title: B1 bradykinin receptor homo-oligomers in receptor cell surface expression and signaling: effects of receptor fragments.
abstract: The human B(1) 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 B(1) receptor homo-oligomerization in cell surface receptor expression. B(1) 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 Leu(26) on top of transmembrane helix 1 and Val(71) at the bottom of helix 2. A receptor construct terminating at Asp(134) 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 B(1) receptor homo-oligomers, and redistributing B(1) receptors from the cell surface to a common intracellular compartment. In contrast, expression of a construct containing the residues downstream of Asp(134), B1del(2-134), was inactive in this regard. Together, these results are consistent with a mechanism where constitutive B(1) 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.
authors.affiliation: Division of Molecular Neurobiology, Wallenberg Neuroscience Center, BMC, A12, Sölvegatan 17, SE-22184 Lund, Sweden.
authors.forename: Dong Soo
authors.initials: DS
authors.lastname: Kang
authors.forename: Caroline
authors.initials: C
authors.lastname: Gustafsson
authors.forename: Matthias
authors.initials: M
authors.lastname: Mörgelin
authors.forename: L M Fredrik
authors.initials: LM
authors.lastname: Leeb-Lundberg
mesh_terms: D001665:Binding Sites; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D006801:Humans; D007930:Leucine; D016253:Microscopy, Immunoelectron; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D043783:Receptor, Bradykinin B1 / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D014633:Valine
chemical_list: D046912:Multiprotein Complexes; D010446:Peptide Fragments; D043783:Receptor, Bradykinin B1; D011994:Recombinant Proteins; D007930:Leucine; D014633:Valine