Human ACE and bradykinin B2 receptors form a complex at the plasma membrane.
To investigate how angiotensin I-converting enzyme (ACE) inhibitors enhance the actions of bradykinin (BK) on B2 receptors independent of blocking BK inactivation, we expressed human somatic ACE and B2 receptors in CHO cells. Bradykinin and its ACE-resistant analog were the receptor agonists. B2 fused with green fluorescent protein (GFP) and ACE were coprecipitated with antisera to GFP or ACE shown in Western blots. Immunohistochemistry of fixed cells localized ACE by red color and B2-GFP by green. Yellow on plasma membranes of coexpressing cells also indicated enzyme-receptor complex formation. Using ACE-fused cyan fluorescent protein donor and B2-fused yellow fluorescent protein (YFP) acceptor, we registered fluorescence resonance energy transfer (FRET) by the enhanced fluorescence of donor on acceptor photobleaching, establishing close (within 10 nm) positions of B2 receptors and ACE. Bradykinin stimulation cointernalized ACE and B2 receptors. We expressed ACE fused to N terminus of B2 receptors, anchoring only receptors to plasma membranes. Here, in contrast to cells, where both ACE and B2 receptors are separately anchored, ACE inhibitors neither enhance activation of chimeric B2 nor resensitize desensitized B2 receptors. Heterodimer formation between ACE and B2 receptors can be a mechanism for ACE inhibitors to augment kinin activity at cellular level.
Chen
Zhenlong
Z
Department of Pharmacology, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Deddish
Peter A
PA
Minshall
Richard D
RD
Becker
Robert P
RP
Erdös
Ervin G
EG
Tan
Fulong
F
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism*; D043782:Receptor, Bradykinin B2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D008164:Luminescent Proteins; D043782:Receptor, Bradykinin B2; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C488034:ACE protein, human; D007703:Peptidyl-Dipeptidase A