The adhesion G-protein-coupled receptor Gpr116 is essential to maintain the skeletal muscle stem cell pool.

Skeletal muscle is populated with a reservoir of quiescent muscle stem cells (MuSCs), which regenerate the tissue after injury. Here, we show that the adhesion G-protein-coupled receptor Gpr116 is essential for long-term maintenance of the MuSC pool. Quiescent MuSCs express high levels of Gpr116, which is rapidly downregulated upon MuSC activation. MuSCs deficient for Gpr116 exhibit progressive depletion over time and are defective in self-renewal. Adhesion G-protein-coupled receptors contain an agonistic peptide sequence, called the "Stachel" sequence, within their long N-terminal ectodomains. Stimulation of MuSCs with the GPR116 Stachel peptide delays MuSC activation and differentiation. Stachel peptide stimulation of GPR116 leads to strong interaction with ß-arrestins. Stimulation of GPR116 increases the nuclear localization of ß-arrestin1, where it interacts with cAMP response element binding protein to regulate gene expression. Altogether, we propose a model by which GPR116 maintains the MuSC pool via nuclear functions of ß-arrestin1.

Structural and Functional Diversity of Animal Toxins Interacting With GPCRs.

Peptide toxins from venoms have undergone a long evolutionary process allowing host defense or prey capture and making them highly selective and potent for their target. This has resulted in the emergence of a large panel of toxins from a wide diversity of species, with varied structures and multiple associated biological functions. In this way, animal toxins constitute an inexhaustible reservoir of druggable molecules due to their interesting pharmacological properties. One of the most interesting classes of therapeutic targets is the G-protein coupled receptors (GPCRs). GPCRs represent the largest family of membrane receptors in mammals with approximately 800 different members. They are involved in almost all biological functions and are the target of almost 30% of drugs currently on the market. Given the interest of GPCRs in the therapeutic field, the study of toxins that can interact with and modulate their activity with the purpose of drug development is of particular importance. The present review focuses on toxins targeting GPCRs, including peptide-interacting receptors or aminergic receptors, with a particular focus on structural aspects and, when relevant, on potential medical applications. The toxins described here exhibit a great diversity in size, from 10 to 80 amino acids long, in disulfide bridges, from none to five, and belong to a large panel of structural scaffolds. Particular toxin structures developed here include inhibitory cystine knot (ICK), three-finger fold, and Kunitz-type toxins. We summarize current knowledge on the structural and functional diversity of toxins interacting with GPCRs, concerning first the agonist-mimicking toxins that act as endogenous agonists targeting the corresponding receptor, and second the toxins that differ structurally from natural agonists and which display agonist, antagonist, or allosteric properties.

Single asparagine to arginine mutation allows PerR to switch from PerR box to fur box.

Fur family proteins, ubiquitous in prokaryotes, play a pivotal role in microbial survival and virulence in most pathogens. Metalloregulators, such as Fur and PerR, regulate the transcription of genes connected to iron homeostasis and response to oxidative stress, respectively. In Bacillus subtilis, Fur and PerR bind with high affinity to DNA sequences differing at only two nucleotides. In addition to these differences in the PerR and Fur boxes, we identify in this study a residue located on the DNA binding motif of the Fur protein that is critical to discrimination between the two close DNA sequences. Interestingly, when this residue is introduced into PerR, it lowers the affinity of PerR for its own DNA target but confers to the protein the ability to interact strongly with the Fur DNA binding sequence. The present data show how two closely related proteins have distinct biological properties just by changing a single residue.

Molecular exploration of the α(1A)-adrenoceptor orthosteric site: binding site definition for epinephrine, HEAT and prazosin.

Despite the physiological and pharmacological importance of the α1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of (125)I-HEAT, (3)H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist.

The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and ßarrestin.

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

Crystallization of recombinant green mamba ρ-Da1a toxin during a lyophilization procedure and its structure determination.

