Hélène Bonin

Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341.

Exposure of beta 2‐adrenergic receptors to agonists causes a rapid desensitization of the receptor‐stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist‐promoted phosphorylation of the beta 2‐adrenergic receptor (beta 2AR) by protein kinase A and the beta‐adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2‐adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide‐sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor‐stimulated adenylyl cyclase response was observed but resulted from the agonist‐induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist‐promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational Rearrangements and Signaling Cascades Involved in Ligand-Biased Mitogen-Activated Protein Kinase Signaling through the β1-Adrenergic Receptor

In recent years, several studies have demonstrated that different ligands can have distinct efficacy profiles toward various signaling pathways through a unique receptor. For example, β1-adrenergic compounds that are inverse agonists toward the adenylyl cyclase (AC) can display agonist activity for the mitogen-activated protein kinase (MAPK) pathway. Such a phenomenon, often termed functional selectivity, has now been clearly established for many G protein-coupled receptors when considering distinct signaling output. However, the possibility that ligands could selectively engage distinct effectors to activate a single signaling output by promoting specific receptor conformations has not been extensively examined. Here, we took advantage of the fact that isoproterenol, bucindolol and propranolol (full, partial, and inverse agonists for the AC pathway, respectively) all activate MAPK through the β1-adrenergic receptor (β1AR) to probe such conformational-biased signaling. Although the three compounds stimulated MAPK in a src-dependent manner, isoproterenol acted through both Gαiβγ- and G protein-independent pathways, whereas bucindolol and propranolol promoted MAPK activation through the G protein-independent pathway only. The existence of such distinct signaling cascades linking β1AR to MAPK activation was correlated with ligand-specific conformational rearrangements of receptor/G protein complexes measured by bioluminescence resonance energy transfer. Taken together, our data indicate that discrete local conformational changes can selectively promote the recruitment of distinct proximal signaling partners that can engage distinct signaling outputs and/or converge on the same signaling output.

Multiplexing of Multicolor Bioluminescence Resonance Energy Transfer

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.

Biochemical characterization of β2-Adrenergic receptor dimers and oligomers

Abstract G Protein-coupled receptor dimerization/oligomerization has been well established during the last several years. Studies have demonstrated the existence of dimers/digomers both in vitro and in living cells. However, a thorough characterization of the biochemical nature of receptor dimers and oligomers as well as their occurrence at the cell surface has not been properly addressed. In this study, we show that both β2-adrenergic receptor (β2AR) dimers and oligomers exist at the plasma membrane and that the detection of such species, following receptor solubilization and resolution by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), does not result from the formation of spurious disulfide bonds during cell lysis. Moreover, our results indicate that the biochemical nature of β2AR dimers is different from that of the oligomers. Although both complexes are partially resistant to SDS denaturation, disulfide bonding is absolutely required for the stability of β2AR oligomers but not dimers in SDS-PAGE. Indeed, dimeric species can be detected even in the presence of high concentrations of reducing and alkylating agents. Although the different biochemical nature of the dimers and oligomers may be indicative of distinct biological roles in cells, additional studies will be required to further elucidate the biosynthesis and function of these receptor forms.

Phorbol-ester-induced phosphorylation of the β2-adrenergic receptor decreases its coupling to Gs

Phorbol‐esters have been shown to modulated the β‐adrenergie‐stimulated adenylyl cyclase in a number of cell lines. Here, using site directed mutagenesis, we investigate the role of the β‐adrenergic receptor phosphorylation by protein kinase C in this regulatory process. Mutation of the serine‐261, ‐262, ‐344 and ‐345 of the β2‐adrenergic receptor prevented the phorbol‐ester‐induced phosphorylation of the receptor. This mutation also abolished the phorbol‐ester‐induced decrease in high‐affinity agonist binding and potency of the β2adrenergic receptor. We suggest that protein kinase C mediated phosphorylation of the receptor promotes its functional uncoupling.

Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy.

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT‐Pep‐27) did not affect basal signaling activity but almost completely abolished agonist‐promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT‐Pep‐27 prevented both agonist‐promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.

Cellular and subcellular context determine outputs from signaling biosensors.

The use of biosensors either individually or as part of panels has now become a common technique to capturing signaling events in living cells. Such biosensors have become particularly important for probing biased signaling and allostery in G protein-coupled receptor drug screening efforts. However, assumptions about the portability of such biosensors between cell types may lead to misinterpretation of drug effects on specific signaling pathways in a given cellular context. Further, the output of a particular biosensor may be different depending on where it is localized in a cell. Here, we discuss strategies to mitigate these concerns which should feed into future biosensor design and usage.