Biomedical subjects

S Conchon

Publications and source records attributed to S Conchon.

9 recordsLinked to original sources

PubMed1999

Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.

Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi.

Alleles↗

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

A noninternalized nondesensitized truncated AT1A receptor transduces an amplified ANG II signal.

The structural determinants of the rat angiotensin (ANG) II AT1A receptor involved in receptor internalization, desensitization, and activation are investigated by producing six mutants that had progressively larger deletions of the cytoplasmic tail (-13, -19, -24, -31, -46, and -56 residues, respectively). After stable transfection of the cDNAs into Chinese hamster ovary cells, all mutants, except the most truncated, exhibit normal [Sar1]ANG II affinities [dissociation constant (Kd) = 0.19-0.70 nM] compared with the wild-type (WT) receptor (Kd = 0.62 nM) and are able to activate a Gq/11 protein and a phospholipase C as measured by the ANG II-induced inositol phosphate (IP) turnover in the different clones. However, one of these mutants, delta 329 (deletion of 31 residues), exhibits a peculiar phenotype. This mutant shows a reduced ligand-induced internalization as measured by the acid-washing procedure (only 32% of receptors are internalized vs. 83% for WT). Moreover, the delta 329 mutant is less desensitized by a pretreatment with either ANG II (15% desensitization of ANG II-stimulated IP turnover vs. 60% for WT receptor) or the phorbol ester phorbol 12-myristate 13-acetate (no desensitization vs. 29% for WT receptor). These functional modifications of the delta 329 mutant are associated with the transduction of an amplified signal as demonstrated on both IP turnover and an integrated physiological effect of ANG II. Taken together, these data indicate that the sequence 329SLSTKMS335 of the rat AT1A receptor is involved in both receptor internalization and desensitization. This is the first demonstration that a desensitization- and internalization-defective AT1A receptor mutant is also hyperreactive and mediates augmented cellular responses.

Amino Acid Sequence↗

PubMed1997

The C-terminal third intracellular loop of the rat AT1A angiotensin receptor plays a key role in G protein coupling specificity and transduction of the mitogenic signal.

To identify the role(s) of the third intracellular loop of the angiotensin II (AngII) type 1A (AT1A) receptor in G protein coupling specificity and receptor activation, several chimerae were constructed and characterized. The cDNA sequence encoding the C-terminal segment of the third intracellular loop of the AT1A receptor (residues 234-240) was replaced with the homologous regions of the alpha1B adrenergic (alpha1B-AR), the beta2 adrenergic (beta2-AR), and the AngII type 2 (AT2) receptors. These chimeric receptors were stably expressed in Chinese hamster ovary cells, and their pharmacological and functional properties were characterized, including AngII-induced inositol phosphate and cyclic AMP (cAMP) productions, [3H]thymidine incorporation into DNA, and internalization. The affinities of these chimeric receptors for [Sar1]AngII, [Sar1,Ile8]AngII, and losartan were essentially normal; however, the affinity of these mutants was increased by a factor of 10-40 for the AT2-specific ligand CGP42112A. The functional properties of the alpha1B-AR chimera were essentially identical to those of the wild type AT1A receptor. On the other hand, replacement with the beta2-AR segment produced a partial reduction of the inositol phosphate production, a measurable AngII-induced cAMP accumulation, a reduced internalization, and a total impairment to transduce the mitogenic effect of AngII. The AT2 chimera presented a normal internalization, but was inactive in all the other functional tests. In conclusion, the distal segment of the third intracellular loop of the rat AT1A receptor plays a pivotal role in coupling selectivity and receptor signaling via G protein(s) as well as in the activation of the specific signaling pathways involved in the mitogenic actions of AngII.

Amino Acid Sequence↗

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↗

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↗