title: Phospholipase D from photoreceptor rod outer segments is a downstream effector of RhoA: evidence of a light-dependent mechanism.
abstract: Photoreceptor cells contain rod outer segments (ROS) which are specialized light-sensitive organelles. The biological function of ROS is to generate a photoresponse, which occurs via the classic transducin-mediated pathway. Moreover, ROS undergo light-regulated membrane turnover and protein translocation whose mechanisms have not been fully elucidated to date. Phospholipase D (PLD) is a key enzyme involved in lipid signal transduction and membrane trafficking. We have previously reported that PLD activity is present in purified ROS (Salvador, G.A., Giusto, N.M., 1998. Characterization of phospholipase D activity in bovine photoreceptor membranes. Lipids 33, 853-860). We now demonstrate that ROS PLD activity is enhanced by phosphatidylinositol bisphosphate (PIP2) and cytosolic factors in a GTP dependent-manner. Western blot analysis demonstrates the presence of PLD1 isoform in purified ROS. In ROS obtained from dark-adapted retinas (DROS), PIP2-dependent PLD activity was higher than that observed in ROS obtained from light-adapted retinas (LROS). In addition, experiments carried out in the presence of C3 toxin inhibited PLD activity from DROS whereas pertussis toxin did not affect the enzyme activity. Western blot analysis demonstrates the presence of RhoA, a PLD upstream-regulator. Moreover, RhoA levels were higher in DROS with respect to those in LROS. The present study reports evidence of the involvement of the small G-protein, RhoA, in ROS PLD regulation. Our data strongly suggest that RhoA regulates ROS PLD activity under a light-dependent mechanism.
authors.affiliation: Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur and Consejo Nacional de Investigaciones Científicas y Técnicas, C.C. 857, B8000FWB Bahía Blanca, Argentina.
authors.forename: Gabriela A
authors.initials: GA
authors.lastname: Salvador
authors.forename: Norma M
authors.initials: NM
authors.lastname: Giusto
mesh_terms: D000221:Adaptation, Ocular; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001905:Botulinum Toxins / Q000494:pharmacology; D002417:Cattle; D003470:Culture Media; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D003623:Dark Adaptation / Q000502:physiology; D005136:Eye Proteins / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis; D012374:Rod Cell Outer Segment / Q000187:drug effects / Q000201:enzymology*; D046509:Tissue Culture Techniques; D020742:rhoA GTP-Binding Protein / Q000032:analysis*
chemical_list: D003470:Culture Media; D005136:Eye Proteins; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; C060980:phosphatidylinositol 3,4-diphosphate; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010739:Phospholipase D; C106801:phospholipase D1; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein