Subtlety of the structure-affinity and structure-efficacy relationships around a nonpeptide oxytocin receptor agonist.

Very few nonpeptide oxytocin agonists have currently been reported, and none of them seem suitable for the in vivo investigation of the oxytocin mediated functions. In an attempt to rationalize the design of better tools, we have systematically studied the structural determinants of the affinity and efficacy of representative ligands of the V(1a), V(2), and OT receptor subtypes. Despite apparently obvious similarity between the ligand structures on one hand, and between the receptor subtypes on the other hand, the binding affinity and the functional activity profiles of truncated and hybrid ligands highlight the subtlety of ligand-receptor interactions for obtaining nonpeptide OT receptor agonists.

Leukotriene BLT2 receptor monomers activate the G(i2) GTP-binding protein more efficiently than dimers.

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.

Solid-phase organic tagging resins for labeling biomolecules by 1,3-dipolar cycloaddition: application to the synthesis of a fluorescent non-peptidic vasopressin receptor ligand.

Two novel solid-phase organic tagging (SPOrT) resins were synthesized to facilitate the labeling of peptides and small organic compounds with a fluorescent probe. Both resins were obtained from the commercially available backbone amide linker (BAL) resin. Following the solid-phase synthesis of model compounds, a tripeptide and benzazepine, the fluorescent probe derived from Lissamine Rhodamine B was incorporated through CuI-catalyzed 1,3-dipolar cycloaddition. Final cleavage in acidic media enabled access to both types of molecules in good yield with high purity. The SPOrT resin was successfully applied to the preparation of the first non-peptidic fluorescent compound with a nanomolar affinity for the human vasopressin V2 receptor (V2R) subtype. This molecule will find application in binding assays that use polarization or fluorescence resonance energy-transfer (FRET) techniques. The SPOrT resins are also well suited for other tags and the parallel synthesis of a fluorescently tagged library for protein screening.

The multifunctional protein GC1q-R interacts specifically with the i3 loop arginine cluster of the vasopressin V2 receptor.

In this study, we identified the multifunctional protein GC1q-R as a novel vasopressin V(2) receptor (V(2)R) interacting protein. For this purpose, we have developed a proteomic approach combining pull-down assays using a cyclic peptide mimicking the third intracellular loop of V(2)R as a bait and mass spectrometry analyses of proteins isolated from either rat or human kidney tissues or the HEK 293 cell line. Co-immunoprecipitation of GC1q-R with the c-Myc-tagged h-V(2)R expressed in a HEK cell line confirmed the existence of a specific interaction between GC1q-R and the V(2) receptor. Then, construction of a mutant receptor in i3 loop allowed us to identify the i3 loop arginine cluster of the vasopressin V(2) receptor as the interacting determinant for GC1q-R interaction. Using purified receptor as a bait and recombinant (74-282) GC1q-R, we demonstrated a direct and specific interaction between these two proteins via the arginine cluster.

Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement.

Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.

Assembly-dependent surface targeting of the heterodimeric GABAB Receptor is controlled by COPI but not 14-3-3.

Cell surface expression of transmembrane proteins is strictly regulated. Mutually exclusive interaction with COPI or 14-3-3 proteins has been proposed as a mechanism underlying such trafficking control of various proteins. In particular, 14-3-3 dimers have been proposed to "sense" correctly assembled oligomers, allowing their surface targeting by preventing COPI-mediated intracellular retention. Here we examined whether such a mechanism is involved in the quality control of the heterodimeric G protein-coupled GABAB receptor. Its GB1 subunit, carrying the retention signal RSR, only reaches the cell surface when associated with the GB2 subunit. We show that COPI and 14-3-3 specifically bind to the GB1 RSR sequence and that COPI is involved in its intracellular retention. However, we demonstrate that the interaction with 14-3-3 is not required for proper function of the GABAB receptor quality control. Accordingly, competition between 14-3-3 and COPI cannot be considered as a general trafficking control mechanism. A possible other role for competition between COPI and 14-3-3 binding is discussed.

Plant-induced cell death in the oomycete pathogen Phytophthora parasitica.

The activation of programmed cell death in the host during plant-pathogen interactions is an important component of the plant disease resistance mechanism. In this study we show that activation of programmed cell death in microorganisms also regulates plant-pathogen interactions. We found that a form of vacuolar cell death is induced in the oomycete Phytophthora parasitica--the agent that causes black shank disease in Nicotiana tabacum--by extracellular stimuli from resistant tobacco. The single-celled zoospores underwent cell death characterized by dynamic membrane rearrangements, cell shrinkage, formation of numerous large vacuoles in the cytoplasm and degradation of cytoplasmic components before plasma membrane disruption. Phytophthora cell death required protein synthesis but not caspase activation, and was associated with the production of intracellular reactive oxygen species. This characterization of plant-mediated cell death signalling in pathogens will enhance our understanding of the biological processes regulating plant-pathogen interactions, and improve our ability to control crop diseases.