Technical Reference #112

112.

Visualization Of Dynamic Trafficking Of A Protein Kinase C B Ii/Green Fluorescent Protein Conjugate Reveals Differences In G Protein-Couples Receptor Activation And Desensitization Feng; X.; Zhang; J.; Barak; L.S.; Meyer; T.; Caron; M.G. and Hannun; Y.A, Duke University, The Journal of Biological Chemistry, 273(112), (1998)
Link To Paper

Abstract:
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 PKCII (GFP-PKCII) we demonstrate a novel approach to study the dynamic redistribution of PKC 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- but not Gs- or Gi-coupled receptors. Interestingly although the stimuli were continuously present only one brief peak of PKC membrane translocation was observed consistent with rapid desensitization of the signaling pathway. Moreover when GFP-PKCII was used to examine cross-talk between two Gq-coupled receptors angiotensin II type 1A receptor (AT1AR) and endothelin A receptor (ETAR) activation of ETARs resulted in a subsequent loss of AT1AR responsiveness whereas stimulation of AT1ARs did not cause desensitization of the ETAR signaling. The development of GFP-PKCII has allowed not only the real time visualization of the dynamic PKC 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.

Microscopic Technique
Fluorescence Microscopy, Immunofluorescence

Cell Type(s)
HEK-293