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Dive into the research topics where Stéphane A. Laporte is active.

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Featured researches published by Stéphane A. Laporte.


Journal of Biological Chemistry | 1999

Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.

Robert H. Oakley; Stéphane A. Laporte; Jason A. Holt; Larry S. Barak; Marc G. Caron

Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of β-arrestin in the resensitization of the β2 adrenergic receptor (β2AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit β-arrestin to the plasma membrane and internalize in a β-arrestin- and clathrin-dependent manner. However, whereas β-arrestin dissociates from the β2AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of β-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of β-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of β-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which β-arrestins regulate the reestablishment of GPCR responsiveness.


Progress in Neurobiology | 2002

Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and ß-arrestin proteins

Audrey Claing; Stéphane A. Laporte; Marc G. Caron; Robert J. Lefkowitz

Sequestration of G protein-coupled receptors from the cell surface is a commonly observed phenomenon following agonist-stimulation. This process is now believed to be important for receptor resensitization as well as for signal transduction. Over the years, numerous studies have aimed at understanding the molecular mechanisms underlying internalization. Proteins such as the G protein-coupled receptor kinases (GRKs) and the beta-arrestins, which were initially characterized as desensitizing molecules, have been shown to be important regulators of the endocytic process. Recently, numerous interacting partners have been identified for each of these two classes of proteins. However, the details regarding the sequence of these interactions and the cross-talk between signaling pathways containing the different protein complexes are just beginning to be uncovered. In this review, we summarize these findings and discuss the role of GRKs and beta-arrestins, two families of key regulatory proteins that regulate G protein-coupled receptor endocytosis.


Journal of Biological Chemistry | 2000

The Interaction of β-Arrestin with the AP-2 Adaptor Is Required for the Clustering of β2-Adrenergic Receptor into Clathrin-coated Pits

Stéphane A. Laporte; Robert H. Oakley; Jason A. Holt; Larry S. Barak; Marc G. Caron

β-Arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires β-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-β-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the β-arrestin C-terminal tail; however, the specific molecular determinants in β-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of β-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg394 and Arg396) in the β-arrestin 2 C terminus that mediate β-arrestin binding to AP-2 and show, in vitro, that these domains in β-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that β2-adrenergic receptor-β-arrestin complexes lacking the β-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-β-arrestin complexes lacking the β-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-β-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of β2-adrenergic receptor to clathrin-coated pits.


Trends in Endocrinology and Metabolism | 2003

The oxytocin receptor

Hans H. Zingg; Stéphane A. Laporte

Novel sites of oxytocin receptor expression have recently been detected, including breast cancer cells, bone cells, myoblasts, cardiomyocytes and endothelial cells. These discoveries have greatly expanded the possible spectrum of oxytocin action beyond its classic role as an inducer of uterine contractions and milk ejection. Additional advances in the understanding of oxytocin receptor structure-function relationships, receptor trafficking and novel receptor-linked signaling cascades have made this receptor an attractive model for the study of G-protein-linked receptor function. Finally, the tocolytic efficiency of the oxytocin receptor antagonist atosiban, recently approved for clinical use in Europe, has opened new avenues for the prevention and treatment of preterm labor.


Journal of Biological Chemistry | 1999

Cellular trafficking of G protein-coupled receptor/beta-arrestin endocytic complexes.

Jie Zhang; Larry S. Barak; Pieter H. Anborgh; Stéphane A. Laporte; Marc G. Caron; Stephen S. G. Ferguson

β-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, β-arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of β-arrestin2 in response to activation of several distinct GPCRs including the β2-adrenergic receptor (β2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to β2AR activation, β-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for β-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed β-arrestins are confined to the periphery of cells and do not traffic along with activated β2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of β-arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the β2AR carboxyl-terminal tail results in a β-arrestin membrane localization pattern similar to that observed in response to β2AR activation. In contrast, the corresponding chimeric β2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate β-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of β-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/βarrestin complexes and cellular distribution of β-arrestins.


