Abstract

The human B₁ bradykinin receptor is an inducible and constitutively active G protein-coupled receptor that is involved in the inflammatory and pain responses to injury. Here, we investigated the role of B₁ receptor homo-oligomerization in cell surface receptor expression. B₁ receptors tagged with either the FLAG or hemagglutinin epitope were monitored immunologically and by radio-ligand binding, biotinylation, and phosphoinositide hydrolysis in human embryonic kidney 293 cells. Selective immunoprecipitation, immunoblotting, and immunoelectron microscopy with epitope-specific antibodies together provided evidence for constitutively formed cell surface receptor homo-oligomers. Truncation of the receptor from the N- and C-terminal ends indicated that the epitope for oligomerization seems to be located between Leu²⁶ on top of transmembrane helix 1 and Val⁷¹ at the bottom of helix 2. A receptor construct terminating at Asp¹³⁴ at the bottom of helix 3, B1stop135, was expressed in the cell. It is interesting that this construct behaved as a dominant-negative mutant by competitively preventing formation of intact B₁ receptor homo-oligomers, and redistributing B₁ receptors from the cell surface to a common intracellular compartment. In contrast, expression of a construct containing the residues downstream of Asp¹³⁴, B1del(2-134), was inactive in this regard. Together, these results are consistent with a mechanism where constitutive B₁ receptor homooligomerization is required for expression of receptors on the cell surface and subsequent constitutive receptor signaling. This may be a novel mechanism by which the cell regulates the presentation of this constitutively highly active receptor at various stages of injury.