PTH–GPCR–arrestin complexes assembly in selective cell membrane spots

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Abstract ID 81135 Poster Board 570 This study addresses a fundamental question in G protein-coupled receptor (GPCR) signaling by determining how a peptide ligand, its receptor, and b-arrestin (barr) assemble in a ternary cell membrane complex. Here, fluorescent moieties were attached to the parathormone (PTH) receptor (PTH₁R) as a prototypical peptide GPCR, PTH, barr and clathrin. By recording dual-color single-molecule imaging at the plasma membrane of live cells, we found that PTH₁R exhibits a near-Brownian diffusion, whereas unbound hormone shows a limited mobilty at the cell surface with minimal lateral diffusion. The PTH–PTH₁R–barr complex assembles through three sequential steps: 1) receptor and ligand collisions in pre-existing clathrin clusters, 2) phosphoinositide (PIP₃) dependent recruitment and conformational change of barr molecules in clathrin clusters, and 3) barr collision with the ligand-bound receptor confined in clathrin clusters. These results provide insight into the cell surface pathway by which a GPCR–barr complex is assembled and unveil a novel role of PIP₃ in regulating GPCR signaling. Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) and the National Institute of Dental and Craniofacial Research (NIDCR) of the US National Institutes of Health (NIH) under grant Award Numbers R01-DK116780, R01-DK122259, and R21-DE032478.