Showing papers by "Michel Bouvier published in 1995"

Agonist-induced desensitization of dopamine D1 receptor-stimulated adenylyl cyclase activity is temporally and biochemically separated from D1 receptor internalization

Abstract: The regulation of the dopamine D1 receptor was investigated by using c-myc epitope-tagged D1 receptors expressed in Sf9 (fall armyworm ovary) cells. Treatment of D1 receptors with 10 microM dopamine for 15 min led to a loss of the dopamine-detected high-affinity state of the receptor accompanying a 40% reduction in the ability of the receptor to mediate maximal dopamine stimulation of adenylyl cyclase activity. After 60 min of agonist exposure, 45 min after the occurrence of desensitization, 28% of the cell surface receptors were internalized into an intracellular light vesicular membrane fraction as determined by radioligand binding and supported by photoaffinity labeling, immunocytochemical staining, and immunoblot analysis. Pretreatment of cells with concanavalin A or sucrose completely blocked agonist-induced D1 receptor internalization without preventing agonist-induced desensitization, indicating a biochemical separation of these processes. Collectively, these findings indicate that the desensitization of D1 receptor-coupled adenylyl cyclase activity and D1 receptor internalization are temporarily and biochemically distinct mechanisms regulating D1 receptor function following agonist activation.

Serotonergic antagonists differentially inhibit spontaneous activity and decrease ligand binding capacity of the rat 5-hydroxytryptamine type 2C receptor in Sf9 cells.

Abstract: The activities of serotonergic antagonists as inverse agonists at the rat 5-hydroxytryptamine (5-HT)2C serotonin receptor were compared with their potencies in promoting receptor "down-regulation," after expression of the recombinant receptor in the baculovirus/Sf9 insect cell system. Baculovirus expression yielded high levels of 5-HT2C receptors (up to 10(6) receptors/cell), which were functionally coupled to polyphosphoinositide turnover in Sf9 cells through a pertussis toxin-insensitive pathway. The expressed receptor exhibited spontaneous activation of inositol phosphate production, which was inhibited in a dose-dependent manner by serotonergic antagonists, consistent with inverse agonist activity. The potencies of antagonists as inverse agonists correlated with their respective binding affinities determined in competition binding studies with membrane preparations. The maximal inhibition of spontaneous activity ranged from 32% inhibition for mianserin to no effect for spiroxatrine, indicating that antagonists differ in their intrinsic inverse efficacies. Antagonist treatment of intact Sf9 cells or membranes containing the 5-HT2C receptor, followed by washout of residual drug, resulted in a decrease (up to 90%) in the number of binding sites for [3H]mesulergine and [3H]5-HT, with no change in the affinity for [3H]mesulergine. The decrease in binding was irreversible, was not due to the presence of residual antagonist, and was not observed after treatment with agonists. This effect of antagonists in membranes was dose dependent, but the rank order of potency was clearly different from that for inverse agonist activity, indicating that the two effects reflect distinct actions of antagonists at the 5-HT2C receptor. The relative abilities of antagonists to produce loss of binding showed a good correlation with their reported abilities to down-regulate 5-HT2 receptors in vivo after chronic treatment, suggesting that these actions reflect the same underlying process.

Mutation of tyrosine-141 inhibits insulin-promoted tyrosine phosphorylation and increased responsiveness of the human beta 2-adrenergic receptor.

Abstract: The ability of insulin to promote phosphorylation of the human beta 2-adrenergic receptor (beta 2AR) was assessed in Chinese hamster fibroblasts transfected with beta 2AR cDNA. Phosphotyrosine residues were detected in purified beta 2AR using a polyclonal anti-phosphotyrosine antibody and by phosphoamino acid analysis following metabolic labelling with inorganic 32P. Treatment of the cells with insulin induced a 2.4-fold increase in the phosphotyrosine content of the receptor. The insulin-promoted phosphorylation of the beta 2AR was accompanied by an increase in the beta-adrenergic-stimulated adenyl cyclase activity. Substitution of a phenylalanine residue for tyrosine-141 completely prevented both the increased tyrosine phosphorylation and the enhanced responsiveness of the beta 2AR promoted by insulin treatment. Mutation of three other tyrosines located in the cytoplasmic domain of the receptor, tyrosine-366, tyrosine-350 and tyrosine-354, did not abolish the insulin-promoted tyrosine phosphorylation. Taken together, these results suggest that insulin promotes phosphorylation of the beta 2AR on tyrosine-141 and that such phosphorylation leads to a supersensitization of the receptor.

Palmitoylation of G-protein-coupled receptors: a dynamic modification with functional consequences.

Abstract: Introduction In recent years, covalent modification of proteins by lipi'ds has been found to be a more frequent modification than originally anticipated [ 11. Three major classes of such modifications have been particularly well characterized. These are: (a) the N-terminal myristoylation of glycine residues through amide linkage; (b) the prenylation of cysteine residues, located in C-terminal domains, via the formation of thioether links to farnesyl or geranylgeranyl moieties; and (c) the palmitoylation which occurs through the thioesterification of cysteine residues, located in various domains of proteins, with palmitic acid. In contrast to myristylation and prenylation, which typically occur cotranslationally, palmitoylation is a genuine post-translational modification which can undergo dynamic regulation and which, in some cases, can be modulated by external stimuli [2-41. This modification has been found to be particularly prevalent for proteins involved in processes such as cell adhesion, cell growth and signal transduction, raising the intriguing possibility that it could play regulatory roles in these processes. Palmitoylated proteins include p2 1 rds [ 51, GAP-43 [ 61, several tyrosine kinases belonging to the p60"" family such as p56"" [7], p59'Y", pSS'gr and ~ 5 6 ' " ~ [8], as well as many G-protein a-subunits [9]. For these proteins, the attachment of the 16-carbon-long fatty acid has been proposed to be essential for their targeting to the inner face of the plasma membrane, the site of their biological functions [ 10-131. However, the observation that integral membrane proteins, which

Dynamic palmitoylation of G-protein-coupled receptors in eukaryotic cells.

