Unveiling the Complexities of GPCR Signaling: Structural and Functional Dynamics of G Protein Receptor Kinase 5 and Neurotensin Recptor1 Interaction

Abstract ID 90578 Poster Board 343 GPCR is the largest family of cell surface receptors and one of the most critical mediators of cell signaling. GPCR kinases (GRKs) are the major regulators of the GPCR signaling pathway by phosphorylating GPCR and thus facilitate the recruitment of arrestin and inhibiting the G proteins binding to the receptor which causes the desensitization of G protein-mediated signaling. Individual GRKs play varying roles in the phosphorylation of a specific GPCR, and each GRK can establish a unique phosphorylation pattern that, in turn, may initiate distinct signaling events. Understanding how GRKs identify and modulate GPCR signaling at the molecular level has been a central area of investigation within GPCR structural biology. However, owing to the substantially diminished binding affinities between GPCRs and GRKs, the attainment of a high-resolution structural depiction of a GPCR-GRK complex poses a formidable technical conundrum. GRK5, as a highly dynamic G-protein receptor kinase, has received comparatively less attention in research compared to other GRKs, despite its significant role in regulating cancer cell cycle progression. GRK5 exhibits considerable instability and dynamism in the context of forming protein-protein complexes. Structural insights into any GRK5 complex with other proteins remain elusive to date. Our proposed structural model aims to offer valuable guidance for future research methodologies focused on understanding GRK5-GPCR interactions. Our study emphasizes the interaction between GRK5 and NTSR1 (a class A GPCR) and its allosteric agonist SBI-553. This compound has been recognized for its ability to selectively reduce addictive behaviors, all while avoiding the undesirable side effects often associated with balanced agonism of NTSR1 induced by neurotensin. Here we show that the GRK5 phosphorating NTSR1 and the phosphorylation can be largely enhanced by SBI-553. Then we propose the structural model of GRK5-NSTR1 with or without SBI-553. Our initial structural data already collected certain classes of GRK5-NTSR1-SBI-553 but not at a high percentage in all images collected. Crosslinking between GRK5 and NTSR1 has been conducted by screening different crosslinkers and a promising cysteine crosslinker BM(PEG)3 has been selected as functional. Further structural study of the crosslinking complex of GRK5-NTST1-SBI-553 and the potential application of NanoBiT technology for increasing the stable complex percentage is being conducted now. Focusing on furnishing structural insights into the GRK5-NTSR1-SBI553 complex will not only innovatively provide a comprehensive model for understanding the intricacies of GPCR-GRK interactions on a broader scale but also potentially pave the way for more promising directions in drug discovery.