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F Fahrenholz

Publications and source records attributed to F Fahrenholz.

At least 55 records · Page 3Linked to original sources
PubMed1994

Lateral mobility of the antagonist-occupied V2 vasopressin receptor in membranes of renal epithelial cells.

The lateral mobility of membrane integral receptors has been implicated as playing a significant role in signal transduction. The adenylate cyclase-coupled vasopressin V2 receptor has been shown to be highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature using a fluorescent vasopressin agonist, with lateral mobility of the V2 receptor proposed to play a role in both adenylate cyclase activation and ligand induced receptor internalization and down-regulation. This study reports the synthesis and characterization of two new fluorescent antagonists [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1,D-Tyr2,Ile4,Lys9(N6-fluoresceinylaminothiocarbonyl )]AVP (FL-AVP-anta) and [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1,D-Tyr2,Ile4,Lys9(N6-tetramethylrhodamylaminothioca rbonyl)]AVP (TR-AVP-anta) for the V2 receptor. The latter was used to determine the parameters of lateral mobility of the V2 receptor in the non-activated antagonist-occupied form. Using fluorescence photobleaching techniques, results were largely comparable to those for agonist-occupied receptor, indicating high mobility at 37 degrees C. Antagonistic properties of the V2 receptor ligands are apparently not related to decreased receptor lateral mobility. Photobleaching measurements, however, did show that in contrast to V2 agonist, V2 antagonist did not induce receptor immobilization due to aggregation with time at 37 degrees C, indicating that this could be of mechanistic importance in the internalization process.

Amino Acid Sequence↗
PubMed1994

Molecular structure analysis of the pituitary adenylate cyclase activating polypeptide type I receptor from pig brain.

In pig brain a type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP) has been identified and structurally characterized. Scatchard analysis of the equilibrium binding data indicates a single class of binding sites with Kd of 0.50 nM and Bmax of 2.52 pmol/mg. The receptors could be efficiently solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonate (Chapso) without altering their ligand binding parameters (Kd = 0.60 nM; Bmax = 2.46 pmol/mg). In both preparations the ligand-receptor complex was identified as a 68 kDa polypeptide by affinity crosslinking with 125I-PACAP-27 and disuccinimidyl suberate. Employment of nonreducing conditions decreased the electrophoretic mobility of the complex to a 60 kDa species suggesting that the native PACAP receptor exists in a compact conformation stabilized by intramolecular disulfide bridges. Disulfide bonds are also important for the ligand binding activity of the PACAP receptor since pretreatment of the membranes with dithiothreitol led to complete inhibition of PACAP binding and to 61% dissociation of the ligand-receptor complex. Among different sulfhydryl selective reagents tested, p-chloromercuriphenylsulfonic acid was found to inhibit PACAP binding to its receptor in a dose-dependent fashion (IC50 = 0.5 mM) suggesting that one or more sulfhydryl groups are located close to the ligand binding domain of the receptor. Desialylation of the affinity-labeled PACAP receptor with neuraminidase revealed a 61 kDa protein, whereas deglycosylation with N-glycosidase F decreased the M(r) to 52,000. Chromatography on a series of lectin agaroses showed the highest affinity for wheat germ agglutinin (WGA). WGA was also effective in dose-dependent inhibition of PACAP binding activity. Our results characterize the pig PACAP receptor as a tri- or tetraantennary non- or low fucosylated complex type glycoprotein with a protein core of about 49 kDa containing several terminal sialyl residues.

Animals↗
PubMed1994

Photocleavable biotinylated ligands for affinity chromatography.

Two derivatives of the peptide hormone cholecystokinin (CCK-8s) containing a photocleavable o-nitrobenzylester group have been synthesized. One analog, [3H]4-(biotin-epsilon-Ahx-oxymethyl)-3-nitrobenzoyl-Gly-Orn (propionyl)-epsilon-Ahx-CCK-8s ([3H]BANA-CCK-8s), was biotinylated, while the other, 4-alanyloxymethyl-3-nitrobenzoyl-epsilon-Ahx-CCK-8s (ANA-CCK-8s) had a free amino group for coupling to amino-reactive affinity matrices. The analogs retained high affinity to both CCK receptors and anti-CCK antibodies. [3H]BANA-CCK-8s was bound to a streptavidin-agarose affinity matrix and was subsequently released by irradiation with uv light of wavelengths > 320 nm. The ability to recover a biotinylated peptide ligand from a streptavidin column under very mild conditions should be useful in the purification of specifically binding proteins. As a demonstration of this approach, [3H]BANA-CCK-8s was incubated with anti-CCK-antiserum and was subsequently passed over a streptavidin-agarose affinity matrix. After washing, the bound antibodies were eluted by photocleavage of the affinity ligand. Forty-six percent of the bound antibodies were recovered after 40 min of irradiation. The eluted antibodies showed essentially unchanged binding characteristics. The suitability of ANA-CCK-8s for the purification of anti-CCK antibodies was also demonstrated. The approach may prove to be generally useful in the isolation of labile proteins in an intact form.

