Biochemical Crosslinking and Liquid Chromatography-Mass Spectrometry Demonstrate a Rhodopsin Dimerization Interface Mediated by Helices 1 and 8

Abstract

Heptahelical G protein-coupled receptors (GPCRs) can exist as monomers and a ternary complex of ligand, GPCR and heterotrimeric G protein is the basic signaling unit. GPCRs also tend to form dimers and higher-order oligomers in membranes, although the functional consequences of these interactions are in most cases unknown. Furthermore, the precise protein-protein interface(s) in receptor dimers and the mechanisms mediating dimerization remain controversial. Two-dimensional and three-dimensional densities obtained from electron microscopy, as well as X-ray data on packing of rhodopsin crystals, suggest that the primary dimer contacts involve transmembrane helix (H) 1 and cytoplasmic H8. Biochemical crosslinking studies with dopamine D2 receptors hint that this interface may be broadly significant. We crosslinked rhodopsin dimers in native rod outer segment disk membranes to demonstrate the proximity of H8 between adjacent receptors. Four homobifunctional cysteine-reactive crosslinkers were used, two with maleimide groups and two with methanethiosulfonate groups. The formation of dimers and oligomers was verified by SDS-PAGE, size-exclusion chromatography, and immunoblot analysis. We used partial proteolysis and high-resolution liquid chromatography-mass spectrometry (LC-MS) to identify the site of a crosslink between Cys316-Cys316. Cys316 is one of two reactive cysteines in rhodopsin and is located in H8. The spacer length of the crosslinkers that formed the intermolecular Cys316-Cys316 crosslink is consistent with the distance predicted in a H1/H8 dimer model. This result corroborates findings from coarse-grained molecular dynamics (CGMD) potential of mean force (PMF) calculations, which show that the H1/H8 orientation is by far the most stable among the possible dimer orientations tested. Together these results strongly suggest the existence of this interface in native membranes. Given the high degree of homology across class A GPCRs, these results may be relevant for other receptors.