Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites.

The adenosine A G protein-coupled receptor (AAR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the AAR orthosteric site is located within the receptor's transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein-coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of AAR-ECL2 to explore the role of this domain on AAR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5'-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in AAR orthosteric ligand binding and receptor activation.

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working relationships. The mutual reliance these health care providers demonstrated led to a great patient outcome a long way from a tertiary health care facility. The feasibility of replicating this model of health care in other sparsely resourced populations in Victoria is currently being examined.http://dx.

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Heart failure remains a leading cause of morbidity and mortality worldwide. Current treatment for patients with heart failure include drugs targeting G protein‐coupled receptors such as β‐adrenoceptor antagonists (β‐blockers) and angiotensin II type 1 receptor antagonists (or angiotensin II receptor blockers). However, many patients progress to advanced heart failure with persistent symptoms, despite treatment with available therapeutics that have been shown to reduce mortality and mortality. GPCR targets currently being explored for the development of novel heart failure therapeutics include adenosine receptor, formyl peptide receptor, relaxin/insulin‐like family peptide receptor, vasopressin receptor, endothelin receptor and the glucagon‐like peptide 1 receptor. Many GPCR drug candidates are limited by insufficient efficacy and/or dose‐limiting unwanted effects. Understanding the current challenges hindering successful clinical translation and the potential to overcome existing limitations will facilitate the future development of novel heart failure therapeutics.

Background: There has been convincing evidence that cardiac α 1A -adrenoceptors (α 1A -ARs) increase in expression and have an enhanced compensatory role during advanced heart failure (O'Connell et al, 2014). Mechanistic target of rapamycin (mTOR) signalling is involved in many critical cellular processes, and implicated in early development, normal function and stress responses of cardiomyocytes. However, it remains unclear whether mTOR is involved in the signalling mechanisms underlying α 1A -AR-mediated cardioprotective effects. This study aims to elucidate the ability of α 1A -ARs to enhance cardiomyocyte viability via mTOR signalling. Methods: Neonatal rat ventricular myocytes (NRVMs) were incubated under normoxic or hypoxic (1% O 2 ) conditions.Whole-cell radioligand binding assay was performed using [ 3 H]-prazosin. Cell viability was measured by propidium iodide staining and caspase 3/7 assay. Glucose uptake was measured using [ 3 H]-2-deoxy-glucose. Activation of cardioprotective signalling proteins were examined using AlphaScreen SureFire and In-Cell Western assays.Results: Stimulation of α 1A -AR with A61603 increased Ca 2+ mobilisation and glucose uptake in NRVMs, but failed to phosphorylate Akt which is a key protein for insulin-mediated glucose uptake. A61603 (100 nM) significantly decreased the percentage of apoptotic cells under hypoxic conditions (Control 22.0 ± 2.0%, A61603 11.9 ± 1.0%). Hypoxia significantly increased the abundance of α 1 -AR protein (Bmax normoxic 62.3 ± 3.2 fmol/mg, hypoxic 102.5 ± 14.6 fmol/mg protein). The general mTOR complex 1/2 inhibitor KU-0063794 (1 µM) significantly inhibited α 1A -ARmediated glucose uptake by 69% and cell survival by 69% in hypoxic NRVMs, whereas the mTOR complex 1 inhibitor rapamycin (10 nM) had no effect, suggesting that mTOR complex 2 has a key role. Conclusion: These findings indicate that α 1A -ARs are proportionally increased in hypoxia, mediate protective signalling in hypoxic NRVMs, increase Akt-independent glucose uptake and promote cell survival via mTORC2.