Neuron | 1999

Muscarinic Supersensitivity and Impaired Receptor Desensitization in G Protein-Coupled Receptor Kinase 5-Deficient Mice

Raul R. Gainetdinov; Laura M. Bohn; Julia K. L. Walker; Stéphane A. Laporte; Alexander D. Macrae; Marc G. Caron; Robert J. Lefkowitz; Richard T. Premont

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.


Journal of Cell Biology | 2002

Phosphoinositide 3-kinase regulates β2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/β-arrestin complex

Sathyamangla V. Naga Prasad; Stéphane A. Laporte; Dean Chamberlain; Marc G. Caron; Larry S. Barak; Howard A. Rockman

Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.


Journal of Biological Chemistry | 2005

C5L2 is a functional receptor for acylation-stimulating protein.

David Kalant; Robin MacLaren; Wei Cui; Ratna Samanta; Peter N. Monk; Stéphane A. Laporte; Katherine Cianflone

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 ± 33%, 5 μm ASP, p < 0.001, where basal = 100%) and glucose transport (168 ± 21%, 10 μm ASP, p < 0.001). C3a similarly stimulates TGS (163 ± 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 ± 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 ± 1.6, with ASP = 21.0 ± 1.4 (144%), with ASP + oligonucleotides = 11.0 ± 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 ± 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 ± 2.9, with ASP = 39.6 ± 8.8 (177%), with ASP + oligonucleotides = 25.3 ± 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged β-arrestin, ASP induced β-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.


Journal of Biological Chemistry | 2005

G Protein-coupled receptor kinase regulates dopamine D3 receptor signaling by modulating the stability of a receptor/filamin/β-arrestin complex: A case of autoreceptor regulation

Kyeong Man Kim; Raul R. Gainetdinov; Stéphane A. Laporte; Marc G. Caron; Larry S. Barak

In addition to its postsynaptic role, the dopamine D3 receptor (D3R) serves as a presynaptic autoreceptor, where it provides continuous feedback regulation of dopamine release at nerve terminals for processes as diverse as emotional tone and locomotion. D3R signaling ability is supported by an association with filamin (actin-binding protein 280), which localizes the receptor with G proteins in plasma membrane lipid rafts but is not appreciably antagonized in a classical sense by the ligand-mediated activation of G protein-coupled receptor kinases (GRKs) and β-arrestins. In this study, we investigate GRK-mediated regulation of D3R·filamin complex stability and its effect on D3 R protein ·G signaling potential. Studies in HEK-293 cells show that in the absence of agonist the D3R immunoprecipitates in a complex containing both filamin A and β-arrestin2. Moreover, the filamin directly interacts with β-arrestin2 as assessed by immunoprecipitation and yeast two-hybrid studies. With reductions in basal GRK2/3 activity, an increase in the basal association of filamin A and β-arrestin2 with D3R is observed. Conversely, increases in the basal GRK2/3 activity result in a reduction in the interaction between the D3R and filamin but a relative increase in the agonist-mediated interaction between β-arrestin2 and the D3R. Our data suggest that the D3R, filamin A, and β-arrestin form a signaling complex that is destabilized by agonist- or expression-mediated increases in GRK2/3 activity. These findings provide a novel GRK-based mechanism for regulating D3R signaling potential and provide insight for interpreting D3R autoreceptor behavior.


Nature | 2016

The conformational signature of β-arrestin2 predicts its trafficking and signalling functions

Mi-Hye Lee; Kathryn M. Appleton; Erik G. Strungs; Joshua Y. Kwon; Thomas A. Morinelli; Yuri K. Peterson; Stéphane A. Laporte; Louis M. Luttrell

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based ‘signalsomes’. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor–arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand–receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

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Robert H. Oakley

National Institutes of Health

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Jason A. Holt

Howard Hughes Medical Institute

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Jie Zhang

Howard Hughes Medical Institute

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Emanuel Escher

Université de Sherbrooke

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May Simaan

McGill University Health Centre

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