Visualization of dynamic trafficking of a protein kinase C βII green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization

Protein kinase C (PKC) links various extracellular signals to intracellular responses and is activated by diverse intracellular factors including diacylglycerol, Ca2+, and arachidonic acid, In this study, using a fully functional green fluorescent protein conjugated PRC beta II (GFP-PKC beta II), we demonstrate a novel approach to study the dynamic redistribution of PRC in live cells in response to G protein-coupled receptor activation. Agonist-induced PKC translocation was rapid, transient, and selectively mediated by the activation of Gq alpha-but not G(s) beta-or G(i) beta-coupled receptors, Interestingly, although the stimuli were continuously present, only one brief peak of PHC membrane translocation was observed, consistent with rapid desensitization of the signaling pathway. Moreover, when GFP-PKC beta II was used to examine cross-talk between two alpha-coupled receptors, angiotensin II type 1A receptor (AT(1A)R) and endothelin A receptor (ETAR), activation of ET(A)Rs resulted in a subsequent loss of AT(1A)R responsiveness, whereas stimulation of AT(1A)Rs did not cause desensitization of the ETAR signaling. The development of GFP-PKC beta II has allowed not only the real time visualization of the dynamic PRC trafficking in live cells in response to physiological stimuli but has also provided a direct and sensitive means in the assessment of activation and desensitization of receptors implicated in the phospholipase C signaling pathway.