title: Visualization of dynamic trafficking of a protein kinase C betaII/green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization.
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 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.
authors.affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: X
authors.initials: X
authors.lastname: Feng
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: L S
authors.initials: LS
authors.lastname: Barak
authors.forename: T
authors.initials: T
authors.lastname: Meyer
authors.forename: M G
authors.initials: MG
authors.lastname: Caron
authors.forename: Y A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: 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
chemical_list: 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