Amino Acid Sequence↗
PubMed1994

Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus.

The gene encoding the human homologue of the guinea pig uterine bombesin receptor [(1992) Eur. J. Biochem. 208, 405] was isolated from a genomic lambda library by the PCR/homology screening approach. The gene spans more than 4 kb and consists of 3 exons and 2 introns. The deduced amino acid sequence shows about 86% identity to that of guinea pig bombesin receptor. This subtype of bombesin receptor is expressed in the pregnant uterus and in two human tumour cell lines, T47D (ductal breast carcinoma) and A431 (epidermal carcinoma). PCR analysis of genomic DNA from human-mouse cell hybrids allows the cloned gene to be localized to the region q26-q28 on chromosome X.

Amino Acid Sequence↗
PubMed1994

Reconstitution of the myometrial oxytocin receptor into proteoliposomes. Dependence of oxytocin binding on cholesterol.

The requirements for regaining high-affinity binding of the myometrial oxytocin receptor after detergent solubilization were investigated by reconstitution experiments. Large unilamellar liposomes were prepared by reverse-phase evaporation from different mixtures of phospholipids, cholesterol and cholesteryl hemisuccinate. In the presence of the oxytocin receptor solubilized from myometrial membranes from pregnant guinea pig uterus, liposomes were treated with 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (Chapso) throughout the range of detergent concentrations that cause the transformation of lamellar structures to mixed micelles. Detergent removal was achieved using bio-beads SM-2 as adsorbent. The presence of cholesterol was a prerequisite for regaining high-affinity binding of [3H]oxytocin and 125I-oxytocin antagonist to reconstituted proteoliposomes. Binding of [3H]oxytocin but not of the antagonist was dependent on the presence of Mn2+ ions. Reconstitution after lectin chromatography and photoaffinity labeling of reconstituted vesicles resulted in the exclusive labeling of the oxytocin receptor with a molecular mass of 68-80 kDa.

Affinity Labels↗
PubMed1994

Enhanced selectivity of oxytocin antagonists containing sarcosine in position 7.

Neurohypophyseal hormone analogues containing sarcosine (Sar) in position 7 were prepared to design more potent and selective oxytocin antagonists. The three analogues (1-3) of [Sar7]arginine-vasopressin ([Sar7]AVP) and six analogues (4-9) of [Sar7]arginine-vasotocin ([Sar7]AVT) had a reduced affinity for antidiuretic V2 receptors. The [Sar7]AVP derivatives (1-3) were potent antiuterotonic (in vitro pA2 = 7.5-8.4, in vivo 6.6-7.1) and antipressor (pA2 = 7.2-8.0) agents. The [Sar7]AVT analogues (4-9) were more potent and selective uterotonic antagonists (in vitro pA2 = 7.9-8.6, in vivo 7.1-7.5); their antipressor potencies were reduced (pA2 = 6.4-7.7). The change of the antagonistic potencies was paralleled by a change in the receptor affinities. Among other antiuterotonic analogues, [Mca1, D-Phe2, Sar7]AVT (4, Mca = beta-mercapto- beta,beta-cyclopentamethyl-enepropionic acid) and [Mca1, D-Tyr(OEt)2,Sar7]AVT (6) were synthesized, two highly potent antiuterotonic compounds (in vitro pA2 = 8.3, in vivo 7.4 and 7.5, respectively) with reduced antipressor activity (pA2 = 6.4) and reduced binding affinity to V2 receptors (Kd = 421 and 35 nM, respectively) and no anti-antidiuretic effect. Another potent antiuterotonic analogue, [Mca1,D-Trp2,-Sar7]AVT (9, in vitro pA2 = 7.9, in vivo 7.5) has virtually no binding capability to V2 receptors (Kd approximately 0.3 mM). These analogues should lead to the design of even more potent and selective oxytocin antagonists.

