PubMed Health⌕ Search

Biomedical subjects

E Escher

Publications and source records attributed to E Escher.

At least 37 records · Page 2Linked to original sources
PubMed1998

N- and C-terminal structure-activity study of angiotensin II on the angiotensin AT2 receptor.

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV >> Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.

Angiotensin II↗
PubMed1998

The angiotensin II binding site on Mycoplasma hyorhynis is structurally distinct from mammalian AT1 and AT2 receptors.

Angiotensin II (AngII) binding sites were characterized on rat pheochromocytoma cells (PC-12) which are known to express exclusively the type-2 (AT2) AngII receptor. Interestingly, we found that, on confluent PC-12 cells, only partial inhibition of 125I-AngII binding was achieved when cells were incubated with a saturating concentration of PD-123 319 (an AT2 selective ligand) suggesting the presence of an atypical binding site. In binding experiments, AngII exhibited high affinity for this atypical binding site with a dissociation constant (Kd) of 16 nM. Moreover, bacitracin potently inhibited PD-123 319-resistant 125I-AngII binding with an IC50 half-maximal inhibitory concentration of 44 microM. Enzyme immunoassay revealed that the cells were contaminated with Mycoplasma hyorhynis. Contaminated PC-12 cells were photolabeled with 125I-[p-benzoylPhe1]AngII and covalently labeled proteins were subjected to polyacrylamide gel electrophoresis followed by autoradiography. Under these conditions, two distinct labeled species of 140 kilodaltons (kDa) and 95 kDa were detected. Deglycosylation of the 140 kDa-labeled AT2 receptor with glycopeptidase-F (PNGase-F) resulted in a 35 kDa protein whereas the 95 kDa band was not affected by digestion with the endoglycosidase. Thus, our results show that the AngII binding site on M. hyorhynis is structurally distinct from mammalian AT1 and AT2 receptors.

Angiotensin II↗
PubMed1998

Influence of surface properties at biodegradable microsphere surfaces: effects on plasma protein adsorption and phagocytosis.

OBJECTIVE: The objective of this work was to determine plasma protein adsorption and macrophage phagocytosis of biodegradable polyanhydride, polylactic acid and polylactic-co-glycolic acid microspheres prepared by both spray-drying and solvent evaporation techniques. METHODS: Microspheres were characterized by scanning electron microscopy (SEM), confocal laser microscopy, particle size distribution and zeta (zeta) potential determination. Plasma protein adsorption onto the microspheres was determined using a fluoroaldehyde reagent. Phagocytosis was evaluated by incubating microspheres containing the angiotensin II antagonist, L-158,809, with the macrophages in the presence or absence of the phagocytosis inhibitor cythochalasin D. The extent of phagocytosis was established by fluorescence determination of L-158,809 and by optical microscopy. The effect of amphiphilic poly(ethylene glycol) (PEG) derivatives on phagocytosis was determined using PEG-distearate incorporated into the microspheres. RESULTS: The average diameter of the microspheres, which depended on the polymer and the initial formulation, ranged from 0.9 to 3.2 micrometers. Zeta potential studies showed strong negative values irrespective of the polymer used for the spray-dried formulations. The zeta potential was masked by the incorporation of PEG 400- or PEG 1,400-distearate in the formulation. Confocal laser microscopy showed a homogenous dispersion of PEG (measured as PEG-fluorescein) in the microspheres. Protein adsorption was not observed for any of the microsphere formulations following incubation with bovine serum. Incubation of microspheres with murine macrophages showed that PEG-distearate inhibited phagocytosis at appropriate levels (0.1% w/w). Higher levels > 1% w/w of PEG-distearate) resulted in enhanced association with macrophages, despite the presence of the phagocytosis inhibitor cytochalasin D, indicating fusion between the microspheres and the plasma membrane. CONCLUSIONS: These results demonstrate that spray-dried PEG-containing microspheres can be manufactured and that an appropriate concentration of this excipient in microspheres results in decreased phagocytosis.

Adsorption↗
PubMed1998

Identification of a pituitary growth hormone-releasing peptide (GHRP) receptor subtype by photoaffinity labeling.

Hexarelin, an analogue of GHRP-6, in which D-Tryptophan has been replaced by its 2-methyl derivative, is known to release growth hormone (GH) in vivo and in vitro by direct action on receptors present in anterior pituitary cells. Measurement of second messengers (c-AMP, Ca++, IP3) upon somatotrophs stimulation, suggests the existence of more than one GHRP receptor subtype. In order to document such an hypothesis, we have used a new photoactivatable derivative of Hexarelin, Tyr-Bpa-Ala-Hexarelin. This derivative was shown to be fully active in the release of GH in vivo with neonate rats. Using this photoactivatable ligand, we have specifically labeled a protein with an apparent Mr of 57,000 in human, bovine and porcine anterior pituitary membranes. Hexarelin and the spiroindoline sulfonamide MK-0677 displaced the Mr-57,000 photolabeled band with an apparent ED50 of 6x10(-7) M and 2x10(-5) M respectively. Taking into account the high efficiency (>60%) of covalent incorporation of the Bpa residue, this photoactivatable Hexarelin derivative has allowed the identification of a pituitary GHRP receptor subtype, which is apparently distinct from the recently cloned GH secretagogue receptor.

Animals↗
PubMed1998

Essential role of leucine222 in mediating signal transduction of the human angiotensin II type 1 receptor.

The angiotensin II (Ang II) type I receptor (AT1) is a member of the superfamily of heptahelical, G protein-coupled receptors (GPCRs) characterized by hydrophobic transmembrane domains connected by extra- and intracellular hydrophilic loops. The third intracellular loop (IC3) of many GPCRs is thought to directly interact with G proteins. We examined the molecular environment of the basic sequence KA221L222KK found in the IC3 of the human AT1 (hAT1) receptor by substituting Ala221 for Glu/Gln or Leu222 for Arg/Gln and determined the pharmacological properties of the resulting mutant receptors. Competitive binding experiments with the antagonist [Sar1,Ile8]Ang II revealed that COS-7 or stably expressing CHO cells transfected with either the wild-type or mutant receptors, produced a single population of high affinity binding sites (Kd of 0.5 nM) but variable receptor levels depending on cell type; Bmax approximately 100,000 sites/cell (COS-7), approximately 20,000 sites/cell (CHO). However, in competitive binding experiments using the agonist Ang II, both the wild type and the Ala-->Glu mutant displayed binding affinities of 1 nM, while the Leu-->Arg mutant had a significantly lower affinity (4 nM). When the functionality of the mutant receptors was examined, a lowered production of inositol-1,4,5-trisphosphate was obtained upon stimulation of the Ala-->Glu and Ala-->Gln mutants when compared to the wild-type receptor. However, no significant production of Ins(1,4,5)P3 was detected for the Leu-->Arg and leu-->Gln mutants. Our results suggest that Leu222 in the KALKK sequence of the IC3 of the hAT1 receptor, through not essential for antagonist binding, has an essential role in mediating interactions with G protein and in signal transduction.

Angiotensin II↗
PubMed1997

Angiotensin analogues palmitoylated in positions 1 and 4.

