Biomedical subjects

C Monnot

Publications and source records attributed to C Monnot.

13 recordsLinked to original sources

PubMed1999

Several interesting phenotypes of the AT1 receptor produced by site-directed mutagenesis.

The angiotensin II (AngII) AT1 receptor is a seven-transmembrane domain receptor coupled to a Gq/11 protein and phospholipase C, but also to other G proteins and to several tyrosine kinase pathways. These signaling pathways transduce inside the cells the classical actions of AngII (vasoconstriction, aldosterone secretion, etc.), but also the mitogenic action of this vasoactive peptide. In the past 5 yr, site-directed mutagenesis has elucidated the molecular determinants of the AngII and nonpeptidic analogue-binding sites together with those of G protein interaction. In addition, these studies have demonstrated that modifications of the specific interactions between transmembrane domains are responsible for the activation of the receptor. Therefore, several mutations of these domains are able to block the receptor in active or inactive states. Finally, these mutagenesis studies identify two interesting phenotypes of the AT1 receptor. (1) A carboxy-terminal truncation of the AT1 receptor produces a mutant that is unable to be internalized and desensitized and therefore is functionally hyper-reactive. (2) A replacement of the distal part of the third intracellular loop of the AT1 receptor by the homologous segment of the beta2-adrenergic receptor produces a mutant coupled to both Gq and Gs proteins, which is unable to transduce the mitogenic action of AngII.

Animals↗

PubMed1998

Functional interactions of L-162,313 with angiotensin II receptor subtypes and mutants.

A nonpeptide ligand, L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-is obutyl-3-thienyl]phenyl]methyl]imidazo[4,5,6]pyridine) was characterized on the angiotensin II receptors. This compound displaced [125I][Sar1]angiotensin II from rat angiotensin AT1A, AT1B or AT2 receptor individually expressed in COS-7 cells (Ki = 207 nM, 226 nM and 276 nM, respectively). In monkey kidney cells expressing angiotensin AT1A or AT1B receptors, it stimulated inositol phosphate accumulation, but the maximal response was 34.9 and 23.3%, respectively, of those of angiotensin II. Furthermore, an antagonist effect of L-162.313 was observed in response to angiotensin II. Single-point substitutions in the second and third transmembrane domains of the rat angiotensin AT1A receptor, which impaired the binding of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1[(1H-tetrazol-5-yl)biphenyl-4 -yl)methyl]imidazole), also affected the binding of L-162,313. Losartan and L-162,313 require some common structural determinants for non-peptide recognition on the angiotensin AT1 receptor. Furthermore, some of these substitutions, which impaired the inositol phosphate accumulation in response to angiotensin II, also impaired the response to L-162,313. Angiotensin II and L-162,313 require common critical residues for angiotensin AT1 receptor activation.

Angiotensin II↗

PubMed1996

Polar residues in the transmembrane domains of the type 1 angiotensin II receptor are required for binding and coupling. Reconstitution of the binding site by co-expression of two deficient mutants.

Type 1 angiotensin receptors (AT1) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT1A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser105, Ser107, and Ser109 are not essential for maintenance of the angiotensin II binding site. Replacement of Asn111 or Ser115 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT1 (DuP753)- or AT2 (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser115 mutant suggests that this residue, in addition to previously identified residues, Asp74 and Tyr292, participates in the receptor activation mechanism. Finally, Lys102 (third helix) and Lys199 (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation.

Amino Acid Sequence↗

PubMed1996

Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements.

Two distinct types of cell-surface angiotensin II receptors (AT1 and AT2) have been defined pharmacologically and cDNAs encoding each type have been identified by expression cloning. These pharmacological studies showed the AT1 receptors to mediate all the known functions of angiotensin II in regulating salt and fluid homeostasis. Further complexity in the angiotensin II receptor system was revealed when homology cloning showed the existence of two AT1 subtypes in rodents and in situ hybridization and reverse transcription-polymerase chain reaction analyses showed their level of expression to be regulated differently in different tissues: AT1A is the principal receptor in the vessels, brain, kidney, lung, liver, adrenal gland and fetal pituitary, while AT1B predominates in the adult pituitary and is only expressed in specific regions of the adrenal gland (zona glomerulosa) and kidney (glomeruli). Expression of AT1A appears to be induced by angiotensin II in vascular smooth-muscle cells but is inhibited in the adrenal gland. Preliminary analysis of the AT1 promoters is also suggestive of a high degree of complexity in their regulation. Investigation of a potential role for altered AT1 receptor function has commenced at a genetic level in several diseases of the cardiovascular system. No mutations affecting the coding sequence have been identified in Conn adenoma and no linkage has been demonstrated with human hypertension by sib-pair analysis. None the less, certain polymorphisms that do not alter the protein structure have been found to be associated with hypertension and to occur at an increased frequency in conjunction with specific polymorphisms in the ACE gene in individuals at increased risk for myocardial infarction. Further characterization of the regions of the AT1 gene that regulate its expression are therefore needed. The physiological importance of the AT2 gene product still remains a matter of debate.

