Fast-diffusing receptor collisions with slow-diffusing peptide ligand assemble the ternary parathyroid hormone–GPCR–arrestin complex

• Published in: Nature communications 2024-12, Vol.15 (1), p.10499-17
• Main Authors: Pacheco, Jonathan, Peña, Karina A., Savransky, Sofya, Gidon, Alexandre, Hammond, Gerald R. V., Janetzko, John, Vilardaga, Jean-Pierre
• Format: Article
• Language: English
• Subjects: 14/19; 14/34; 14/35; 631/57/2265; 631/80/86/2363; 631/92/609; 9/10; 96; 96/106; 96/109; 96/95; Animals; Arrestin; Assembly; beta-Arrestins - metabolism; Cell Membrane - metabolism; Cell membranes; Cell surface; Clathrin; Clathrin - metabolism; Collisions; Diffusion; Diffusion rate; Fluorescence; G protein-coupled receptors; HEK293 Cells; Humanities and Social Sciences; Humans; Lateral diffusion; Ligands; Membranes; multidisciplinary; Parathyroid hormone; Parathyroid Hormone - metabolism; Peptides; Peptides - chemistry; Peptides - metabolism; Phosphatidylinositol 3,4,5-triphosphate; Phosphatidylinositols - metabolism; Protein Binding; Receptor, Parathyroid Hormone, Type 1 - metabolism; Receptors; Receptors, G-Protein-Coupled - metabolism; Science; Science (multidisciplinary); Signal Transduction; Single Molecule Imaging
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-54772-3

The assembly of a peptide ligand, its receptor, and β-arrestin (βarr) into a ternary complex within the cell membrane is a crucial aspect of G protein-coupled receptor (GPCR) signaling. We explore this assembly by attaching fluorescent moieties to the parathyroid hormone (PTH) type 1 receptor (PTH 1 R), using PTH as a prototypical peptide hormone, along with βarr and clathrin, and recording dual-color single-molecule imaging at the plasma membrane of live cells. Here we show that PTH 1 R exhibits a near-Brownian diffusion, whereas unbound hormone displays limited mobility and slow lateral diffusion at the cell surface. The formation of the PTH–PTH 1 R–βarr complex occurs in three sequential steps: (1) receptor and ligand collisions, (2) phosphoinositide (PIP 3 )-dependent recruitment and conformational change of βarr molecules at the plasma membrane, and (3) collision of most βarr molecules with the ligand-bound receptor within clathrin clusters. Our results elucidate the non-random pathway by which PTH–PTH 1 R–βarr complex is formed and unveil the critical role of PIP 3 in regulating GPCR signaling. The mechanisms underlying the formation of the GPCR ligand-receptor-arrestin complex remain poorly characterized. Here, the authors elucidate some of the molecular events that facilitate the assembly of the ternary complex at the plasma membrane.