Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton.
Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.
Vaziri
C
C
Department of Biochemistry, University of Dundee, United Kingdom.
Downes
C P
CP
D000818:Animals; D002458:Cell Fractionation; D002852:Chromatography, Ion Exchange; D003599:Cytoskeleton / Q000201:enzymology / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008823:Micelles; D008970:Molecular Weight; D011983:Receptors, Purinergic / Q000378:metabolism*; D014422:Turkeys; D010738:Type C Phospholipases / Q000097:blood* / Q000302:isolation & purification
D007295:Inositol Phosphates; D008823:Micelles; D011983:Receptors, Purinergic; D003850:Deoxyribonuclease I; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins