Luc Ménard

Role of β-Arrestin in Mediating Agonist-Promoted G Protein-Coupled Receptor Internalization

β-Arrestins are proteins that bind phosphorylated heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) and contribute to the desensitization of GPCRs by uncoupling the signal transduction process. Resensitization of GPCR responsiveness involves agonist-mediated receptor sequestration. Overexpression of β-arrestins in human embryonic kidney cells rescued the sequestration of β2-adrenergic receptor (β2AR) mutants defective in their ability to sequester, an effect enhanced by simultaneous overexpression of β-adrenergic receptor kinase 1. Wild-type β2AR sequestration was inhibited by the overexpression of two β-arrestin mutants. These findings suggest that β-arrestins play an integral role in GPCR internalization and thus serve a dual role in the regulation of GPCR function.

Dynamin and beta-arrestin reveal distinct mechanisms for G protein-coupled receptor internalization.

The process of agonist-promoted internalization (sequestration) of G protein-coupled receptors (GPCRs) is intimately linked to the regulation of GPCR responsiveness. Following agonist-mediated desensitization, sequestration of GPCR is presumably associated with the dephosphorylation and recycling of functional receptors. However, the exact mechanisms responsible for GPCR sequestration, even for the prototypic β2-adrenergic receptor (β2AR), have remained controversial. We demonstrate here that dynamin, a GTPase that regulates the formation and internalization of clathrin-coated vesicles, is essential for the agonist-promoted sequestration of the β2AR, suggesting that the β2AR internalizes via the clathrin-coated vesicle-mediated endocytic pathway. In contrast, internalization of the angiotensin II type 1A receptor (AT1AR), another typical GPCR, does not require dynamin. In addition, the AT1AR internalizes independent of the function of β-arrestin, a critical component for β2AR cellular trafficking, but additional AT1ARs are mobilized to the dynamin-dependent pathway upon overexpression of β-arrestin. These findings demonstrate that GPCRs can utilize distinct endocytic pathways, distinguishable by dynamin and β-arrestin, and that β-arrestins function as adaptor proteins specifically targeting GPCRs for dynamin-dependent endocytosis via clathrin-coated vesicles.

Molecular and biochemical features of poly (ADP-ribose) metabolism

In the past five years, poly(ADP-ribosyl)ation has developed greatly with the help of molecular biology and the improvement of biochemical techniques. In this article, we describe the physico-chemical properties of the enzymes responsible for the synthesis and degradation of poly(ADP-ribose), respectively poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase. We then discuss the possible roles of this polymer in DNA repair and replication as well as in cellular differentiation and transformation. Finally, we put forward various hypotheses in order to better define the function of this polymer found only in eucaryotes. (Mol Cell Biochem122: 171–193, 1993)

THE BRET2/ARRESTIN ASSAY IN STABLE RECOMBINANT CELLS: A PLATFORM TO SCREEN FOR COMPOUNDS THAT INTERACT WITH G PROTEIN-COUPLED RECEPTORS (GPCRS)*

ABSTRACT In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC™, RLuc emits blue light at 395u2005nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510u2005nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395u2005nm) using appropriate dual channel luminometry instruments (e.g., Fusion™ Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein–Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of β-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2 : β-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2 : β-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R : RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand–GPCR interactions which can be applied to ligand identification for orphan receptors.

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.

Both stimulatory and inhibitory GDPGTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins☆

Six peaks of small GTP-binding proteins (G proteins) were separated by column chromatographies from the cytosol fraction of the differentiated HL-60 cells: two peaks of rho p21, one peak of smg/rap1 p21, two peaks of rac1 p21, and one peak of an unidentified small G protein with a Mr of about 20,000 (20 KG). smg GDS, previously thought to be a stimulatory GDP/GTP exchange protein for smg p21, Ki-ras p21, and rho p21, but not for Ha-ras p21 or smg p25A, was also active on rac1 p21. rho GDI, previously thought to be an inhibitory GDP/GTP exchange protein specific for rho p21, was also active on rac1 p21. These results indicate that both smg GDS and rho GDI are active on multiple small G proteins.

Enzymological properties of poly(ADP-ribose)polymerase: characterization of automodification sites and NADase activity

We have characterized the effect of poly(ADP-ribose) polymerase automodification on the enzymes activities, which include poly(ADP-ribose) synthesis and NADase activity. The apparent Km of the enzyme for NAD+ during polymer synthesis is higher than the one measured for alternate NADase activity. Furthermore, we have found that there are 28 automodification sites, in contrast to the 15 sites (postulated to be on the 15 glutamic acids) reported to be present in the automodification domain. For the first time, we show that some of these acceptor sites are outside the reported automodification domain (15 kDa); we demonstrate automodification in the NAD+ binding domain (55.2 kDa) and the DNA binding domain (42.5 kDa). We have analyzed the relationship between the number of sites modified on poly(ADP-ribose) polymerase and its effect on the polymerization activity and its alternate NADase activity. Automodification greatly altered both enzyme activities, decreasing both polymer synthesis and alternate NADase activity.

Expression and recovery of functional G-protein-coupled receptors using baculovirus expression systems.

Baculovirus expression systems have been used for more than ten years as the tool of choice to over-express G-protein-coupled receptors. Although this expression system has also been used to study the signaling mechanisms of the receptors at the cellular level, it was found to be a most useful method to produce large quantities of receptors for biochemical and biophysical studies. Methods that allow easy and selective recovery of properly folded and mature receptors in viral particles open new perspectives for such applications.

Poly(ADP-ribosyl)ation of chromatin in an in-vitro poly(ADP-ribose)-turnover system

This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.

Poly(ADP-ribose) catabolism in mammalian cells

Poly(ADP-ribose) catabolism is a complex situation involving many proteins and DNA. We have developed anin vitro turnover system where poly(ADP-ribose) metabolism is monitored in presence of different relative amounts of two principal enzymes poly(ADP-ribose) transferase and poly(ADP-ribose) glycohydrolase along with other proteins and DNA. Our current results reviewed here show that the quality of polymer, i.e. chain length and complexity, as well as preference for the nuclear substrate varies depending upon the availability of poly(ADP-ribose) glycohydrolase. These results are interpreted in the light of the recent data implicating poly(ADP-ribose) metabolism in DNA-repair. (Mol Cell Biochem 138: 45–52 1994)