Abstract

The inducible kinin B₁ receptor is emerging as an attractive therapeutic target for the treatment of pain and inflammation. Although many studies described its regulation at the transcriptional level, little is known about the maturation of the B₁ receptor. Using two human embryonic kidney (HEK) 293 cell lines stably expressing rabbit B₁ receptors tagged with the yellow fluorescent protein at the C terminus (B₁R-YFP) or the N-terminal myc epitope (myc-B₁R), we showed that receptors are mainly retained in a perinuclear compartment and detectable as low-glycosylated species under control conditions. Interference with the ubiquitin-proteasome pathway function (proteasome inhibitors, coexpression with dominant-negative ubiquitin) blocked B₁ receptor degradation and amplified its intracellular accumulation. A potent nonpeptide antagonist specifically increased the abundance of highly glycosylated B₁R-YFP forms at the cell surface (accessible to chymotrypsin digestion in intact cells); this compound augmented low-glycosylated receptors in brefeldin A-treated cells, supporting the hypothesis that it reaches a newly synthesized receptor in the endoplasmic reticulum. Cell-impermeant peptide or low-affinity nonpeptide B₁ receptor antagonists failed to influence the level of highly glycosylated receptors. Chemical chaperones stabilized all B₁R-YFP species and up-regulated endogenous B₁ receptors expressed at the surface of rabbit smooth muscle cells. Although myc-B₁Rs behaved similarly to B₁R-YFP in most aspects, antibody-based detection assays failed to reveal highly glycosylated species of this construct. Taken together, these results show that B₁ receptors overexpressed in HEK 293 cells are degraded by the proteasome. Furthermore, a pharmacological chaperone highlights the existence of a highly N-glycosylated form of the rabbit kinin B₁ receptor at the cell surface.