PhD Scientific Days 2025

Poster Session III. - C: Molecular Medicine

CXCR-Mediated β-Arrestin Recruitment - Identifying Non-Receptor Interactions

Name of the presenter

Kis Katalin

Institute/workplace of the presenter

Semmelweis Egyetem Élettani Intézet

Authors

Dr. Katalin Kis¹

1: Semmelweis Egyetem Élettani Intézet

Text of the abstract

Introduction
β-arrestin recruitment to G protein-coupled receptors (GPCRs) is governed by specific phosphorylation patterns. High-affinity interactions between AT1R and β-arrestin2 rely on 'stability locks' (Tóth et al., 2018), where β-arrestin2 residues K11 and K12 engage phosphorylated Ser/Thr sites within the receptor's C-terminal domain. We identified a conserved motif—ArreSTick (Tóth et al. 2024.)—that facilitates stable β-arrestin interactions. This motif is also present in numerous non-receptor proteins. We found that HTSF1, a transcription factor linked to HIV replication, binds β-arrestin2 via phosphorylation. Expanding this research, we now investigate HIV-related receptors, CXCR4 and CCR5 which contains the conserved Ser/Thr pattern.
Aims
Our goals were to identify non-receptor β-arrestin partners and determine how CXCR activation influences their interaction. We also aimed to optimize β-arrestin membrane recruitment to reduce background and enable downstream proteomic analysis.
Methods
We applied BRET assays and confocal microscopy to study β-arrestin binding in CXCR4- or CXCR7-expressing cells, with or without CXCL12 stimulation. We assessed the effect of co-expressing various GRKs and a dominant-negative dynamin mutant on phosphorylation and β-arrestin recruitment, comparing unstimulated and stimulated states.
Results
CXCR4 showed constitutive β-arrestin interaction, absent in the K2A β-arrestin mutant, indicating basal phosphorylation. CXCR7 lacked basal activity but recruited β-arrestin upon ligand binding. The strongest recruitment occurred with dynamin co-expression, suggesting increased internalization and signaling potential.
Conclusion
The CXCR7–dynamin system offers low baseline and strong stimulus-dependent β-arrestin recruitment, making it ideal for mass spectrometry and downstream signaling studies.
Funding
This work was supported by the Hungarian National Research, Development, and Innovation Fund (NKFI FK 138862, K 139231, ADVANCED_2024 151284) and by the SE 250+ Excellence PhD Scholarship.

kis.katalin@stud.semmelweis.hu
Semmelweis Egyetem
Dr. Turu Gábor