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Query: UNIPROT:P01350 (gastrin)
 9,683 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Double-tailed lipo-tetragastrin derivatives of increasing fatty acid chain length were used to identify the minimum size of the fatty acid moieties (> or = C10) that restricts the access to the CCK-B/gastrin (CCK: cholecystokinin) receptor via a membrane-bound pathway. Then dimyristoyl-mercaptoglycerol/maleoyl-gastrin adducts of increasing peptide chain length were synthesized to define the minimal peptide size required for receptor binding affinities comparable, to those of underivatized gastrin peptides despite anchorage of the lipid tails in the membrane bilayer. The experimental results indicated that most of the little-gastrin sequence, i.e., 2-17, is needed for optimal interaction of the molecule with the binding cleft of the receptor. From these data experimentally based restraints could be derived for docking of lipo-gastrin onto a CCK-B/gastrin receptor model applying molecular dynamics simulations and energy minimizations. In the receptor-bound state some of the secondary structure elements of gastrin as determined by nmr analysis of gastrin-peptides in low dielectric constant media are retained. The N-terminal gastrin portion interacts in a more or less extended conformation with the receptor surface, and upon a sharp kink at the Ala-Tyr dipeptide portion the C-terminal pentapeptide amide part inserts deeply into the helix bundle. Besides Arg-57 on top of helix 1 of the receptor, for which no potential interaction with the ligand could be detected, the other amino acid residues identified by mutagenesis studies as involved in gastrin recognition were found to interact with the C-terminal portion of gastrin. Even taking into account the strong limitations of such a model system, it represents an interesting tool for rationalizing the experimental results of the extensive structure-function studies performed previously on gastrin and to delineate more precisely the putative ligand binding site on the extracellular face of the receptor.
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PMID:Mapping of ligand binding sites of the cholecystokinin-B/gastrin receptor with lipo-gastrin peptides and molecular modeling. 912 41

The cholecystokinin (CCK) receptor types A and B (CCKAR and CCKBR) are G protein-coupled receptors with approximately 50% amino acid identity; both have high affinity for the sulfated CCK octapeptide (CCK-8), whereas only the CCKBR has high affinity for gastrin. Previously, we identified five amino acids in the second extracellular loop (ECL) of the CCKBR that were essential for gastrin selectivity. Subsequent mutagenesis of one of these five amino acids (H207F) resulted in the loss of radiolabeled CCK-8 binding. CCK-8 stimulated total inositol phosphate accumulation in COS-1 cells transiently expressing the CCKBR-H207F with full efficacy and a 3044-fold reduced potency, which suggests that the loss of radioligand binding was caused by a loss in affinity. Alanine scanning mutagenesis was performed on the amino terminus near the top of transmembrane domain I (TMI) and on ECL1, two extracellular domains implicated in ligand binding by previous mutagenesis studies. 125I-Bolton-Hunter-CCK-8 binding to mutant receptors transiently expressed in COS-1 identified one nonconserved amino acid, R57A, at the top of TMI that caused a 21-fold reduction in CCK-8 affinity and four conserved amino acids, N115A, L116A, F120A and F122A, in the ECL1 that caused a 15.6-, 6-, 440-, and 8-fold reduction in affinity or efficacy. Alanine substitution of the equivalent amino acids in the CCKAR corresponding to each of the five amino acids in ECL1 and ECL2 affecting CCK-8 affinity for the CCKBR revealed only two mutations, L103A and F107A, that decreased CCK-8 affinity (68- and 2885-fold, respectively). These data suggest that CCK-8 interacts at multiple contact points in the extracellular domains of CCK receptors and that the CCKAR and CCKBR have distinct binding sites despite their shared high affinity for CCK-8.
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PMID:Role of the extracellular domains of the cholecystokinin receptor in agonist binding. 968 78

To understand molecular basis of Gs coupling to cholecystokinin (CCK)-A and CCK-B receptor subtypes, we examined cAMP responses in three sets of human CCK receptor mutants expressed in human embryonic kidney (HEK)293 cells. Single or double substitutions of the four nonconserved amino acids in the first intracellular loop of the CCK-BR were made with their CCK-AR counterparts to determine which residues are critical in Gs coupling. Single substitution of Ser82 to Asn, produced maximal cAMP responses comparable with the chimeric CCK-BR containing the entire first intracellular loop of the CCK-AR. Two other single substitutions, Leu81 to Arg and Leu85 to Met, produced significant but smaller cAMP responses. Ser82 was further changed into Asp, Thr, or Ala to determine the specificity of this position in Gs coupling by the CCK-BR. Replacements of Ser to Asp or Thr showed significant cAMP increases but the stimulatory effects were smaller than Ser to Asn, whereas Ser to Ala did not enhance any cAMP response to either CCK or gastrin. Finally, CCK-AR reverse mutants were studied to compare them with their corresponding CCK-BR mutants that showed increased cAMP responses. Substitution of CCK-AR residue Arg68 to Leu resulted in a complete loss of cAMP response, whereas Asn69 to Ser or Met72 to Leu showed markedly diminished cAMP responses. These data identify that specific residues in the first intracellular loop of both CCK receptor subtypes are critical for Gs coupling. Substitution of a single residue Ser82 to Asn in the CCK-BR is sufficient to confer full cAMP responses to agonist stimulation.
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PMID:Single amino acid substitution of serine82 to asparagine in first intracellular loop of human cholecystokinin (CCK)-B receptor confers full cyclic AMP responses to CCK and gastrin. 1022 May 57

