The pleckstrin homology domain of human beta I sigma II spectrin is targeted to the plasma membrane in vivo.
We have examined the in vivo targeting potential of the Pleckstrin Homology (PH) domain from human beta I sigma II spectrin using a novel Aequoria victoria green fluorescent protein (GFP) fusion vector constructed from a human codon optimized cDNA. This vector efficiently expresses both GFP and the GFP spectrin fusion protein in COS7 and other cell lines. GFP expressed alone shows only diffuse cytoplasmic staining which is not associated with the plasma membrane. In contrast the GFP-beta I sigma II spectrin PH domain fusion protein localizes under the plasma membrane of transfected COS7 cells in vivo. Fixation of cells transfected with GFP alone in -20 degrees C methanol results in the removal of all specific fluorescence. In contrast cells transfected with the GFP-beta I sigma II spectrin construct and fixed -20 degrees C methanol continue to show strong membrane fluorescence, consistent with a role for the spectrin PH domain in membrane localization in vivo.
Wang
D S
DS
Department of Neuroscience, University of Florida College of Medicine, Gainesville 32610, USA.
Miller
R
R
Shaw
R
R
Shaw
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G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000235:genetics* / Q000378:metabolism
D001798:Blood Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D013049:Spectrin; D049452:Green Fluorescent Proteins