Amino Acid Sequence↗
PubMed1993

Direct identification of an extracellular agonist binding site in the renal V2 vasopressin receptor.

To purify the renal V2 receptor and identify domains involved in hormone binding, photoaffinity labeling of the membrane-bound bovine V2 receptor with a tritium-labeled photoreactive vasopressin agonist was performed. The labeled 30,000 M(r) protein was purified to homogeneity by anion-exchange chromatography, isoelectric focusing, gel filtration, gel electrophoresis, and reversed-phase HPLC. N-terminal sequencing showed that the isolated protein which contains the covalently bound hormonal ligand, represents an N-terminal truncated bovine V2 receptor. The purified labeled V2 vasopressin receptor protein was digested with V8 protease, and peptide fragments were isolated. Protein microsequencing and comparison with the cDNA sequence of a cloned PCR product identified two extra- and two intracellular peptides of the bovine V2 receptor. Radioactivity was incorporated into two amino acid residues localized in the second extracellular domain. Our results indicate that this extracellular domain is involved in peptide agonist binding of the V2 receptor.

Affinity Labels↗
PubMed1993

Renal and central vasopressin receptors: immunocytochemical localization.

Employing an anti-vasopressin monoclonal antibody for immunization, anti-idiotypic monoclonal antibodies were obtained which induced plasminogen activator production in the renal epithelial cell line LLC-PK1. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 smooth muscle cells by immunofluorescence. All results indicated specificity of the anti-idiotypes for both V1 and V2 vasopressin receptor subtypes. These antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed. On rat brain tissue sections, dense staining was observed within various circumventricular organs. The staining pattern corresponded to that obtained in autoradiographic studies with labeled AVP(4-9) fragment peptide and differed from the distribution of binding sites for labeled vasopressin or V1 antagonists.

Animals↗
PubMed1993

Molecular cloning and functional characterization of V2 [8-lysine] vasopressin and oxytocin receptors from a pig kidney cell line.

[Arg8]vasopressin and oxytocin are the two main members of the neurohypophysial hormone family found to be present in nearly all mammals. [Lys8]vasopressin ([Lys8]VP) has been identified as the antidiuretic hormone in pig and some marsupial families. The porcine-derived kidney epithelial cell line, LLC-PK1, expresses both [Lys8]VP receptors coupled to the activation of adenylate cyclase (V2 receptors) and oxytocin receptors. Here we report the molecular cloning of the V2 [Lys8]VP receptor and the oxytocin receptor from LLC-PK1 cells. The cloned V2 [Lys8]VP receptor differs from human and rat V2 [Arg8] receptors mainly in its N-terminal region, in residues located in the extracellular loops and in intracellular phosphorylation sites. When expressed in COS7 cells, the V2 [Lys8]VP receptor exhibits the relative order of ligand affinity [Lys8]VP = [Arg8]VP >> 1-deamino[D-Arg8]VP > or = oxytocin and adenylate-cyclase stimulation, expected for the porcine V2 [Lys8]VP receptor but different from V2 [Arg8]VP receptors. Adenylate-cyclase activation by [Lys8]VP was inhibited in COS7 cells by a V2 antagonist. The cloned oxytocin receptor exhibits in COS7 cells a ligand specificity typical of mammalian oxytocin receptors. mRNA-distribution analysis revealed a single 5.5-kb transcript in the uterus from pregnant guinea pig.

Amino Acid Sequence↗
PubMed1993

Photoaffinity labeling of central cholecystokinin receptors with high efficiency.

A new photoreactive tritiated cholecystokinin (CCK) analogue was synthesized which contains the p-benzoylbenzoyl moiety linked to an ornithine residue at the N-terminus of the sulfated CCK octapeptide (CCK-8s). p-Benzoylbenzoyl-Orn(propionyl)-CCK-8s bound specifically and with high affinity to CCK binding sites in membranes both from pig cerebral cortex and from rat pancreatic membranes. The apparent dissociation constants KD were 1.2 nM and 0.5 nM, respectively. [3H]-p-Benzoylbenzoyl-Orn(propionyl)-CCK-8s was incubated with CCKB receptor preparations enriched by lectin chromatography and subsequently photoactivated. A polypeptide migrating with an apparent molecular weight M(r) of 56,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was specifically labeled. The labeling was suppressed by the CCKB receptor agonist pentagastrin. The efficiency of incorporation of radioactivity was high, reaching up to 70% of specifically bound radioactivity. After treatment with trifluoromethanesulfonic acid, the molecular weight of the labeled protein decreased to 45,000, indicating that the receptor is glycosylated. Photoaffinity labeling of CCKA receptors from rat pancreas resulted in the specific labeling of a protein band with M(r) of 80,000-100,000. Our synthetic approach should be useful for the design of photoreactive analogues of a variety of peptides. The high efficiency photolabeling of the CCKB receptor will be valuable for further characterization and purification of this receptor.

