A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002)

CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling

Stress and anxiety disorders are risk factors for depression and these behaviours are modulated by corticotropin releasing factor (CRFR1) and serotonin (5-HT2R) receptors. However, the potential behavioral and cellular interaction between these two receptors is unclear. Here, we showed that pre-administration of CRF into the prefrontal cortex of mice sensitized 5-HT2R-mediated anxiety behaviours in response to 2,5-dimethoxy-4-iodoamphetamine. In both heterologous cell cultures and mouse cortical neurons, the activation of CRFR1 also sensitized 5-HT2 receptor-mediated inositol phosphate formation. CRFR1-mediated increases in 5-HT2R signaling were dependent upon receptor internalization and receptor recycling via rapid recycling endosomes resulting in increased cell surface 5-HT2R expression. The sensitization of 5-HT2R signaling by CRFR1 required intact PDZ domain binding motifs at the end of the C-terminal tails of both receptor types. These data reveal a novel mechanism by which CRF, a peptide known to be released by stress, sensitized anxiety-related behaviour via sensitization of 5-HT2R signaling

Structure-Based Design of PDZ Ligands as Inhibitors of 5-HT2A Receptor/PSD-95 PDZ1 Domain Interaction Possessing Anti-hyperalgesic Activity

International audienceDisrupting the interaction between the PDZ protein PSD-95 and the C-terminal domain of the 5-HT2A serotonin receptor has been shown to reduce hyperalgesia in a rodent model of neuropathic pain. Here, we designed and synthesized PDZ ligands capable of binding to the first PDZ domain (PDZ1) of the PSD-95 protein and evaluated their biological activity in vitro and in vivo. A series of substituted indoles was identified by docking simulations, and six novel analogues were synthesized. Three analogues displayed strong interactions with the first PDZ domain (PDZ1) of PDZ-95 in 1H-15N heteronuclear single-quantum coherence (HSQC) experiments and two of them were able to inhibit the interaction between PSD-95 and the 5-HT2A receptor in vitro. We identified compound 8b as the analogue able to significantly suppress mechanical hyperalgesia in an experimental model of traumatic neuropathic pain in the rat. This effect was suppressed by the coadministration of the 5-HT2A receptor antagonist M100907, consistent with an inhibitory effect upon 5-HT2A receptor/PSD-95 interaction. Finally, we determined an NMR-restraint driven model structure for the PSD95 PDZ1/8b complex, which confirms that indole 8b binds to the putative PDZ-ligand binding site

Opposite Effects of PSD-95 and MPP3 PDZ Proteins on Serotonin 5-Hydroxytryptamine(2C) Receptor Desensitization and Membrane Stability

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons

Living with Long-Term Pain after a Stroke

The general aim of this thesis was to classify and describe long-term pain two years after a stroke and to describe the experiences of pain, and the consequences it has on the persons’ lives. The studies were conducted from a multidimensional perspective on pain, combining quantitative and qualitative methods. Three types of long-term pain were classified and described among the 43 participants included, aged 33-82 years. These were central post-stroke pain, nociceptive, mainly shoulder pain, and tension-type headache. Pain onset, within one to six months in most of the cases was after discharge from the hospital. Continuous pain or pain almost every day was reported by nearly two-thirds. The pain was mostly described as troublesome, annoying and tiring in all pain groups. The rating of pain intensity revealed individual differences among the participants within the pain groups. In addition to long-term pain, the participants suffered several impairments and nearly half of them were dependent on others, and two-thirds on assistive devices. Several coping strategies were described, most often problem-focused. Their health-related quality of life was decreased, mostly related to their long-term pain and physical impairments. Their experiences of caring revealed the need of improvements in knowledge about longterm pain, attention and understanding among the professionals, and continuity in the contacts.On the day of the public defence the status of article III was: Accepted for publication and the status of article IV was: In press.</p