Optical biosensor provides insights for bradykinin B(2) receptor signaling in A431 cells.
The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B(2) receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B(2) specific antagonist HOE140, suggested that the optical signature is a direct result of B(2) receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B(2) receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B(2) signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B(2) receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B(2) signaling. The roles of endocytosis and cytoskeleton modulation in B(2) signaling were also demonstrated.
Fang
Ye
Y
Biochemical Technologies, Science and Technology Division, Corning Incorporated, Sullivan Park, Corning, NY 14831, USA. fangy2@corning.com
Li
Guangshan Gary
GG
Peng
Jinlin
J
D015374:Biosensing Techniques; D001920:Bradykinin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D065094:Bradykinin B2 Receptor Antagonists; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology; D004705:Endocytosis; D066246:ErbB Receptors / Q000502:physiology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology; D006801:Humans; D055095:Optics and Photonics; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000494:pharmacology
D065094:Bradykinin B2 Receptor Antagonists; D043782:Receptor, Bradykinin B2; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C065679:icatibant; D002784:Cholesterol; D066246:ErbB Receptors; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D001920:Bradykinin