Showing papers by "Michel Bouvier published in 2004"

Roles of G-protein-coupled receptor dimerization.

Abstract: The classical idea that G-protein-coupled receptors (GPCRs) function as monomeric entities has been unsettled by the emerging concept of GPCR dimerization. Recent findings have indicated not only that many GPCRs exist as homodimers and heterodimers, but also that their oligomeric assembly could have important functional roles. Several studies have shown that dimerization occurs early after biosynthesis, suggesting that it has a primary role in receptor maturation. G-protein coupling, downstream signalling and regulatory processes such as internalization have also been shown to be influenced by the dimeric nature of the receptors. In addition to raising fundamental questions about GPCR function, the concept of dimerization could be important in the development and screening of drugs that act through this receptor class. In particular, the changes in ligand-binding and signalling properties that accompany heterodimerization could give rise to an unexpected pharmacological diversity that would need to be considered.

Pharmacological chaperones: potential treatment for conformational diseases

Abstract: Increasing numbers of inherited diseases are found to result from mutations that lead to misfolded proteins. In many cases, the changes in conformation are relatively modest and the function of the protein would not be predicted to be affected. Yet, these proteins are recognized as ‘misfolded’ and degraded prematurely. Recently, small molecules known as chemical and pharmacological chaperones were found to stabilize such mutant proteins and facilitate their trafficking to their site of action. Here, we review the recent published evidence suggesting that pharmacological chaperones represent promising avenues for the treatment of endocrine and metabolic diseases such as hyperinsulinemic hypoglycemia, hypogonadotropic hypogonadism and nephrogenic diabetes insipidus, and might become a general therapeutic stategy for the treatment of conformational diseases.

Mutant Frizzled 4 associated with vitreoretinopathy traps wild-type Frizzled in the endoplasmic reticulum by oligomerization.

Abstract: nt signalling pathways regulate cell proliferation, cell fate and morphogenetic movements. Here, we demonstrate that the Frizzled (Fz) family of Wnt receptors1,2,3,4, similarly to G-protein-coupled receptors (GPCRs)5,6,7, form specific homo- and hetero-oligomers. Two lines of evidence suggest that oligomerization occurs in the endoplasmic reticulum: first, a mutant allele of Fz4, encoding a truncated protein that is retained in the endoplasmic reticulum, is linked to the autosomal-dominant retinal degenerative disease, familial exudative vitreoretinopathy (FEVR)8. We show that this mutant form of Fz4 oligomerizes with wild-type Fz4, retains it in the endoplasmic reticulum and inhibits its signalling. Second, a derivative of Fz1 targeted to the endoplasmic reticulum traps wild-type Fz1 in the endoplasmic reticulum and blocks its signalling. These data support the hypothesis that oligomerization of mutant and wild-type Fz proteins occurs in the endoplasmic reticulum and may explain the genetic dominance of this FEVR allele.

Heterodimerization of V1a and V2 vasopressin receptors determines the interaction with β-arrestin and their trafficking patterns

Abstract: V1a vasopressin receptor (V1aR) and V2 vasopressin receptor (V2R) present distinct mechanisms of agonist-promoted trafficking. Although both receptors are endocytosed by way of β-arrestin-dependent processes, β-arrestin dissociates rapidly from V1aR, allowing its rapid recycling to the plasma membrane while β-arrestin remains associated with V2R in the endosomes, leading to their intracellular accumulation. Here, we demonstrate that, when coexpressed, the two receptors can be endocytosed as stable heterodimers. On activation with a nonselective agonist, both receptors cotrafficked with β-arrestin in endosomes where the stable interaction inhibited the recycling of V1aR to the plasma membrane, thus conferring a V2R-like endocytotic/recycling pattern to the V1aR/V2R heterodimer. Coexpression of the constitutively internalized R137HV2R mutant with V1aR was sufficient to promote cointernalization of V1aR in β-arrestin-positive vesicles even in the absence of agonist stimulation. This finding indicates that internalization of the heterodimer does not require activation of each of the protomers. Consistent with this notion, a V1aR-selective agonist led to the coendocytosis of V2R. In that case, however, the V1aR/V2R heterodimer was not stably associated with β-arrestin, and both receptors were recycled back to the cell surface, indicating that the complex followed the V1aR endocytotic/recycling path. Taken together, these results suggest that heterodimerization regulates the endocytotic processing of G protein-coupled receptors and that the identity of the activated protomer within the heterodimer determines the fate of the internalized receptors.

