Scarpelli Pereira, P. H. | Search bioRxiv

bioRxiv2026

GRK-dependent ACKR3 endocytosis and chemokine scavenging is independent of receptor phosphorylation and beta-arrestin

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting {beta}-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and {beta}-arrestin interaction. However, the respective roles of {beta}-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)-based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with {beta}-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in {beta}-arrestin-deficient ({Delta}{beta}arr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. {beta}-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired {beta}-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in {Delta}GRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G{beta}{gamma} was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of {beta}-arrestins but requires GRKs. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

cell biology↗

bioRxiv2026

Unconventional Interplay Between GPCRs and RTKs Signaling Pathways Through SH2 Domain-Containing Proteins

Crosstalk across two major receptor families involved in signal transduction, namely receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), have been observed at different levels of their signaling cascades. Using newly developed enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors that monitor the recruitment of SH2 domains to activated RTKs, we assessed the ability of GPCRs to modulate cellular localization of SH2 domains. Receptor-mediated activation of either Gq/11 or G12/13 but not Gs or Gi/o (e.g., thromboxane A2 receptor, TP, and type-2 protease activated receptor, PAR2) resulted in the plasma membrane (PM) dissociation of SH2 domains derived from RTKs effectors such as GRB2, STAT5 and PLC{gamma}1. The role of Gq/11, G12/13, Rho and downstream kinases in the subcellular SH2 domain redistribution was further confirmed using both pharmacological and genetic approaches. BRET imaging and spectrometric analyses showed that the dissociation of SH2 domains from the PM was accompanied by their accumulation in the nucleus and a reduction in RTK signaling activity, as determined using a STAT5 transcriptional assay. The effect of Gq/11 and G12/13 activation on STAT5 transcriptional activity was observed both in engineered systems and in HeLa cells endogenously expressing all the components of the regulatory mechanism. The Gq/11 / G12/13-mediated redistribution of SH2 domain-containing proteins represents an undescribed mechanism through which GPCRs regulate RTKs activity. Significance StatementThis study reveals a novel crosstalk mechanism between G protein coupled receptors and receptor tyrosine kinases showing that Gq/11 and G12/13 activation triggers Rho-dependent translocation of SH2-containing effector proteins, such as GRB2, PLC{gamma}1 and STAT5. This process causes compartmentalization inside the nucleus and thus reduces their availability at the plasma membrane, leading to attenuated RTK responses.

cell biology↗