Lipidated angiotensin II (Ang) agonists and antagonists were synthesized and evaluated for their biological activities for eventual use an antimyoproliferative agents. Solid phase peptide synthesis was used for the assembly of the peptides with the Fmoc protection scheme. N-Acetyl-Ser1 Ang was palmitoylated on the serine hydroxyl function. The nonpalmitoylated analogue retained one-third of Ang's affinity toward the AT1 receptor on bovine adrenal cortex membranes, and the palmitoylated analogue was essentially inactive. Upon enzymatic lipolysis or mild saponification of the palmitoylated peptide, biological activity was restored. An analogous compound of Ang, N-acetyl-Ser1,beta-D-naphthylalanine8 ([NAcSer1,D-Nal8]Ang), was a pure antagonist on rabbit aorta but with lower affinity. Its O-palmitoylated form was inactive as well but was easily converted to the nonlipidated active form by lipolysis or saponification. Direct palmitoylation of [sarcosine1]Ang with palmitoyl chloride was obtained on the free phenolic hydroxyl of Tyr4 on solid phase on an otherwise fully protected peptide. This lipopeptide was fully active, was comparable to [Sar1]Ang, and exhibited strongly prolonged activity. Lipolysis and saponification under mild conditions yielded standard [Sar1]Ang. The corresponding [Sar1,D-Nal8]Ang was a potent and very long-lasting antagonist (pA2 = 8.1), and its analogous palmitoyl phenyl ester in position 4 was active in its palmitoylated form (antagonist) and, again, returned to the nonlipidated form upon saponification or lipolysis. [Sar1,Tyr4(O-octadecyl)]Ang, an analogue to Tyr-palmitoylated [Sar1]Ang with an octadecyl phenyl ether in position 4, was also prepared. Surprisingly, the ether compound was inactive. Premature hydrolysis of the palmitoyl phenyl ester peptide was excluded by HPLC analysis, and the activity of the ester peptide is attributed to a putative hydrogen bond that may be critical for biological activity. The discovery of potent biologically active lipidated antagonists of Ang gives access to potential antimyoproliferative agents with numerous application possibilities.

Adrenal Cortex↗
PubMed1997

Identification of angiotensin II-binding domains in the rat AT2 receptor with photolabile angiotensin analogs.

To identify binding domains between angiotensin II (AngII) and its type 2 receptor (AT2), two different radiolabeled photoreactive analogs were prepared by replacing either the first or the last amino acid in the peptide with p-benzoyl-L-phenylalanine (Bpa). Digestion of photolabeled receptors with kallikrein revealed that the two photoreactive analogs label the amino-terminal part of the receptor within the first 182 amino acids. Digestion of 125I-[Bpa1]AngII.AT2 receptor complex with endoproteinase Lys-C produced a glycoprotein of 80 kDa. Deglycosylation of this 80-kDa product decreased its apparent molecular mass to 4.6 kDa and further cleavage of this 4.6-kDa product with V8 protease decreased its molecular mass to 3.6 kDa, circumscribing the labeling site of 125I-[Bpa1]AngII within amino acids 3-30 of AT2 receptor. Treatment of 125I-[Bpa8]AngII.AT2 receptor complex with cyanogen bromide produced two major receptor fragments of 3.6 and 2.6 kDa. Cyanogen bromide hydrolysis of a mutant AT2 receptor produced two major fragments of 12.6 kDa and 2.6 kDa defining the labeling site of 125I-[Bpa8]AngII within residues 129-138 of AT2 receptor. Our results indicate that the amino-terminal tail of the AT2 receptor interacts with the amino-terminal end of AngII, whereas the inner half of the third transmembrane domain of AT2 receptor interacts with the carboxyl-terminal end of AngII.

Affinity Labels↗
PubMed1997

Use of LiCl in phospholipase C assays masks the impaired functionality of a mutant angiotensin II receptor.

We recently reported that replacement of Tyr302 for Ala in the human angiotensin II type 1 receptor (hAT1) severely impaired its ability to activate phospholipase C (PLC). Another study demonstrated that the same mutation in the rat AT1 receptor only slightly impaired its ability to activate PLC. The most striking difference between the two studies was the use of LiCl in the experimental conditions. Thus, in the present report we evaluated the effect of LiCl on the rate of accumulation of inositol trisphosphate (IP3) in transfected cells stimulated with angiotension II (Ang II). In the presence of LiCl, Ang II caused a significant accumulation of IP3 in COS-7 cells transfected with the hAT1Y302A mutant receptor. In stably expressing CHO cells, stimulation of hAT1Y302A did not induce any IP3 elevation even in the presence of LiCl whereas the hAT1 wild-type receptor increased the production of IP3 exclusively in the presence of LiCl. These results show that LiCl is a convenient tool to enhance the sensitivity of PLC assays. However, in structure-activity relationship studies, it may underestimate or mask the debilitating effect of some mutations.

