Flores-Espinoza, E. | Search bioRxiv

bioRxiv2026

A conserved hydrophobic interaction governs GPCR-transducer association

A central feature of G protein-coupled receptor (GPCR) desensitization is the direct competition between heterotrimeric G proteins and {beta}-arrestins ({beta}arrs) for an overlapping binding site within the intracellular receptor cavity. Although numerous high-resolution structures of GPCR-transducer complexes exist, the exact nature of this shared site and the molecular basis of transducer competition remain unclear. To investigate this, we employed an interdisciplinary approach integrating systemic mutational mapping, bioinformatics, and structural analysis across multiple classes of GPCRs and their transducers and regulators. We identified two highly conserved leucine residues within both the {beta}arr finger loop and the G C-terminal -helix, which engage a hydrophobic patch on GPCRs formed by TM3, TM5, and TM6 in a nearly identical manner, thereby stabilizing the complexes. Notably, the GPCR kinase N-terminal -helix also contains hydrophobic residues that associate with this same receptor patch and are vital for the GPCR-GRK engagement. Our findings reveal a conserved hydrophobic interface that mediates direct competition among GPCR transducers and regulators suggesting a universal mechanism that governs receptor access and desensitization.

biochemistry↗

bioRxiv2025

The Lysosome Surface is an Unappreciated Hub for Vasopressin V2 Receptor Signaling

G protein-coupled receptors (GPCRs) have traditionally been understood to signal through heterotrimeric G proteins exclusively from the cell surface followed by {beta}-arrestin ({beta}arr)-mediated desensitization and receptor internalization into endosomes. However, this view has evolved significantly with growing evidence showing that some GPCRs continue to signal from endosomes after their internalization as well as from other intracellular organelles. The vasopressin V2 receptor (V2R) exemplifies this paradigm shift as it promotes robust endosomal G protein before being sorted to lysosomes for degradation. Intriguingly, recent observations suggest that the lysosomal surface itself holds a substantial pool of heterotrimeric G proteins, raising the possibility that GPCRs such as the V2R may stimulate signaling from this subcellular region. To investigate this, we here employed a NanoBiT bystander approach to track intracellular V2R trafficking and transducer activation in real-time. Our results show that activated V2R is trafficked relatively fast to lysosomes where it retains the ability to couple to both G proteins and {beta}arrs. Applying nanobody/intrabody biosensors, we further demonstrated that the V2R activates endogenous G proteins and {beta}arrs at the lysosomal surface and that inhibition of V2R translocation to endolysosomal compartments blunts its ability to stimulate G protein signaling. Together, these findings suggest that the lysosomal surface serves as an unappreciated hub for signaling by some GPCRs before they eventually are engulfed into the lysosomal lumen for degradation. One-sentence summaryUpon activation, the vasopressin V2 receptor is internalized and sorted to the lysosomal membrane, where it activates G proteins and {beta}-arrestins.

cell biology↗