Affinity Labels↗
PubMed1993

Synthesis and binding characteristics of two sulfhydryl-reactive probes for vasopressin receptors.

The present study describes the synthesis and receptor binding affinities of the sulfhydryl-reactive vasopressin analogs deamino[Dab(N delta-N-maleoyl-beta-alanin e)4]AVP (1a) and deamino[Lys(N epsilon-N-maleoyl-beta-alanine)8VP (2a). The analogs were obtained by introducing the sulfhydryl-reactive maleoyl-beta-analyl group at the delta-amino group of Dab4 in deamino[Dab4]AVP (1) and at the epsilon-amino group of Lys8 in deamino[Lys8]VP (2), which were synthesized by the solid-phase method. Furthermore, the analog modified at Dab4 was prepared as tritium labeled compound (1b) after catalytic iodine tritium exchange at Tyr2 in deamino[Dab4]AVP. The sulfhydryl-reactive vasopressin analogs retained high binding affinity for the V2 vasopressin receptor in membranes derived from bovine kidney inner medulla. Apparent dissociation constants Kd of 45 nM (compound 1a) and 15 nM (compound 2a) were determined. Incubation of the ligand receptor complexes at pH 5.5 resulted in dissociation of the sulfhydryl-reactive vasopressin analogs from the V2 receptor. No indications of a covalent reaction between analogs 1a, 2a and 1b and sulfhydryl groups in or close to the hormone binding site of the V2 receptor were found.

Amino Acid Sequence↗
PubMed1993

Oxytocin induced cAMP-dependent protein kinase activation and urokinase-type plasminogen activator production in LLC-PK1 renal epithelial cells is mediated by the vasopressin V2-receptor.

Using a variety of peptide analogues of oxytocin (OT) and Arg8-vasopressin (AVP), OT-mediated induction of urokinase-type plasminogen activator (uPA) was examined in LLC-PK1 renal epithelial cells, which possess distinct high-affinity receptors of both the OT- and vasopressin renal (V2-) types. OT or OT-receptor specific agonists induced concentration-dependent cAMP synthesis, activation of the cAMP-dependent protein kinase (cAMP-PK) and uPA production consistent with their respective binding affinities for the V2- and not the OT-receptor. OT-mediated uPA induction could be inhibited in a concentration-dependent fashion by coincubation with a V2/V1-receptor specific antagonist, but not by an OT-receptor specific antagonist. Results implied that stimulation of cAMP- and uPA responses in LLC-PK1 cells by OT was V2-receptor-mediated.

Amino Acid Sequence↗
PubMed1993

S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium.

The marine sponge Geodia cydonium contains several lectins. The main component, called lectin-1, is composed of three to four identical subunits. The subunits of the lectins were cloned from a cDNA library; two clones were obtained. From the deduced aa sequence of one clone, LECT-1, a mol. wt of 15,313 Da is calculated; this value is in good agreement with mass spectrometric analysis of 15,453 +/- 25 Da. The sequence of another clone, LECT-2, was analysed and the aa sequence was deduced (15,433 Da). The two subunits have a framework sequence of 38 conserved aa which are characteristic for the carbohydrate-binding site of vertebrate S-type lectins. Clustering of lectin sequences of various species following their pairwise comparison establishes a dendrogram, which reveals that the sponge lectin could be considered as the ancestor for vertebrate S-type lectins.

Amino Acid Sequence↗
PubMed1993

Vasopressin anti-idiotypic antibody staining in the rat brain: colocalization with [35S] [pGlu4, Cyt6]AVP(4-9) binding sites.