Amino Acid Sequence↗

PubMed1994

Internalization of the rat AT1a and AT1b receptors: pharmacological and functional requirements.

The capacity of the angiotensin II (AngII) agonist [Sar1]AngII, the antagonist [Sar1-Ile8]AngII and the non-peptidic antagonist DuP753 to undergo receptor internalization were studied in Chinese hamster ovary cells expressing rat AngII type 1a or 1b receptors (AT1a or AT1b) or a mutant of AT1a (Asn74) unable to couple G-protein. In this expression system, the ligand-induced internalization of rat AT1a and AT1b are similar. Moreover, peptidic ligands, either the agonist or antagonist, induce a significant internalization of AT1 receptors, but the non-peptidic antagonist DuP753 is far less potent. Finally, the normal internalization of the mutant Asn74 demonstrates that receptor activation and G-protein coupling are not required for AT1a internalization.

Angiotensin II↗

PubMed1994

Synthetic cDNA encoding the rat AT1a receptor: a useful tool for structure-function relationship analysis.

To carry out systematic structure-function studies of the rat angiotensin II receptors by site directed mutagenesis, or production of chimeric receptors, we have produced a synthetic cDNA coding for the AT1a receptor. The synthetic cDNA is 1101 base pairs long, and contains 49 unique restriction sites that are on the average 23 base pairs apart, allowing replacement of specific restriction fragments by synthetic counterparts containing the desired modified sequence. The total cDNA was assembled in the expression vector pECE. After stable expression in Chinese Hamster Ovary cells, the protein encoded by this synthetic cDNA presents a pharmacological profile and a signal transduction mechanism indistinguishable from the wild type rat AT1a receptor.

Angiotensin II↗

PubMed1994

Distribution of type 1 angiotensin II receptor subtype messenger RNAs in the rat fetus.

The localization of the two type 1 angiotensin II receptor subtype (AT1A and AT1B) messenger RNAs in the 19-day-old rat fetus was studied by in situ hybridization. AT1 receptor mRNAs were detected in target organs of the renin-angiotensin system such as the kidney, adrenal gland, liver, heart, large arteries, and pituitary gland. In addition, angiotensin II receptors were present in specialized mesenchymal cells surrounding the cartilage, in the pericardium, in the lung, and in the undifferentiated mesenchymal tissue. The AT1A subtype was predominant in all tissues and organs except the adrenal cortex and glomeruli in the kidney, which expressed both AT1A and AT1B mRNAs. The widespread distribution of AT1 receptors in tissues and organs involved in hydromineral equilibrium and blood pressure regulation shows that during fetal development angiotensin II may already act as a regulator of the cardiovascular system. An effect on cellular differentiation and/or proliferation via AT1 receptors is also suggested by their location in several mesenchymes.

Angiotensin II↗

PubMed1993

Mutation of Asp74 of the rat angiotensin II receptor confers changes in antagonist affinities and abolishes G-protein coupling.

Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G protein-coupled receptors and is functionally important for agonist binding and G-protein coupling in beta 2-adrenergic and luteinizing hormone receptors. To determine whether this aspartic acid is also involved in the function of the rat vascular angiotensin II receptor subtype 1 (AT1a), Asp74 was replaced either by asparagine or by glutamic acid. When expressed in CHO cells, the two mutants and the wild-type receptor displayed similarly high affinities for the agonist [Sar1, Tyr(125I)4]angiotensin II [where Sar is sarcosine and Tyr(125I) is monoiodinated tyrosine] and the other agonists: ([Sar1]angiotensin II > angiotensin II > angiotensin III >> angiotensin I). However, the Asn74 mutant shows striking differences in its affinity for some antagonists when compared with the wild-type receptor: the affinity for DUP753 was decreased 10-fold, whereas it was increased 6-fold for [Sar1,Ala8]angiotensin II and 20-fold for CGP42112A. These pharmacological changes were associated with a major defect in transmembrane signaling, since angiotensin II was unable to stimulate inositol phosphate production and increase cytosolic Ca2+ concentration through the two mutated receptors, whereas a clear dose-dependent stimulation was observed in cells expressing the wild-type receptor. Angiotensin II was able to promote DNA synthesis through the wild type but not through the mutated receptors. These data indicate that the conserved Asp74 residue of the AT1a receptor is important for the binding of angiotensin II antagonists and is essential for the transmembrane signaling cascade.