Amino Acid Substitution↗
PubMed1997

Improved activity of a new angiotensin receptor antagonist by an injectable spray-dried polymer microsphere preparation.

PURPOSE: To characterize and evaluate in vitro and in vivo the release mechanisms involved in spray-dried biodegradable microspheres having different Poly(D,L-lactide) blend formulations and containing an antihypertensive drug (L-158,809). METHODS: Microspheres and blended polymers were characterized by DSC, SEM, confocal laser microscopy and size analysis. In vitro release studies were evaluated by using microspheres made from various blends of high and low molecular weight polymer. In vivo studies were evaluated by L-158,809 antagonist AT1 function versus the shift of the normal dose-response curve of blood pressure induced by Angiotensine II. RESULTS: The average yield of L-158,809 microspheres (10% (w/w)) was 95% of the theoretical loading. The average diameter of the microspheres was from 1 to 3 micrometers. In all release experiments, a significant burst effect (< 15%) was observed followed by a near zero-order release kinetics. In vivo studies with two different formulations show a strong shift of angiotensin II dose-response curve. CONCLUSIONS: The release kinetics and photomicrographs suggest that the system is best described as a multi-parameter controlled released system in which the drug is molecularly dispersed. In vivo results demonstrating the controlled release of L-158,809.

Angiotensin II↗
PubMed1997

Characterization of a specific binding site for angiotensin II in chicken liver.

We have characterized a specific binding site for angiotensin II (AngII) in chicken liver membranes. Pseudo-equilibrium studies at 22 degrees C for 30 min have shown that this binding site recognizes AngII with a high affinity (pKD of 8.13 +/- 0.21). The binding sites are saturable and relatively abundant (maximal binding capacity varies from 0.318 to 0.88 pmol/mg of protein). Nonequilibrium kinetic analyses at 22 degrees C revealed a calculated kinetic pKD of 8.77 +/- 0.20. The binding site is pharmacologically distinct from the classic AngII receptors AT1 and AT2. Competitive binding studies with chicken liver membranes demonstrated the following rank order of effectiveness: AngII (human; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > AngI(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) > AngIII(Arg-Val-Tyr-Ile-His-Pro-Phe) > AngIV (Val-Tyr-Ile-His-Pro-Phe) > Ang(1-7) (Asp-Arg-Val-Tyr-Ile-His-Pro) > PD123319 (1-[4(dimethylamino)3-methylphenyl] methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-6-carboxylic acid) > DuP753 (2-n-butyl-4-chloro-5 hydroxymethyl-1-[(2'-1H-tetrazol-5-yl)biphenyl-4-yl)methyl] imidazole. This atypical AngII binding site (chicken AT) was sensitive to increasing concentrations of DTT and Mn2+. The structure-activity relationship on position 1 of AngII showed that the primary N-terminal amine was essential for binding affinity ([Asp1]AngII > [Suc1]AngII > or = [Sar1]AngII), but modifications of the side chain in position 1 had less influence on the affinity ([Gly1]AngII > [Cys1]AngII approximately [aminoisobutyryl1]AngII approximately [Ser1]AngII > > > [Sar1]AngII). The presence of substantial quantities of this binding site in chicken liver membranes suggests the possibility that the chicken AT may play an important, yet unrecognized, role in the renin-angiotensin system.

Angiotensin II↗
PubMed1997

Expression of the five somatostatin receptor (SSTR1-5) subtypes in rat pituitary somatotrophes: quantitative analysis by double-layer immunofluorescence confocal microscopy.

Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 +/- 9.7% of GH cells followed by SSTR2 in 42 +/- 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 +/- 4.7% and 18 +/- 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5 +/- 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

Animals↗
PubMed1997

Epitope tagging and immunoreactivity of the human angiotensin II type 1 receptor.

