Cross-talk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling |
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Authors: | Zhang Xianming Tan Fulong Brovkovych Viktor Zhang Yongkang Skidgel Randal A |
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Affiliation: | Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA. |
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Abstract: | G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys(9)-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase. |
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Keywords: | Carboxypeptidase G Protein-coupled Receptors (GPCR) Peptidases Peptide Hormones Protein/Protein Interactions Bradykinin Receptors |
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