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

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 (PTH₁R), 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 PTH₁R exhibits a near-Brownian diffusion, whereas unbound hormone displays limited mobility and slow lateral diffusion at the cell surface. The formation of the PTH-PTH₁R-βarr complex occurs in three sequential steps: (1) receptor and ligand collisions, (2) phosphoinositide (PIP₃)-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-PTH₁R-βarr complex is formed and unveil the critical role of PIP₃ in regulating GPCR signaling.