Binding affinities and activation of Asp712Ala and Cys100Ser mutated kinin B1 receptor forms suggest a bimodal scheme for the molecule of bound-DABK

AbstractMutant forms of kinin B1 receptor (B1R) and analogs of the full agonist des-Arg9-bradykinin (DABK) were investigated aiming to verify the importance of selected receptor residues and of each agonist-peptide residue in the specific binding and activation. Linked by a specific disulfide bond (Cys100–Cys650), the N-terminal (Nt) and the EC3 loop C-terminal (Ct) segments of angiotensin II (AngII) receptor 1 (AT1R) have been identified to form an extracellular site for binding the agonist Nt segment (Asp1 and Arg2 residues). Asp712 residue at the receptor EC3 loop binds the peptide Arg2 residue. By homology, a similar site might be considered for DABK binding to B1R since this receptor contains the same structural elements for composing the site in AT1R, namely the disulfide bond and the EC3 loop Asp712 residue. DABK, Alan-DABK analogs (n=Ala1-, Ala2-, Ala3-, Ala4-, Ala5-, Ala6-, Ala7-, Ala8-DABK), and other analogs were selected to binding wild-type, Asp712Ala and Cys100Ser mutated B1R receptors. The results obtained suggested that the same bimodal scheme adopted for AngII-AT1R system may be applied to DABK binding to B1R. The most crucial similarity in the two cases is that the Nt segments of peptides equally bind to the homologous Asp712 residue of both AT1R and B1R extracellular sites. Confirming this preliminary supposition, mutation of residues located at the B1R extracellular site as EC3 loop Asp712 and Cys100 caused the same modifications in biological assays observed in AT1R submitted to homologous mutations, such as significant weakening of agonist binding and reduction of post-receptor-activation processes. These findings provided enough support for defining a site that determines the specific binding of DABK to B1R receptors

Evidence that kinin B(2) receptor expression is upregulated by endothelial overexpression of B(1) receptors

Bradykinin (BK) and des-Arg(9)-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B(2) (B(2)R) and B(1) (B(1)R) receptors, respectively. It was already shown that the deletion of kinin B(1)R or of B(2)R induces upregulation of the remaining receptor subtype [10,12,16,28,36]. However studies on overexpression of B(1)R or B(2)R in transgenic animals have supported the importance of the overexpressed receptor but the expression of the another receptor subtype has not been determined [17,19,33]. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B(1)R (TGR(Tie(2)B(1))) exclusively in the endothelium. In another study, mice overexpressing B(1)R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B(2)R was investigated in the thoracic aorta isolated from TGR(Tie(2)B(1)) rats overexpressing the B(1)R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B(2)R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie(2)B(1)) rats could be due to the enhanced expression of B(2)R associated with the overexpression of the B(1)R

Angiotensin II binding to angiotensin I-converting enzyme triggers calcium signaling

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system