Structural analysis of G-protein-coupled receptors has largely been limited to photoaffinity labeling and site-directed mutagenesis. This is primarily due to the difficulty in the production of antibodies against this class of receptors. We were therefore interested in tagging the amino-terminal side of the human angiotensin II (AngII) type 1 receptor (AT1) with the FLAG epitope DYKDDDDK. Competitive binding experiments with [125I][Sar1,Ile8]AngII revealed that stably transfected Chinese hamster ovary (CHO) cells express 37,300 hAT1-FLAG receptors/cell with a high affinity of 0.53 nM, comparable with that of the wild-type hAT1. After photolabeling and solubilization, a significant proportion of hAT1-FLAG specifically immunoprecipitated with anti-FLAG M5 and M2 antibodies. The immunoprecipitated receptor comigrated on SDS-PAGE with photolabeled wild-type hAT1. Immunofluorescence studies by FACS scan analysis revealed that 11.9% of CHO cells expressing hAT1-FLAG receptor significantly increased their fluorescence level as a result of M5 specific reactivity. Western blot analysis failed to show any specific interaction between M5 antibody and denatured hAT1-FLAG receptor. These results demonstrate the efficiency of the epitope tagging approach for specific immunoreactivity against AT1 receptor. Appropriate refinements of this approach could improve the level of immunoreactivity.

Affinity Labels↗
PubMed1996

Identification of transmembrane domain residues determinant in the structure-function relationship of the human platelet-activating factor receptor by site-directed mutagenesis.

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.

Amino Acid Sequence↗
PubMed1996

Novel cyclic analogs of angiotensin II with cyclization between positions 5 and 7: conformational and biological implications.

To study the conformational features of molecular recognition of angiotensin II (Asp1-Arg2-Val3-Tyr4-Val/IIe5-His6-Pro7-Phe8, AII), the synthesis and biological testing of several cyclic analogs of AII cyclized between positions 5 and 7 have been performed. The synthesized analogs were Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Pen7)-Phe8 (3), Sar1-Arg2-Val3-Tyr4-cyclo(Asp5-His6-Apt7)-Phe8 (4), Sar1-Arg2-Val3-Tyr4-cyclo(Glu5-His6-Apt7)-Phe8 (5), Sar1-Arg2-Val3-Tyr4-cyclo-(Cys5-His6-Mpt7)-Phe8 (6), Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Mpc7)-Phe8 (7), Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpt7)-Phe8 (8), and Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpc7)-Phe8 (9), where Apt stands for 4-amino-trans-proline, and Mpt and Mpc for 4-mercapto-trans- and -cis-prolines, respectively. Compound (9) showed good affinity at AT-1 receptors, namely a KD = 20 nM. In functional assays, it showed the characteristics of a weak partial agonist with a relative affinity of 0.26% of that for AII and an intrinsic efficacy, alpha E, of 0.42. Molecular modeling suggested a possible explanation for this finding: the relatively strong binding and the weak partial agonistic activity of compound 9 are due to interaction with AT-1 receptor of only two functionally important groups, namely, the side chains of the His6 and Phe8 residues.

Amino Acid Sequence↗
PubMed1996

Analysis of the role of N-glycosylation in cell-surface expression and binding properties of angiotensin II type-2 receptor of rat pheochromocytoma cells.