Abstract: Publisher Summary This chapter discusses the dynamics of palmitoylation of G-protein-coupled receptors in eukaryotic cells. Studying the dynamics of receptor palmitoylation is essential for elucidating the role of the modification in regulating signaling. Acylation of the G-protein subunits and regulatory kinases plays an important role in the targeting to proper membrane locations and may influence the ability to form functional multimeric complexes. For the receptors that are integral membrane proteins composed of seven transmembrane domains, palmitoylation is proposed to promote the formation of an additional cytoplasmic loop. It affects the coupling, phosphorylation, and even internalization of some receptors. Palmitoylation is truly posttranslational rather than cotranslational, and is reversible. This suggests the possibility of regulatory cycles of palmitoylation–depalmitoylation that may be involved in signal transduction.

Functional effects of long-term activation on human β2- and β3-adrenoceptor signalling

Abstract: The functional effects of long-term activation of beta-adrenoceptors were investigated by measuring adenylyl cyclase activity, cyclic AMP accumulation and cyclic AMP-dependent protein kinase activity in CHW and L cells expressing either human beta 2- or beta 3-adrenoceptors. Pre-incubation of CHW and L cells expressing beta 2-adrenoceptors with 10 microM isoprenaline for 24 h produced a marked reduction in the total receptor number and dramatically reduced the capacity of the receptor to stimulate adenylyl cyclase maximally. In contrast, the ability of beta 3-adrenoceptors number was observed in L but not in CHW cells. Maximal levels of intracellular cyclic AMP concentrations were reached during the first hour of receptor activation with isoprenaline in all four cell lines. In the absence of phosphodiesterase inhibitors, cyclic AMP decreased to basal levels within 24 h of continuous stimulation. This phenomenon occurred more rapidly in cells expressing the beta 2- than the beta 3-adrenoceptors. These results confirm that, at the level of adenylyl cyclase stimulation and cyclic AMP accumulation, the beta 3-adrenoceptor is more resistant than the beta 2-adrenoceptor to long-term desensitization. However, when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and beta 3-adrenoceptor expressing cells led to the desensitization of the kinase in L but not in CHW cells. In conclusion, long-term desensitization may have distinct functional effects on cell signalling depending on the receptor subtype and the cell type considered.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the vasorelaxant activity of tyramine and other phenylethylamines in rat aorta

Abstract: We recently reported that tyramine caused concentration-dependent relaxation of rat aorta, which was endothelium independent and was not exerted via α1-adrenoceptors (AR), α2AR, β1AR, β2AR, or receptors for 5-hydroxytryptamine, histamine, and adenosine. The present studies were done on endothelium-denuded strips to determine structure – vasorelaxant activity after blockade of βAR by propranolol plus irreversible blockade of α1AR with benextramine. Vasorelaxation under these conditions was limited to noncatecholamines, and their vasorelaxant potencies were methoxyphenamine > tyramine > p-hydroxyephedrine > L-amphetamine > L-ephedrine > phenylethylamine > synephrine > methoxamine > octopamine. β3AR agonists (BRL 37344 and CGP 12177A) did not produce vasorelaxation, although tyramine could compete for cyanopindolol binding to murine L cells expressing human β2AR or β3AR. There was no significant specific binding of [3H]tyramine to aortic membrane preparations after the inhibition of monamine oxidase. Yohimbi...

Le récepteur β2-adrénergique. Un modèle d'étude des mécanismes moléculaires de la désensibilisation

Abstract: La stimulation prolongee d'un recepteur membranaire par son ligand induit souvent un etat de desensibilisation interrompant la transmission du signal. Ainsi, le recepteur β 2 adrenergique couple a la proteine Gs et a l'adenylyl cyclase est-il tout d'abord phosphoryle par la proteine kinase dependante de l'AMPc (PKA) et, a forte concentration en ligand, par la βARK (β adrenergic receptor kinase) qui est recrutee par l'activation du recepteur et la dissociation des sous-unites βγ de la proteine Gs. La phosphorylation du recepteur par la βARK, sur des sites differents des cibles de la PKA, permet la fixation d'une proteine inhibitrice denommee arrestine qui bloque l'interaction entre le recepteur et la proteine Gs. A plus long terme, l'internalisation du recepteur dans les endosomes, sa possible degradation dans les lysosomes et la modification de l'abondance du messager peuvent contribuer a la regulation de la sensibilite au ligand. L'importance de ces phenomenes est bien mise en evidence par la comparaison entre les recepteurs β 2 et β 3 adrenergiques ; ces derniers ne sont phosphoryles, ni par la PKA, ni par la βARK, et ne sont pas desensibilises lors d'une stimulation prolongee.

Methods of screening compounds for their pharmacological relevance based on downregulation of recombinant receptors

Abstract: This invention relates to the use of reagents in a novel manner to screen compounds in vitro for their ability to interact with and modulate the functional properties of receptors coupled to guanyl nucleotide-binding regulatory proteins (G-proteins).Methods are described herein for determining the ability of compounds to act as antagonists by observing their ability to downregulate recombinant G-protein coupled receptors in membrane preparations. The methods described permit the downregulation effect to be observed within a time frame that is dramatically accelerated compared to other known methods of testing the same effect, thereby providing novel and improved efficient means of screening compounds for their probable pharmaceutical efficacy. This invention also relates to the use of such reagents and methods in kits.