synthesized together with the hormone pancreatic polypeptide in a common precursor in the major endocrine cell type of the duodenal pancreas. This processing has been previously demonstrated in man and in the dog. In the present study the cat pancreatic eicosapeptide and a C-terminally extended form of this were isolated and characterized from acid/ethanol extracts of pancreas by gel filtration and reverse-phase h.p.l.c. The sequence homology in the C-terminal part of the eicosapeptides from different species was shown to continue to the other side of the monobasic cleavage site in the extended intermediate form, whereas the end of the extension differed both in chain length and amino acid sequence. It is concluded that the processing site in the intermediate form of the pancreatic eicosapeptide is an example of a proline-directed monobasic cleavage site that has been conserved during evolution.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of ligand-induced conformational states in the beta 2 adrenergic receptor.

Drugs acting at G protein coupled receptors can be classified in biological assays as either agonists, partial agonists, neutral antagonists, or as inverse agonists. Very little is known about the actual molecular events and structural changes that occur in the receptor following ligand binding and during transmission of a signal across the membrane. Therefore, the structural basis for the biological classification of drug action remains unknown. To date, the conformational state of G protein coupled receptors has been inferred from the activity of the effector enzyme modulated by the G protein. We have used two different approaches to monitor conformational changes in beta 2 adrenergic receptor. Fluorescence spectroscopy can be used to directly monitor structural changes in purified beta 2 adrenergic receptor in real-time. The emission from many fluorescent molecules is strongly dependent on the polarity of the environment in which they are located. Thus, fluorescent probes covalently bound to proteins can be used as sensitive indicators of conformational changes and protein-protein interactions. In addition, we examined functional differences between agonists and partial agonists using fusion proteins between wild-type beta 2 receptor or a constitutively active beta 2 receptor mutant and Gs alpha. These receptor-G protein fusion proteins guarantee highly efficient coupling with a defined stoichiometry. The results of these experiments will be discussed in the context of current models of G protein coupled receptor activation.

Animals↗