ρ-Da1a toxin from eastern green mamba (Dendroaspis angusticeps) venom is a polypeptide of 65 amino acids with a strong affinity for the G-protein-coupled α(1A)-adrenoceptor. This neurotoxin has been crystallized from resolubilized lyophilized powder, but the best crystals grew spontaneously during lyophilization. The crystals belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 37.37, c = 66.05 Å, and diffracted to 1.95 Å resolution. The structure solved by molecular replacement showed strong similarities to green mamba muscarinic toxins.

G protein-coupled receptors, an unexploited animal toxin targets: Exploration of green mamba venom for novel drug candidates active against adrenoceptors.

At a time when pharmaceutical companies are having trouble finding new low MW drugs and when biologics are becoming more common, animal venoms could constitute an underexploited source of novel drug candidates. We looked for identifying novel animal toxins active against G protein-coupled receptors (GPCR), the most frequently exploited class of treatment targets, with the aim to develop novel research tools and drug candidates. Screening of green mamba (Dendroaspis angusticeps) venom against adrenoceptors identified two novel venom peptides. ρ-Da1a shown an affinity of 0.35 nM for the α1a-AR while ρ-Da1b displayed affinities between 14 and 73 nM for the three α2-ARs. These two venom peptides have sequences similar to those of muscarinic toxins and belong to the three-finger-fold protein family. α1a-AR is the primary target for the treatment of prostate hypertrophy. In vitro and in vivo tests demonstrated that ρ-Da1a reduced prostatic muscle tone as efficiently as tamsulosin (an antagonist presently used), but with fewer cardiovascular side effects. α2-ARs are the prototype of GPCRs not currently used as treatment targets due to a lack of specific ligands. Blockage of these receptors increases intestinal motility, which may be compromised by abdominal surgery and reduces orthosteric hypotension. In vitro and in vivo tests demonstrated that ρ-Da1b antagonizes α2-ARs in smooth muscles and increased heart rate and blood catecholamine concentrations. These results highlight possible exploitation of ρ-Da1a and ρ-Da1b in important pathologies.

Pharmacological characterization of zinc and copper interaction with the human alpha(1A)-adrenoceptor.

Metal ions have a major role in human health, and interact with many classes of receptors including the G-protein coupled receptors. In the peripheral system, zinc mainly accumulates in the soft prostate organ and, with copper, influences prostate disease progression, from normal to hypertrophic or cancerous states. The development of these pathologies may be influenced by the α(1A)-adrenoceptor, the principal regulator of prostate tonicity. There is currently no information on possible interactions between metals and the α(1A)-adrenoceptor. We therefore studied the effects of several mono- and divalent ions on this receptor subtype using binding and functional experiments performed on expressed cloned human α(1A)-adrenoceptor. Regardless of the counter anion used, Zn(2+) and Cu(2+) interact with α(1A)-adrenoceptor with apparent affinities in the low micromolar range. In addition, using specific binding experiments, we established that these ions acted as negative allosteric ligands on prazosin/α(1A)-adrenoceptor interaction, but in a different manner from the allosteric modulator 5-(N-ethyl-N-isopropyl)-amiloride, suggesting distinct mode of interaction. In addition, the presence of Cu(2+) weakly decreased epinephrine affinity, whereas the addition of Zn(2+) shifted to the left the epinephrine binding curve, revealing a positive allosteric effect but only on half of the binding site. Finally, cell-based functional experiments demonstrated that Zn(2+) and Cu(2+) antagonized epinephrine activation in an insurmountable manner, by reducing agonist efficacy without any shift in the epinephrine activation curves. This study shows the interactions between metal ions and the α(1A)-adrenoceptor with affinities compatible with physiological concentrations and suggests that zinc and copper may have a biological role in prostate function.

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2-adrenoceptors.

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACH In binding experiments with (3) H-rauwolscine, we studied the interactions of green mamba venom fractions with α(2) -adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α(2)-adrenoceptors expressed in mammalian cells. KEY RESULTS ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of (3) H-rauwolscine binding to the three α(2)-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α(2A) -adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA(2) values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively. CONCLUSIONS AND IMPLICATIONS ρ-Da1b is the first toxin identified to specifically interact with α(2)-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α(2)-adrenoceptor subtypes.