We previously demonstrated that the AT2 receptor is a glycoprotein containing N-linked oligosaccharide side chains and that the marked disparity between the sizes of AT2 receptors from different tissues was related to different degrees of N-glycosylation. In the present study, we used an inhibitor of N-glycosylation, tunicamycin, as well as an endoglycosidase, glycopeptidase-F, to examine the contribution of carbohydrate moieties to the ligand-binding properties, cell-surface expression and apparent molecular mass of AT2 receptors of rat pheochromocytoma cells (PC-12 cells). Photoaffinity labelling of cell-surface AT2 receptors revealed that PC-12 cells grown in the presence of tunicamycin expressed, in addition to the previously described 140 kDa receptor, lower-molecular-mass receptors of 63 kDa, 47 kDa and 32 kDa. Lectin affinity chromatography revealed that the 63 kDa and the 47 kDa receptors are partially glycosylated and that the 32 kDa receptor is completely deglycosylated. Competitive binding experiments were carried out on tunicamycin-treated cells that expressed predominantly the 63 kDa or the 47 kDa receptors. Both receptor forms exhibited a high affinity for angiotensin II, although a slight decrease (of about 2-fold) was consistently observed on tunicamycin-treated cells as compared with control cells. Endoglycosidase digestion of AT2 receptors of PC-12 cells also yielded smaller receptor forms of 47 kDa and 32 kDa. Similarly, angiotensin II showed a high but slightly decreased binding affinity (of about 2-fold) for deglycosylated membranes as compared with control membranes. In conclusion, the stepwise action of tunicamycin suggests the presence of at least three N-linked oligosaccharide side chains on the AT2 receptor of PC-12 cells. These oligosaccharide side chains have a minor contribution to the affinity of the receptor. Interestingly, glycosylation is not an essential requirement for the expression of AT2 receptor at the surface of PC-12 cells.

Amidohydrolases↗
PubMed1996

Molecular weight determination of the hepatic vasopressin receptor with a high-affinity photoprobe.

We report here a study of photoaffinity labeling of the V1a-vasopressin receptor with high-affinity, V1-specific radioiodinated antagonist ligands: one containing an azidophenylalanine residue ([beta,beta-dimethyl-beta-mercaptopropionyl(1), p-azido-Phe2,Val4,Lys8,D-Tyr9] vasopressin), two others containing nitrophenylalanine, and one, highly similar but without a photosensitive function, as control. All analogues competed in the dark for the same binding site with vasopressin. Long-wavelength UV irradiation of rat liver membranes incubated in presence of the radio-iodinated azido photolabel produced a specifically labeled protein band at 53 kDa in SDS-PAGE. Identical experiments with the nitrophenylalanyl peptides produced only non-specific labeling and control experiments with the non-photosensitive analogue produced no labeling at all. Chemical crosslinking of 3H-VP to the same membrane preparation produced a result identical to that of the azido photolabel, confirming the receptor nature of the labeled protein. Deglycosylation of the labeled receptor with endoglycosidase F reduced the observed molecular weight of 53 kDa to 43 kDa. The molecular parameters reported herein of the presumed hepatic vasopressin receptor confirm the values deduced from the molecular cloning of the rat V1a receptor.

Affinity Labels↗
PubMed1996

The tyrosine within the NPXnY motif of the human angiotensin II type 1 receptor is involved in mediating signal transduction but is not essential for internalization.

The NPXnY motif is involved in the internalization process of several types of receptors, including lipoprotein receptors and G protein-coupled receptors. We replaced Tyr302 with either phenylalanine or alanine in the NPLFY site of the human angiotensin II receptor type 1 and determined the pharmacological properties of the resulting mutant receptors. Competitive binding experiments revealed that COS-7 cells transfected with either the wild-type or mutant receptors expressed approximately the same amount of high affinity binding sites (Bmax 70,000 sites/cell and Kd approximately 2 nM). Photoaffinity labeling of both native and mutant receptors revealed apparent molecular masses of 110 kDa. Incubation of transfected cells with 0.2 nM [125I]Ang II at 37 degrees revealed an efficient internalization of the wild-type receptor and the mutant receptors, although the mutant receptors were internalized at a slower rate. Interestingly, however, the transmembrane signaling was severely impaired in transfected cells expressing mutant receptors. No significant production of inositol-1,4,5-trisphosphate was observed when these cells were challenged for 3 min with a concentration of angiotensin II as high as 1 microM. This is in contrast to the dose-dependent stimulation of inositol-1,4,5-trisphosphate production in cells expressing the wild-type receptor. Thus, our results show that the Tyr302 in the NPXnY motif of the human angiotensin II receptor type 1 is not essential for agonist binding properties or for internalization of the receptor but plays an important role in transmembrane signaling.

Affinity Labels↗