Functional and structural insights into interactions between β-Arrestin 1 and Gαs or Gαi1

G proteins and arrestins are key transducers for G protein-coupled receptor (GPCR) signaling, mediating distinct downstream pathways. Recent evidence suggests that G proteins and β-arrestins (βarrs) can directly or functionally interact. However, the molecular details and functional consequences of Gα–βarr interactions remain poorly understood. Here, we quantify the binding affinities between βarr1 and Gαs or Gαi1 in various activation states using microscale thermophoresis (MST). βarr1 in the active conformational ensemble state favors binding, whereas Gα activation status is less determinant. Hydrogen/deuterium exchange mass spectrometry reveals distinct conformational changes between Gαs versus Gαi1 upon βarr1 binding, suggesting differential binding mechanism between Gαs–βarr1 and Gαi1–βarr1 complexes. Both the Ras-like domain and the α-helical domain of Gα contribute to complex formation. Functionally, a BODIPY-FL–GTPγS assay shows that βarr1 does not alter GDP/GTP turnover of Gαs or Gαi1, whereas β-strand XX (βXX) release assays demonstrate that Gαs enhances βarr1 C-tail release. Together, these results propose molecular mechanism of the interaction and asymmetric functional coupling within Gα–βarr complexes and uncover a previously underappreciated layer of GPCR signal transduction. G proteins and arrestins, regarded as two transducers in GPCR signaling, have been suggested to interact directly or coordinate functionally. Here, authors provide evidence for direct, conformation-specific interactions between these two effectors, accompanied by domain-level mapping of contact sites and functional consequences.