Visualization of dynamic trafficking of a protein kinase C betaII/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 PKCbetaII (GFP-PKCbetaII), 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 Gqalpha- but not Gsalpha- or Gialpha-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-PKCbetaII was used to examine cross-talk between two Gqalpha-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-PKCbetaII 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.
Feng
X
X
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Zhang
J
J
Barak
L S
LS
Meyer
T
T
Caron
M G
MG
Hannun
Y A
YA
D001692:Biological Transport; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D064546:Protein Kinase C beta; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction
D007527:Isoenzymes; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D019204:GTP-Binding Proteins