Sun, Y.-E. | bioRxiv Science

bioRxiv2026

Unraveling β2-Adrenergic Receptor Signaling Bias Between Gαs and Gα15 in Shaping Macrophage Function

Myocardial infarction and heart failure are leading global causes of mortality. Chronic {beta}-adrenergic receptor ({beta}AR) activation in cardiomyocytes promotes heart failure via Gs signaling after myocardial infarction, whereas {beta}2AR activation may also provide cardiac protection and repair through alternative pathways. Macrophages play a pivotal role in cardiac repair, and {beta}2AR has been reported to signal via the hematopoietic-specific G15 in these cells. We aimed to characterize signaling bias between Gs and G15 downstream of {beta}2AR and to elucidate their roles in macrophage polarization. Using TRUPATH triple assays, we observed that several {beta}2AR agonists activate G15 with at least an order of magnitude greater potency than Gs in this system. In addition, clinically used {beta}-blockers may exhibit differential inhibition on these two pathways. Macrophages are briefly classified into M1 and M2 polarization according to their activating stimuli and functional properties. Transcriptomic profiling of THP-1-derived macrophage-like cells treated with the {beta}2AR agonist clenbuterol revealed enrichment of M1 transcriptional profile and repair-related hallmarks. Knockdown of Gs showed M1 enrichment, whereas G15 knockdown was associated with negative M2 enrichment as well as M1 enrichment. Loss of either Gs or G15 negatively affected repair-associated hallmarks. In contrast, pharmacological intervention of the Gs-cAMP signaling produced opposing M1/M2 transcriptional responses, while suppressing repair-associated hallmarks. These in vitro findings explore the distinct pharmacological profiles of {beta}2AR ligands toward Gs and G15 and reveal how {beta}2AR agonism may modulate macrophage function through dual-transducer signaling.

cell biology↗

bioRxiv2026

Designing Rigid Protein Fiducials to Visualize GPCR Conformational States

G-protein coupled receptors (GPCRs) mediate precise ligand-specific signaling profiles, yet structural visualization of how ligands alter receptor conformational landscapes in the absence of signaling partners or mimetics has proven incredibly challenging. Here we show that by combining generative protein design with deep-learning based conformational ensemble prediction we can reliably design fiducial markers to facilitate cryogenic electron microscopy (cryoEM) of GPCRs at arbitrary fusion points, enabling the visualization of previously intractable states. We validate the approach with high-throughput determination of inactive state structures of four pharmaceutically relevant GPCRs, allowing for key details of receptor pharmacology to be resolved in each case. We then engineered an extracellular fiducial marker for the prototypical {beta}2-adrenergic receptor that enabled direct structural characterization of the rearrangement of key intracellular motifs in the absence of G-protein. Comparison with recent co-folding models highlights gaps in current methods for predicting ligand-induced GPCR conformational changes. These results present a generalizable framework for accessing traditionally inaccessible structural states of small, dynamic proteins.

biophysics↗