Human B1 and B2 bradykinin receptors and their agonists target caveolae-related lipid rafts to different degrees in HEK293 cells.
To address the targeting of G protein-coupled receptors to caveolae-related lipid rafts (CLR), we studied the human B2 (B2R) and B1 (B1R) bradykinin receptor subtypes in HEK293 cells. CLR were enriched on the basis of their unique buoyant density and composition of cholesterol, caveolin-1, and flotillin-1 but not clathrin. CLR contained B2R and B1R as determined by both receptor immunoblotting and the increase in specific activity of receptor agonist binding to cells at both 4 and 37 degrees C when binding was followed by CLR enrichment. B2R was highly enriched in this fraction, whereas B1R was not enriched. Furthermore, acid washing of cells prior to cell disruption minimally affected the CLR-associated B2R agonist binding, whereas it dissociated a major portion of the CLR-associated B1R agonist binding. In addition, when agonist binding at 4 degrees C was followed by an increase in the temperature to 37 degrees C, B2R agonist binding in CLR transiently increased, and this increase was dependent on the C-terminal domain. On the other hand, B1R agonist binding remained unchanged and was independent of the C-terminal domain. Our results show that B2R is constitutively targeted to CLR in HEK293 cells and appears to shuttle the agonist through these domains, whereas B1R may be there by default.
Lamb
Maria E
ME
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Zhang
Chongwu
C
Shea
Thomas
T
Kyle
Donald J
DJ
Leeb-Lundberg
L M Fredrik
LM
D000595:Amino Acid Sequence; D000818:Animals; D021941:Caveolae / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D006801:Humans; D007700:Kinetics; D021962:Membrane Microdomains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists* / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D010446:Peptide Fragments; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin