Qiuyan Chen (Purdue University )| Manolo Plasencia (University of Michigan )| Zhuang Li (Purdue University )| Somnath Mukherjee (University of Chicago )| Dhableswar Patra (Purdue University )| Chun-Liang Chen (Purdue University )| Thomas Klose (Purdue University )| Xin-Qiu Yao (Georgia State University )| Anthony Kossiakoff (University of Chicago )| Leifu Chang (Purdue University )| Philip Andrews (University of Michigan )| John Tesmer (Purdue University )
G protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate GPCRs in their activated states, priming them for arrestin binding and internalization. How GRKs recognize these receptors and how receptors turn on the kinase activity of GRKs is debated. Here we trapped a light activated rhodopsin (Rho*) in complex with rhodopsin kinase (GRK1) using a novel crosslinker and determined a 4.0 Å cryo-EM structure of this complex with the aid of a GRK1-binding Fab. GRK1-bound Rho* adopts unique active conformations, distinct from arrestin-1-bound or transducin-bound Rho*. GRK1 interacts with Rho* entirely via its N-terminal helix and kinase small lobe, and the kinase domain overall assumes a closed, active conformation. The regulator of G protein signaling homology domain is detached from the kinase domain and is not visible in this complex. The most prominent interaction is the docking of the GRK1 N-terminus into the cytoplasmic cleft that forms upon activation of rhodopsin. Crosslinking and functional studies validate GRK specific interactions in the structure and reveal dynamics consistent with the ability of GRK1 to phosphorylate multiple sites in the C-terminus of Rho*. We also identify residues required for the activation of GRK1 by acidic phospholipids that would reside adjacent to the visible receptor–GRK interface. Our data provides not only a model for how GRKs engage and are synergistically activated by receptors and anionic phospholipids, but also a potential mechanism for how arrestin biased siganling could occur in some GPCRs.