Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors

<div><p>GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance <i>in vivo</i>. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure–function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated “GASP motif” of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.</p> </div

Study of proton exchange membrane fuel cell safety procedures in case of emergency shutdown

International audienc

Structure of the Toll/interleukin 1 receptor (TIR) domain of the immunosuppressive Brucella effector BtpA/Btp1/TcpB.

International audienceBtpA/Btp1/TcpB is a virulence factor produced by Brucella species that possesses a Toll interleukin-1 receptor (TIR) domain. Once delivered into the host cell, BtpA interacts with MyD88 to interfere with TLR signalling and modulates microtubule dynamics. Here the crystal structure of the BtpA TIR domain at 3.15 Å is presented. The structure shows a dimeric arrangement of a canonical TIR domain, similar to the Paracoccus denitrificans Tir protein but secured by a unique long N-terminal α-tail that packs against the TIR:TIR dimer. Structure-based mutations and multi-angle light scattering experiments characterized the BtpA dimer conformation in solution. The structure of BtpA will help with studies to understand the mechanisms involved in its interactions with MyD88 and with microtubules

A purified truncated form of yeast Gal4 expressed in <em>Escherichia coli</em> and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active <em>in cellulo</em>

International audienceGal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticies. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models

Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain

Abstract Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy

Purified full-length GPCRs dose-dependently bind to the central domain of GASP-1 in SPR experiments.

<p><i>A</i>, Interaction of the central domain of GASP-1 compared to the full-length protein with GST-fused ADRB2 and CNR2 C-tails by GST pull down experiments. The results show that both receptors interact <i>in vitro</i> with GASP-1 and that the central part of GASP-1 is strongly involved in the interaction with ADRB2 and CNR2. <i>B</i>, Binding of a range of concentrations of ADRB2 to the central domain of GASP-1. <i>C</i>, Binding of a range of concentrations of CNR2 to the central domain of GASP-1. Overall, we observed a dose-dependent binding of ADRB2 and CNR2 with the central domain of GASP-1. The receptor concentrations are indicated on the figures. All curves are double referenced and corrected for changes in capture density of the central domain of GASP-1. ADRB2, β₂ adrenergic receptor; CNR2, cannabinoid receptor type 2.</p

A small synthetic peptide derived from the GASP motif of GASP-2 blocks the interaction between GASPs and GPCR C-tails in GST-pull down experiments.

<p>A, GASP peptide competes for the interaction between GASP-2 and GST-fused ADRB1 C-tail. The scrambled peptide displayed no significant effect on the interaction between GASP-2 and ADRB1. <i>B,</i> A fixed concentration of GASP peptide (150 µM) inhibits the interaction between GASP-1, -2 or -7 with ADRB1 C-tail, but not the scrambled peptide. <i>C,</i> Phosphor-imaging quantification of the competition experiments for the interaction between GASP-1, -2 and -7 and four different receptor C-tails with GASP peptide. A fixed concentration of GASP peptide (150 µM) strongly inhibited interactions of GASPs with ADRB1, M₁, CALCR and TXA₂ C-tails. Results are represented as percent of the interaction between the corresponding GASPs and GPCRs in absence of peptide (mean ± S.E.M of three independent experiments).</p

GST-pull down experiments with radiolabelled GASP-1, -2, -3, -6, -7 and -9 and GST-fused receptor C-tails.

<p><i>A,</i> GASP-1, -2 and -3 showed medium to strong interactions with some GPCR C-tails but no interaction was detected with the two one-transmembrane receptor C-tails (TGF_β and IGF₁). <i>B,</i> GASP-7 showed weak to medium interactions with some GPCR C-tails. GASP-6 and -9 showed very weak interactions with all tested receptors. No interaction was detected with TGF_β and IGF C-tails. Data were quantified by Phosphor-imaging. Results are shown as percent of the [³⁵S]-GASPs input retained by the GST-fused receptor C-tails and correspond to the mean ± S.E.M of three independent experiments. Lower panels correspond to representative gel images. 5HT₇, 5-hydroxytryptamine 7 receptor; ADRB1, β₁ adrenergic receptor; CALCR, calcitonin receptor; DOR, δ-opioid receptor; FZ₄, frizzled 4 receptor; H₂, histamine 2 receptor; IGF₁, insulin growth factor I receptor; KOR, κ-opioid receptor; M₁, muscarinic M₁ acetylcholine receptor; M₂, muscarinic M₂ acetylcholine receptor; MOR, µ-opioid receptor; ORL₁, opioid receptor-like 1; TXA₂, α isoform of the thromboxane A₂ receptor; TGF_β, type III transforming growth factor β receptor.</p

The GASP motif is critical for the interaction of GASP-2 with GPCRs.

<p><i>A,</i> GST-pull down experiments with two truncated mutants of GASP-2 and ADRB1, M₁ and CALCR C-tails. Grey boxes represent the 15 AA GASP motifs. Deletion analysis revealed that the central domain of GASP-2, which contains the two GASP motifs, is critical for the interaction between GASP-2 and ADRB1, M₁ and CALCR C-tails. <i>B,</i> GST-pull down experiments with full-length GASP-2 where one (GASP2-m1 and GASP2-m2) or both GASP motifs (GASP2-dm) were mutated. Grey boxes represent the wild-type motifs and X represent the mutant motifs. Consensus sequences are given for wild-type and mutant motifs. Mutated amino acids are underlined. Site directed mutagenesis analysis of these two repeated motifs showed that they played a crucial role in the interaction of GASP-2 with the three receptor C-tails tested here. Results are shown as percent of the wild-type GASP-2 interaction and correspond to the mean ± S.E.M of three independent experiments.</p

Schematic comparison of GASP family members.

<p>Black boxes represent the conserved carboxyl-terminal domain of 250 amino acids. The percentage of identical amino acids shared with GASP-1 is indicated within each box. Small grey boxes represent a highly conserved motif of 15 amino acids that is repeated 22 times in GASP-1 and two times in GASP-2 to -5. The consensus sequence of this motif is: (E/D/G) (E/D) E X (I/L/V/S/T) (I/V/A/F) (G/N) (S/T) W F W (A/V/T/S/D/E) (G/E/R) (E/D/K) (E/D/K/A/Q). For GASP-2, two regions showing significant sequence homology with GASP-1 are separated by a gap represented by dotted lines. GASPs accession numbers from SPtrEMBL database are indicated on the left of the figure.</p