Amino Acid Sequence↗

PubMed1993

Co-expression of type 1 angiotensin II receptor (AT1R) and renin mRNAs in juxtaglomerular cells of the rat kidney.

Physiological and ligand binding studies have shown that Angiotensin II (AII) exerts various functions along different segments of the nephron, via the type-1 receptor (AT1R), resulting in the control of glomerular filtration rate (GFR) and water and salt homeostasis. We have used the recently cloned AT1R cDNA to localize, by in situ hybridization, the cells expressing AT1R mRNA in the rat kidney. On serial sections, juxtaglomerular (JG) renin secreting cells, identified by hybridization with a renin cRNA probe, also co-express AT1R mRNA. The co-expression of AT1R and renin mRNAs in the same cells documents visually the direct feedback control of AII on renin secretion. AT1R mRNA was also present in known target cells for AII: proximal convoluted tubule, mesangium and vasa recta.

Animals↗

PubMed1992

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.

Angiotensin II↗

PubMed1992

[Is angiotensin II a growth factor?].

Angiotensin II is an octapeptide resulting from the enzymatic cascade of the renin-angiotensin system and involved in vasoconstriction and aldosterone secretion. The extensive use of converting enzyme inhibitors recently suggested that angiotensin II may have a specific action on growth of its target tissues. Cellular models confirm that angiotensin II is able to produce in vitro a cellular hypertrophy of many cell types. Nevertheless a controversy was developed on the real possibility for angiotensin II to act on cell division. Some cells, such as adrenocortical cells, present a clear induction of their division by angiotensin II, but contradictory results were obtained on vascular smooth muscle cells. The mechanism by which angiotensin II induces hypertrophy of its target tissues, is largely unknown but may involve a direct action on proto-oncogene synthesis, or an indirect action on growth factor secretion. The nature of the angiotensin II receptor involved in these mechanisms has to be identified.

Angiotensin II↗

PubMed1991

Cloning and functional characterization of a novel mas-related gene, modulating intracellular angiotensin II actions.

The mas oncogene codes for a GTP binding protein-coupled receptor that determines a physiological response to angiotensin when expressed in Xenopus laevis oocytes or in the neuronal cell line NG115-401L. However, another gene, rat thoracic aorta gene, structurally related to mas, is devoid of any functional similarity with the angiotensin receptor(s). The relationships between the mas-related proteins and the angiotensin receptors were investigated by identifying and characterizing new members of the mas gene family. A new mas-related gene (mrg) was cloned in a human genomic library at low stringency using the mas cDNA as probe. Mrg codes for a seven-hydrophobic-segment receptor that is 35% identical to the mas product and 29% identical to the rat thoracic aorta gene product. Mrg mRNA was not detected in several rat and human adult tissues that normally express the angiotensin II (AII) receptor, and transfections of COS and CHO cells with the mrg gene did not modify the number of AII binding sites. These results indicate that mrg and the human AII receptor genes are not identical. However, injection of mrg mRNA into Xenopus oocytes markedly increased the electrophysiological response to angiotensin peptides, indicating some functional similarities with the mas product. The reduction of the response after defolliculation of the oocyte, together with the full agonist effect of Sar1IIe8AII and the partial agonist effect of Sar1Ala8AII, seem to indicate that mrg interacts with the signaling pathways of the endogenous Xenopus angiotensin receptor to potentiate the response to AII.

Amino Acid Sequence↗

PubMed1990

The difficult challenge of cloning the angiotensin II receptor.

The vasopressor peptide angiotensin II exerts its cellular effects through a membrane-bound receptor coupled to a G protein. Biochemical and pharmacological analyses of this receptor already identify two different membrane-bound receptors and one cytosoluble angiotensin-II-binding protein. Nevertheless, the purification of the membrane-bound form(s) appears to be difficult. In the absence of purified protein, two cloning strategies of the gene have been explored: (1) expression cloning, identifying the functions of the protein expressed from a cDNA library in COS cells or Xenopus oocytes, has been unsuccessful until now; (2) analogical cloning, trying to identify related members of the seven transmembrane segment receptor family, which could be related to angiotensin receptors, identifies the mas oncogene and two related genes. However, there are accumulating data to exclude their involvement in angiotensin binding.

Animals↗