Determining the Requirements for Gαi:β‐arrestin Complex Formation at G Protein‐Coupled Receptors

G protein‐coupled receptors (GPCRs) comprise the largest class of transmembrane receptors and are targeted by nearly a third of prescription drugs. Canonical GPCR signaling involves pathways mediated by G proteins and β‐arrestins (βarrs). While these have historically been thought of as separate, our lab has demonstrated that the inhibitory G protein Gα subunit (Gαi) and βarr form complexes following receptor activation. We have also shown that Gαi:βarr complex formation occurs even if the receptor signals through other classes of G proteins or lacks G protein activity altogether, as in the case of endogenously βarr‐biased Atypical Chemokine Receptor 3 (ACKR3). To expand on this work, we used a split nano‐luciferase assay to examine Gαi:βarr complex formation at βarr‐biased mutants of two ‘balanced’ receptors, which exhibit both G protein and βarr signaling— the type 1 Angiotensin II Receptor (AT1R) and D2 dopamine Receptor (D2R). Consistent with our results with ACKR3, we found that mutation of these other receptors to abrogate G protein‐mediated signaling did not eliminate Gαi:βarr association. Observing this at multiple additional receptors further supported the idea that G protein activity is not required for Gαi:βarr complex formation. To add to our characterization of the determinants of Gαi:βarr association, we then sought to explore how different mechanisms of βarr activation may contribute to complex formation at ACKR3. To accomplish this, we utilized previously described polar core, proximal finger loop, and lipid mutant βarr constructs. The polar core and proximal finger loop mutants achieve constitutive activation by different mechanisms, while the lipid mutant’s ability to be recruited to and bind at the plasma membrane is deficient; these result in decreased Gαi:βarr association at Vasopressin Receptor 2 (V2R). We compared these mutants’ ability to associate with Gαi at ACKR3. Our constitutively active βarr constructs showed differential abilities to form Gαi:βarr complexes compared to wild‐type βarr, suggesting that certain methods of βarr activation may be more relevant in Gαi:βarr complex formation than others. Additional insights into the determinants of Gαi:βarr association in the context of receptor, G protein, and βarr activity will enhance our understanding of the Gαi:βarr complex and its importance in GPCR signaling, potentially informing future development of therapeutic agents targeting GPCRs.

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