Beyond providing novel insight into the physiology of this system, re-annotation of proapelin to the bioactive apelin-55 isoform adds to the molecular toolkit for dissection of apelin-AR interactions and expands the repertoire of therapeutic targets for the apelinergic system.
Apela (also referred to as ELABELA and toddler) is a peptide hormone that activates the apelin receptor (AR or APJ) to regulate cardiovascular system development and function. Here, we report the first biophysical characterization of three apela isoforms, apela-54, -32, and -11, alongside a monomeric C1S-apela-11 mutant, using circular dichroism (CD) spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. The behaviour of apela-54 is consistent with a preprotein containing a hydrophobic, N-terminal signal peptide. The potential for apela-membrane binding, leading to membrane catalyzed interactions with AR, was tested comprehensively for apela-32 and -11 in the presence of membrane-mimetic dodecylphosphocholine (DPC), sodium dodecyl sulfate (SDS), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) micelles. According to pulsed-field gradient diffusion NMR experiments, apela-32 interacts with all three micelles. Chemical shift perturbations indicate widespread interactions along apela, with DPC and LPPG micelles inducing short segments with α-helical character at distinct regions. Consistent with these data, ps-ns dynamics along the peptide backbone appear decreased in the presence of micelles. Apela-11 and C1S-apela-11, alternatively, interact preferentially with SDS and LPPG micelles, promoting β-turn character observable by CD. Distinct differences in membrane-interaction propensity are therefore apparent both as a function of apela isoform and of detergent headgroup. These results imply the potential for cell membrane involvement in apela-AR recognition and binding, with the implication that membrane catalysis has distinct functional and regulatory roles throughout the apelinergic system.
Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor’s active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.
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