| The bradykinin (BK) B2 receptor (BKB2R) and angiotensin II (AngII) 1a receptor (AT1aR) are generally opposed in their physiological functions. This study illustrates that global sequence exchanges between these unrelated G-protein coupled receptors (GPCR) is possible. This study also identifies key residues and motifs within the intracellular face (IC) of the BKB2R which are important for signaling and self maintenance.; At first, the serine/threonine cluster within the distal C-terminus of the BKB2R (dCt, 335–366) was examined. Results showed that S 348 promotes receptor internalization, S341 downregulates it, and that S341, S350 and T344 are crucial for signaling. Replacement of the entire dCt with that of AT1aR resulted in normal endocytosis and signaling. Receptor modeling and double mutations indicated that this region interacts with the IC2.; Replacement of the BKB2R IC2 with the AT1aR resulted in a receptor with normal signaling capacity. IC3 exchange abolished signal transduction. However, exchange of IC2 and IC3 simultaneously resulted in a “super-active” receptor, indicating interaction of the IC2 and IC3. Replacement of all three IC domains (IC2/IC3/dCt) resulted in a hybrid which bound BK normally, mediated BK-dependent signaling and receptor internalization. This hybrid upregulated connective tissue growth factor mRNA in response to BK as AT1aR does in response to AngII. This showed that this hybrid acquired at least a part of AT1aR function.; When the entire BKB2R C-terminal tail was replaced with AT1aR, the resulting hybrid, while binding BK, lost its signaling ability. Smaller motif exchanges revealed that the charged amino acids K317, R319 and E320 are crucial for BKB2R signaling.; The role of the IC1 was also investigated. The whole IC1 of BKB2R could only be exchanged with BK B1 receptor (BKB1R), not AT1aR. When replaced by AT1aR IC1, BKB2R ligand binding was compromised. N63 was identified as critical for this loss of binding. E68, located just distal to the IC1, was also identified as important for ligand binding. These results show promise for future successful hybrid formation between unrelated GPCR with the ultimate goal of developing gene therapy modalities for cardiovascular disease. |
