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Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay
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Fast-diffusing receptor collisions with slow-diffusing peptide ligand assemble the ternary parathyroid
Jonathan Pacheco1, Karina A Peña1, Sofya Savransky1,2
1Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA.
Nature Communications
|December 3, 2024
Summary
The assembly of G protein-coupled receptor (GPCR) signaling complexes involves sequential steps, including ligand-receptor interactions and β-arrestin recruitment. Phosphoinositide (PIP3) is critical for regulating this process at the cell membrane.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Signaling
Background:
- G protein-coupled receptor (GPCR) signaling is initiated by the assembly of peptide ligands, receptors, and β-arrestin (βarr) into ternary complexes.
- Understanding the dynamics of this assembly at the plasma membrane is crucial for deciphering GPCR signal transduction pathways.
Purpose of the Study:
- To investigate the step-by-step assembly of the parathyroid hormone (PTH) type 1 receptor (PTH1R) with its ligand (PTH) and βarr.
- To elucidate the role of phosphoinositide (PIP3) in regulating this complex formation.
Main Methods:
- Utilized dual-color single-molecule imaging on live cells to observe fluorescently labeled PTH1R, PTH, βarr, and clathrin.
- Analyzed the diffusion patterns of individual molecules and tracked complex formation dynamics at the plasma membrane.
Main Results:
- Demonstrated that PTH1R exhibits near-Brownian diffusion, while unbound PTH has limited mobility.
- Identified a three-step assembly process: ligand-receptor collision, PIP3-dependent βarr recruitment and conformational change, and βarr-receptor complex formation within clathrin clusters.
- Unveiled a critical role for PIP3 in modulating GPCR signaling complex assembly.
Conclusions:
- The formation of the PTH-PTH1R-βarr complex follows a non-random, sequential pathway.
- PIP3 acts as a key regulator, influencing βarr dynamics and subsequent complex assembly, thereby controlling GPCR signaling outcomes.
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