Vasopressin and its fragment peptides such as [pGlu4, Cyt6]AVP(4-9) (AVP(4-9) represent putative neuromodulators within central nervous homeostatic, memory and behavioural circuits. To localize their central receptor systems, the previously characterized monoclonal anti-idiotypic antibody mAb 237 was employed in immunocytological investigations of rat brain tissue sections. This antibody was raised to the monoclonal idiotypic anti-AVP antibody mAb 113 which preferentially binds to the acyclic C-terminal portion of the AVP molecule and is therefore also capable of binding the naturally occurring AVP(4-9) fragment. Immunoreactive magnocellular neurones were detected in the AVP-synthesizing supraoptic but not paraventricular nuclei. Dense staining was observed within circumventricular organs lacking a blood-brain barrier (BBB). These structures include the subfornical organ, the organum vasculosum laminae terminalis, the internal layer of the median eminence, the body of the pineal gland, the choroid plexus and the area postrema, where immunoreactivity was found on capillaries, neurones and fibres. Further staining was found in the nucleus of the solitari tract and the arcuate nucleus, endowed with a leaky BBB. Distinct cell patches in the ependymal lining of the third ventricle as well as dendritic processes of juxtaependymal neurones were labelled by the anti-idiotypic antibody mAb 237. The observed staining pattern did not parallel that obtained in autoradiographic studies performed using either radiolabelled AVP or a V1-receptor antagonist, but that found with the [35S]-labelled AVP(4-9) fragment. Using [35S]-labelled AVP(4-9) fragment, specific high density binding sites could be localized autoradiographically in structures within and outside the BBB, in complete agreement with the anti-idiotypic immunoreactivity. Since the anti-idiotypic methodology is based on transfer of complementary structures, and the epitope recognized by the corresponding idiotypic antibody resembles the sequence of AVP(4-9), the anti-idiotypic antibodies might recognize the AVP(4-9) receptor with high affinity.

Animals↗
PubMed1993

Lack of interaction of vasopressin with its antisense peptides: a functional and immunological study.

The peptide encoded in the 5' to 3' direction by rat vasopressin complementary RNA, rat PVA (H-Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala- OH) and the corresponding bovine PVA (H-Ala-Pro-Trp-Ala-Val-Leu-Glu-Val-Ala-OH) were investigated with respect to their interaction with [8-arginine] vasopressin (AVP) and V2 vasopressin receptor binding and function. Rat or bovine PVA did neither affect the binding of the hormone to the V2 receptor of bovine kidney membranes and LLC-PK1 pig kidney cells nor influence the AVP-induced cAMP-production in LLC-PK1 cells. Rat PVA was further investigated by the use of vasopressin-specific polyclonal and monoclonal antibodies with different affinity and epitope specificity. Consistent with receptor binding studies no inhibition of [3H]AVP-binding in fluid- or solid-phase antibody binding tests after preincubation with PVA was found. Direct interaction of rat PVA and [3H]AVP measured on solid surface was not observed in contrast to specific binding of the hormone with NP II and antibodies. In our study no evidence for an interaction of AVP and its antisense peptides was found.

Amino Acid Sequence↗
PubMed1993

Difference between male and female rats in vasopressor response to arginine vasopressin.

A study was carried out how the sexual difference influences the increase in blood pressure (BP) induced by arginine vasopressin (AVP), and how the binding characteristics of 3H-labelled AVP on membranes prepared from the vascular bed were affected. After the administration of various doses of AVP, a significantly higher BP increase was observed in male rats than in females. The vasopressor effect of AVP was reduced in males following orchidectomy or administration of the antiandrogen cyproterone acetate. The vasopressin (VP) antagonist d(CH2)5Tyr(Me)AVP diminished the BP response to AVP in both sexes. The plasma AVP level was found to be much higher in males than in females, but it was decreased to the level of females after orchidectomy. The density of AVP-binding sites in the aorta membrane preparation was smaller in females, and in orchidectomized or cyproterone acetate-treated male rats than in the control males. The results demonstrate that testosterone upregulates the number of AVP-binding sites, leading to an increase in the pressor response to AVP in the rat vascular bed.

Animals↗
PubMed1992

Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy.

The homology screening approach has been used to clone a new member of the guanine-nucleotide-binding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Binding experiments with the stably transfected LLC-PK1 cell line expressing the new receptor protein confirmed the bombesin-like nature of the cloned receptor. The relative order of ligand affinity, GRP = neuromedin C much greater than neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.

Amino Acid Sequence↗