Determinants of GPCR-G protein complex assembly and selectivity

G-protein-coupled-receptors (GPCRs), the largest and most diverse group of cell surface receptors in eukaryotes, regulate an incredible range of bodily functions, from sensation to growth to hormone responses. This occurs due to the binding of an external signaling molecule to the GPCR, which undergo conformational changes in order to trigger its specific interaction with nearby G protein, thus activating it. The mechanism governing the assembly of the GPCR-G protein complex and how selective coupling is encoded remains relatively elusive. In this project, which was carried out at the University of Copenhagen in the Rasmussen’s lab, residues believed to be involved in the assembly mechanism were investigated. Specifically, a conserved leucine in the α5 helix of Ga proteins forms a hydrophobic interaction with hydrophobic residues in TM5 and TM6, usually residue positions 5.61 and 6.37, when forming the nucleotide-free GPCR-G protein complex as observed in crystal and cryo-EM structures. Also, the C-terminus of the Ga protein forms an ionic interaction with a positively charged residue in position 6.32 of the GPCR in several but not all solved GPCR-G protein complex structures. Site-directed mutagenesis was performed to generate constructs that prevented these two interactions from forming, and their effects on G protein coupling were subsequently measured by radioligand competition binding assays. The results suggest that these interactions occur early in the coupling process to anchor the G protein in a position allowing other specific interactions to occur between the GPCR and G protein. This hypothesis was investigated in the dopamine D1 and D2, muscarinic acetylcholine M1, µ opioid, α2A and β2 adrenergic receptors.

Determinanti dell'assemblaggio e della selettività del complesso proteico GPCR-G