Christian Bergmayr

Insights into the molecular evolution of oxytocin receptor ligand binding.

The design and development of selective ligands for the human OT (oxytocin) and AVP (arginine vasopressin) receptors is a big challenge since the different receptor subtypes and their native peptide ligands display great similarity. Detailed understanding of the mechanism of OTs interaction with its receptor is important and may assist in the ligand- or structure-based design of selective and potent ligands. In the present article, we compared 69 OT- and OT-like receptor sequences with regards to their molecular evolution and diversity, utilized an in silico approach to map the common ligand interaction sites of recently published G-protein-coupled receptor structures to a model of the human OTR (OT receptor) and compared these interacting residues within a selection of different OTR sequences. Our analysis suggests the existence of a binding site for OT peptides within the common transmembrane core region of the receptor, but it appears extremely difficult to identify receptor or ligand residues that could explain the selectivity of OT to its receptors. We remain confident that the presented evolutionary overview and modelling approach will aid interpretation of forthcoming OTR crystal structures.

Recruitment of a cytoplasmic chaperone relay by the A2A-adenosine receptor.

Background: The A2A receptor is known to accumulate in the endoplasmic reticulum. Results: Mass spectrometry identified molecular chaperones (HSP90 and HSP70) bound to the A2A receptor. Conclusion: Sequential recruitment of chaperones to the cytosolic face of the A2A receptor is consistent with a heat-shock protein relay assisting folding. Significance: The observations are consistent with a chaperone/COPII exchange model, where heat-shock proteins bound to the receptor preclude its premature ER export. The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.

Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species

An oxytocin derivative that may not trigger adverse side effects has been generated. A more selective oxytocin receptor agonist Oxytocin is clinically used to induce labor, and there is interest in using this peptide to treat social disorders. However, oxytocin triggers adverse cardiovascular side effects because it activates the vasopressin receptor and the oxytocin receptor. Muttenthaler et al. generated ligands based on oxytocin with subtle modifications, yielding a lead compound that was more selective for the oxytocin receptor than for the vasopressin receptors. It reduced social fear in mice and induced contractile activity in human myometrial strips without affecting cultured cardiomyocytes. Given the cross-talk between oxytocin, vasopressin, and their receptors, this compound will also be helpful in identifying effects that are solely mediated by the oxytocin receptor. Oxytocin and vasopressin mediate various physiological functions that are important for osmoregulation, reproduction, cardiovascular function, social behavior, memory, and learning through four G protein–coupled receptors that are also implicated in high-profile disorders. Targeting these receptors is challenging because of the difficulty in obtaining ligands that retain selectivity across rodents and humans for translational studies. We identified a selective and more stable oxytocin receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin and vasopressin. [Se-Se]-oxytocin-OH displayed similar potency to oxytocin but improved selectivity for OTR, an effect that was retained in mice. Centrally infused [Se-Se]-oxytocin-OH potently reversed social fear in mice, confirming that this action was mediated by OTR and not by V1a or V1b vasopressin receptors. In addition, [Se-Se]-oxytocin-OH produced a more regular contraction pattern than did oxytocin in a preclinical labor induction and augmentation model using myometrial strips from cesarean sections. [Se-Se]-oxytocin-OH had no activity in human cardiomyocytes, indicating a potentially improved safety profile and therapeutic window compared to those of clinically used oxytocin. In conclusion, [Se-Se]-oxytocin-OH is a novel probe for validating OTR as a therapeutic target in various biological systems and is a promising new lead for therapeutic development. Our medicinal chemistry approach may also be applicable to other peptidergic signaling systems with similar selectivity issues.

