Proximity Labeling Reveals a Multivalent Scaffold that Targets GRK5 to a Signaling Complex with Beta-Adrenergic Receptors

Abstract ID 101448 Poster Board 363 Agonist binding to a G protein-coupled receptor (GPCR) promotes activation of heterotrimeric G-proteins, which mediate downstream signaling and ultimately a physiological response. GPCR kinases (GRKs) play a central role in GPCR desensitization and trafficking by facilitating arrestin recruitment to the GRK-phosphorylated receptor. While numerous GRK complexes with different regulators and substrates like GPCRs have been characterized, no systematic screening of GRK interactions has been conducted. Additionally, it is of interest to identify novel interactors that may regulate GRK localization or facilitate recruitment to the receptor following stimulation. GRK5 is predominantly plasma membrane localized under basal conditions and might be targeted to receptor microdomains via a protein scaffold. Here, we utilized the proximity labeling method BioID with biotin ligase miniTurbo tethered to GRK5 to map the molecular context of GRK5 in unstimulated HEK293 cells. We identified multiple unique high-confidence GRK5 interactors. One of the top hits was the multivalent scaffold protein SAP97 (DLG1) that has been previously shown to associate with signaling proteins such as the b1-adrenergic receptor (b₁AR), phosphodiesterase 4D (PDE4D) and the L-type Ca²⁺ channel (LTCC), thus enabling context-specific signaling. We confirmed binding between GRK5 and SAP97 in HEK cells using co-immunoprecipitation and mapped the C-terminal region of SAP97 as the primary site of GRK5 binding. Chemical cross-linking and surface plasmon resonance analysis revealed a weak interaction between the purified proteins, however, we found that GRK5 could stoichiometrically phosphorylate SAP97. Mapping of GRK5 phosphorylation sites on SAP97 using MS/MS analysis revealed a cluster of phosphorylation sites within an intrinsically disordered region near the PDZ1 domain of SAP97. Since the b₁AR and b₂AR are both substrates for GRK5 and also interact with SAP97 via a C-terminal PDZ binding motif, we next assessed the effects of b₁AR and b₂AR on GRK5-SAP97 binding. Under basal conditions, the b₁AR and b₂AR both had a positive effect on SAP97 binding to GRK5 and this effect was transiently enhanced by agonist stimulation. Taken together, our BioID studies suggest a role for the protein scaffold SAP97 in targeting GRK5 to plasma membrane microdomains before GPCR activation. Since GRK5 and bARs are coupled to SAP97 through distinct sites, SAP97 might act as a bridge between the kinase and the receptor, forming a complex that directs kinase activity towards the receptor or other components of the SAP97 complex. Targeting GRK5 to the SAP97 signaling network can provide an additional layer of regulation to ensure spatiotemporal fidelity in signaling.