Translocation of phosducin in living neuroblastoma x glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein.
Activation of G protein-coupled receptors triggers translocation of certain proteins from cytoplasm to cell membrane located targets. One of these cytosolic proteins is phosducin (Phd) which has been described to compete with G protein-coupled receptor kinases for Gbetagamma dimers attached to the cell membrane, thereby attenuating desensitization of activated receptors. These features of protein redistribution prompted us to examine whether stimulation of membrane associated E-prostaglandin receptors coupled to Gs causes Phd to migrate towards the plasma membrane. We made use of enhanced green fluorescence protein (EGFP), a reporter protein, to follow redistribution of Phd both by means of confocal microscopy and biochemical techniques in living neuronal NG 108-15 hybrid cells challenged with prostaglandin E1 (PGE1). The cells were transiently transfected to express Phd fused to the C-terminus of EGFP, or to express EGFP only. Overexpression of the proteins is implied by FACS analysis as well as by western blot technique, and the functional integrity of EGFP-tagged Phd was confirmed by its ability to elevate cAMP accumulation. Time-lapse imaging of single living cells by means of confocal microscopy revealed that exposure to prostaglandin causes EGFP/Phd, which is evenly spread throughout the cell, to relocate towards the membrane within few minutes. Fluorescence associated with the cell nucleus displayed little rearrangement. The principle finding that prostaglandin triggers translocation of Phd from cytosol to the cell periphery was verified with membranes prepared from EGFP/Phd expressing cells. We found maximal concentrations of membrane associated fluorescent material 5 to 7 min upon prostaglandin exposure. The present study reports for living NG 108-15 hybrid cells that PGE1 stimulation causes cytosolic Phd to translocate towards the membrane, where it is believed to bind to G protein subunits such as Gbetagamma and Galphas.
Schulz
R
R
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Königinstr. 16, D-80539 München, Germany. schulz@pharmtox.vetmed.uni-muenchen.de
Schulz
K
K
Wehmeyer
A
A
Murphy
J
J
D000527:Alprostadil / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003116:Color; D000242:Cyclic AMP / Q000378:metabolism; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D020555:GTP-Binding Protein Regulators; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D005910:Glioma; D049452:Green Fluorescent Proteins; D006822:Hybrid Cells / Q000737:chemistry / Q000201:enzymology*; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D009447:Neuroblastoma; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000493:pharmacokinetics; D014162:Transfection
D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C065631:phosducin; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D000527:Alprostadil