Repurposing treprostinil for enhancing hematopoietic progenitor cell transplantation

Activation of Gs-coupled receptors enhances engraftment of hematopoietic stem and progenitor cells (HSPCs). We tested the hypothesis that treprostinil, a prostacyclin analog approved for the treatment of pulmonary hypertension, can be repurposed to improve hematopoietic stem cell transplantation. Murine and human HSPCs were isolated from bone marrow and umbilical cord blood, respectively. Prostanoid receptor agonists and the combination thereof with forskolin were tested for their capacity to stimulate [3H]cAMP accumulation in HSPCs. Three independent approaches were employed to verify the ability of agonist-activated HSPCs to reconstitute the bone marrow in lethally irradiated recipient mice. The underlying mechanism was explored in cellular migration assays and by blocking C-X-C motif chemokine receptor 4 (CXCR4). Among several prostanoid agonists tested in combination with forskolin, treprostinil was most efficacious in raising intracellular cAMP levels in murine and human HPSCs. Injection of murine and human HSPCs, which had been pretreated with treprostinil and forskolin, enhanced survival of lethally irradiated recipient mice. Survival was further improved if recipient mice were subcutaneously administered treprostinil (0.15 mg kg−1 8 h−1) for 10 days. This regimen also reduced the number of HSPCs required to rescue lethally irradiated mice. Enhanced survival of recipient mice was causally related to treprostinil-enhanced CXCR4-dependent migration of HSPCs. Treprostinil stimulates the engraftment of human and murine hematopoietic stem cells without impairing their capacity for self-renewal. The investigated dose range corresponds to the dose approved for human use. Hence, these findings may be readily translated into a clinical application.

Pharmacological stimulation of murine and human hematopoetic stem cells

Background Hematopoietic stem cell (HSC) transplantation is a standard procedure in the treatment of hematological disorders and also applicable to support aggressive chemotherapy in cancer. In practice, the clinical outcome is often limited by inefficient bone marrow (BM) engraftment or by low numbers of available stem cells. It would therefore be desirable to enhance engraftment by pharmacological stimulation. HSC require several signals for successful migration into the bone marrow. One of these signals is provided by stimulation of Gas[1]. Pretreatment with prostaglandin (PG) E2 enhances engraftment via activation of Gas-coupled EP2 and EP4 receptors [2]. Treprostinil is a stable analogue of prostacyclin/PGI2, which acts via IP, EP2 and EP4 receptors and is approved for treatment of pulmonary hypertension. Here we tested the hypothesis that treprostinil stimulates stem cell engraftment.

Novel sources for potent and selective GPCR modulators

No abstract is available for this article.

The folding interactome of GPCRs

Background The A2A adenosine receptor is a prototypical G proteincoupled receptor. It is expressed in a wide variety of cells including as different types as neurons, platelets, cells of the immune system and muscle. The A2A receptor has an unusually long C-terminus (of >120 residues), which for the most part is dispensable for coupling to Gs. This C-terminus turned out to be the docking site for other proteins. Using a yeast-2-hybrid screen we have previously identified proteins interacting with the C-terminus including ARNO/cytohesin2, SAP102 and USP4.

Treprostinil stimulates the engraftment of haematopoetic stem cells

Background Successful transplantation of haematopoetic stem cells (HSC) is often limited by low transplantation efficiency. This may be enhanced by pharmacological means. In fact, HSCs require a Gas-transduced signal to re-populate the bone marrow [1]. Pretreatment with prostaglandin E2 (PGE2) enhances engraftment via activation of Gascoupled EP2 and EP4 receptors [2]. Treprostinil is a stable analogue of prostacyclin/PGI2. It predominantly acts via EP2 and EP4 receptors. Treprostinil is approved for treatment of pulmonary hypertension. Here we test the hypothesis that treprostinil may also be useful to promote engraftment of HSC.

The interactome of the A2A adenosine receptor in vitro and in vivo

Background The A2A adenosine receptor is a prototypical G proteincoupled receptor. It is expressed in a wide variety of cells including as different types as neurons, platelets, cells of the immune system and muscle. The cell-specific expression of the A2A adenosine receptor is controlled by at least three different upstream non-coding exons and their corresponding promotors. Compared to other G protein-coupled receptors the A2A receptor holds an unusually long intracellular carboxy terminus, which consists of 122 amino acids. This C-terminus turned out to be the docking site for other proteins. Using a yeast-2hybrid screen we have previously identified proteins interacting with the C-terminus including ARNO/cytohesin-2, SAP102 and USP4.