Regulation of membrane raft recruitment of the bradykinin B2 receptor by close association with the ATP/UTP receptor P2Y2

Highlights: • P2Y₂R recruits bradykinin B2R to lipid rafts. • B2R does not affect lipid raft distribution of P2Y₂R. • Signal crosstalk between P2Y₂R and B2R occurs in lipid rafts. • Association of P2Y₂R with both lipid rafts and B2R occurs in the ER. Several G protein-coupled receptors are present in lipid rafts. We have shown that most of the P2Y₂ receptor (P2Y₂R) protein is fractionated into lipid rafts in COS 7 cells. In the same cells, about 25–30% of the bradykinin B2 receptor (B2R) protein is also fractionated into lipid rafts. When both P2Y₂R and B2R are co-expressed, the distribution of P2Y₂R remained unchanged, but more B2R shifted into the raft fraction. This indicates that the interaction between both receptors recruited B2R into the lipid rafts. After 15 min of UTP stimulation, both receptors almost completely disappeared from the cell surface by endocytosis as observed with a confocal fluorescence microscope. Furthermore, with bradykinin stimulation for 15 min, portions of both receptors disappeared from the cell surface and were endocytosed. As we reported previously with both CHO-K1 cells and HEK 293 cells, continuous stimulation of COS7 cells with GT1b and CSC resulted in the disappearance of both P2Y₂R and B2R from the cell membrane surface. Thus, both P2Y₂R and B2R migrate into membrane rafts and are endocytosed in parallel with signal crosstalk, clearly indicating that both closely interact on membrane rafts. The P2Y₂R N-glycosylation deficient mutant does not migrate to the cell surface. It remains predominantly in the endoplasmic reticulum and is fractionated into raft fractions. In the presence of this glycosylation mutant, most of B2R remains in the endoplasmic reticulum, and is fractionated into the raft fraction. These findings demonstrate that in the membrane rafts of the endoplasmic reticulum, both receptors are already closely associated, and B2R shifts into the rafts by affinity with P2Y₂R.