Functional Rescue of the Constitutively Internalized V2 Vasopressin Receptor Mutant R137H by the Pharmacological Chaperone Action of SR49059

Abstract: In most cases, nephrogenic diabetes insipidus results from mutations in the V2 vasopressin receptor (V2R) gene that cause intracellular retention of improperly folded receptors. We previously reported that cell permeable V2R antagonists act as pharmacological chaperones that rescue folding, trafficking, and function of several V2R mutants. More recently, the vasopressin antagonist, SR49059, was found to be therapeutically active in nephrogenic diabetes insipidus patients. Three of the patients with positive responses harbored the mutation R137H, previously reported to lead to constitutive endocytosis. This raises the possibility that, instead of acting as a pharmacological chaperone by favoring proper maturation of the receptors, SR49059 could mediate its action on R137H V2R by preventing its endocytosis. Here we report that the β-arrestin-mediated constitutive endocytosis of R137H V2R is not affected by SR49059, indicating that the functional rescue observed does not result from a stabilization of the re...

Real-time monitoring of ubiquitination in living cells by BRET.

Abstract: Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of beta-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.

Receptor activity-independent recruitment of βarrestin2 reveals specific signalling modes

Abstract: The roles of βarrestins in regulating G protein coupling and receptor endocytosis following agonist stimulation of G protein-coupled receptors are well characterised. However, their ability to act on their own as direct modulators or activators of signalling remains poorly characterised. Here, βarrestin2 intrinsic signalling properties were assessed by forcing the recruitment of this accessory protein to vasopressin V1a or V2 receptors independently of agonist-promoted activation of the receptors. Such induction of a stable interaction with βarrestin2 initiated receptor endocytosis leading to intracellular accumulation of the βarrestin/receptor complexes. Interestingly, βarrestin2 association to a single receptor protomer was sufficient to elicit receptor dimer internalisation. In addition to recapitulating βarrestin2 classical actions on receptor trafficking, the receptor activity-independent recruitment of βarrestin2 activated the extracellular signal-regulated kinases. In the latter case, recruitment to the receptor itself was not required since kinase activation could be mediated by βarrestin2 translocation to the plasma membrane in the absence of any interacting receptor. These data demonstrate that βarrestin2 can act as a ‘bonafide' signalling molecule even in the absence of activated receptor.

Determination of Protein-Bound Palmitate Turnover Rates Using a Three-Compartment Model That Formally Incorporates [3H]Palmitate Recycling†

Abstract: The observation that the palmitoylation state of certain proteins can be biologically modulated led to the proposal that it could, much like phosphorylation, be an important dynamic regulator of protein function However, based on single-phase exponential decay analysis of data from [(3)H]palmitate pulse/chase experiments, the measured protein-bound palmitate turnover rates were often found to be too slow to account for rapid physiological responses This paper reports that exponential decay does not adequately describe the results of such experiments because it fails to account for the recycling of [(3)H]palmitate from cellular lipids to palmitoyl CoA Taking this recycling into account, a three-compartment model was used to deduce the time-dependent changes of cellular [(3)H]palmitoyl CoA and to infer the time course for the incorporation of [(3)H]palmitate into proteins The validity of the inferences made by the model was checked against data obtained by metabolic labeling of endogenous HEK293 cell proteins In addition, the model could account for reported anomalies, discrepancies, and apparently paradoxical observations obtained by traditional analysis of data from pulse/chase experiments Including the recycling of cellular palmitate in the formal description of the system offers a new tool for quantitative assessment of protein-bound palmitate turnover rates Through the re-evaluation of these rates, the model provides a means for the reassessment of the potential physiological implications of dynamic palmitoylation The model may also be generally applicable to other areas of research where recycling of tracer is a concern