Carboxypeptidase M and kinin B1 receptors interact to facilitate efficient b1 signaling from B2 agonists.
Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg(9)-bradykinin or des-Arg(10)-kallidin. Carboxypeptidase M (CPM) is a membrane protein potentially well suited for this function. Here we show that CPM expression is required to generate a B1R-dependent increase in [Ca(2+)](i) in cells stimulated with B2R agonists kallidin or bradykinin. CPM and the B1R interact on the cell membrane, as shown by co-immunoprecipitation, cross-linking, and fluorescence resonance energy transfer analysis. CPM and B1R are also co-localized in lipid raft/caveolin-enriched membrane fractions, as determined by gradient centrifugation. Treatment of cells co-expressing CPM and B1R with methyl-beta-cyclodextrin to disrupt lipid rafts reduced the B1R-dependent increase in [Ca(2+)](i) in response to B2R agonists, whereas cholesterol treatment enhanced the response. A monoclonal antibody to the C-terminal beta-sheet domain of CPM reduced the B1R response to B2R agonists without inhibiting CPM. Cells expressing a novel fusion protein containing CPM at the N terminus of the B1R also increased [Ca(2+)](i) when stimulated with B2R agonists, but the response was not reduced by methyl-beta-cyclodextrin or CPM antibody. A B1R- and CPM-dependent calcium signal in response to B2R agonist bradykinin was also found in endothelial cells that express both proteins. Thus, a close relationship of B1Rs and CPM on the membrane is required for efficiently generating B1R signals, which play important roles in inflammation.
Zhang
Xianming
X
Department of Pharmacology, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Tan
Fulong
F
Zhang
Yongkang
Y
Skidgel
Randal A
RA
D001920:Bradykinin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism*; D058851:GPI-Linked Proteins; D015870:Gene Expression; D006801:Humans; D007609:Kallidin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D021962:Membrane Microdomains / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D043783:Receptor, Bradykinin B1 / Q000819:agonists* / Q000378:metabolism; D043782:Receptor, Bradykinin B2 / Q000819:agonists* / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
D022461:Caveolins; D058851:GPI-Linked Proteins; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D014665:Vasodilator Agents; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D007609:Kallidin; D002784:Cholesterol; C058479:carboxypeptidase M; D008666:Metalloendopeptidases; D001920:Bradykinin; D002118:Calcium