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.
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PMID:A synthetic glycine-extended bombesin analogue interacts with the GRP/bombesin receptor. 1096 39

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.
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PMID:Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity. 1127 2

Neuroendocrine peptides mature partly through endoproteolytic processing of long precursor forms. Best characterised is cleavage at mono- and dibasic residues, but additional sites also exist. Among these is post-Phe cleavage, first suggested to participate in the processing of chicken progastrin. In order to characterise this new mechanism, antibodies recognising the processing products of post-Phe cleavage of chicken progastrin were produced for radioimmunoassay measurements and immunocytochemistry. High concentrations of the carboxyamidated C-terminus and the N-terminus of gastrin-53 were measured in extracts of the antrum. In addition, significant amounts were detected using an assay specific for the N-terminus of gastrin-30 and with another assay for the C-terminus of the corresponding peptide, gastrin-53(1-23), obtained after cleavage at the Phe(23)-Ala(24) bond of gastrin-53. Colocalisation in antral G-cells of the N-termini of gastrin-53 and gastrin-30 and of the C-terminus of gastrin-53(1-23) was confirmed by immunohistochemistry. Finally, we identified the intact N-terminal 1-23 fragment of gastrin-53 complementary to gastrin-30, verifying endoproteolytic cleavage at the Phe(23)-Ala(24) bond. Taken together, the results support the existence of vertebrate endoprotease cleaving hormone precursors at post-Phe sites.
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PMID:A processing enzyme cleaving avian progastrin at post-Phe bonds. 1134 92

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.
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PMID:Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties. 1252 98

The cholecystokinin 2 receptor (CCK2R) increases proliferation of normal and neoplastic gastrointestinal cells and activates various mitogenic signaling pathways when stimulated by gastrin. To study the incidence of permanent activation of this receptor in tumorigenicity, a constitutively active mutant was generated by replacing residue Glu151 in the conserved E/DRY motif by Ala. Expression of the E151A-CCK2R mutant in NIH-3T3 cells causes ligand-independent activation of phospholipase C and ornithine decarboxylase, two enzymes critical for mitogenesis. Strikingly, the constitutive activity of this mutant was associated with dramatic alteration of NIH-3T3 cell morphology, enhanced cell proliferation and invasion. Moreover, injection of cells expressing E151A-CCK2R in nude mice resulted in the development of large and rapidly growing tumors. By contrast, none of these effects was observed with cells expressing the wild-type CCK2R, indicating that the tumorigenic properties of the E151A-CCK2R mutant is the result of its constitutive activation. To date, this is the first report that provides evidence for the high tumorigenic effect of a constitutively active CCK2R mutant, thus raising a potential role of the CCK2R in human cancer.
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PMID:High tumorigenic potential of a constitutively active mutant of the cholecystokinin 2 receptor. 1295 87

Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-beta homoglutaminyl residue in the peptide H-beta homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo(N epsilon-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates [Gln1]-gastrin, [Gln1]-neurotensin and [Gln1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.
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PMID:Substrate specificity of glutaminyl cyclases from plants and animals. 1471

Amidated forms of the peptide hormone gastrin act via the cholecystokinin-2 receptor to stimulate gastric acid secretion, whereas non-amidated forms stimulate colonic mucosal proliferation via a novel, as yet uncharacterised, receptor. Nuclear magnetic resonance (NMR) and fluorescence spectroscopic studies have revealed that glycine-extended gastrin17 bound two ferric ions, and that ferric ion binding was essential for biological activity. We have therefore investigated the role of ferric ions in the biological activity of amidated gastrin17. As with glycine-extended gastrin17, fluorescence quenching experiments indicated that Glu7 Ala and Glu8,9 Ala mutants of amidated gastrin17 each bound only one ferric ion. The affinity of the mutant peptides for the cholecystokinin-2 receptor on transfected COS-7 cells or on Tlymphoblastoid Jurkat cells, and their potency in stimulation of proliferation in Jurkat cells and inositol phosphate production in transfected COS-7 cells, were similar to the values obtained for amidated gastrin17. In addition, the iron chelator desferrioxamine did not significantly inhibit either binding of amidated gastrin17 to the cholecystokinin-2 receptor, or stimulation of inositol phosphate production by amidated gastrin17 in transfected COS-7 cells. We conclude that, in contrast to glycine-extended gastrin17, binding of ferric ions is not essential for the biological activity of amidated gastrin17. Our results support the concept of distinct modes of action for amidated and non-amidated gastrins, and raise the possibility of developing selective antagonists of the actions of non-amidated and amidated gastrins.
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PMID:Divergent roles for ferric ions in the biological activity of amidated and non-amidated gastrins. 1512 80


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