Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments.
Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of rhodopsin kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca2+ concentrations. Inhibition of rhodopsin kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca2+ sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca2+ concentration in comparison with the initial rod outer segment membranes (IC50=0.76 microm in DRMs and 1.91 microm in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-beta-cyclodextrin yielded a similar shift of the Ca2+-dependent dose-response curve of rhodopsin kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca2+-dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.
Senin
Ivan I
II
A. N. Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov Moscow State University, 119992 Moscow, Russia.
Höppner-Heitmann
Doris
D
Polkovnikova
Olga O
OO
Churumova
Valeriya A
VA
Tikhomirova
Natalya K
NK
Philippov
Pavel P
PP
Koch
Karl-Wilhelm
KW
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D051606:G-Protein-Coupled Receptor Kinase 1; D020128:Inhibitory Concentration 50; D008055:Lipids / Q000737:chemistry; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051603:Recoverin; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D020349:Surface Plasmon Resonance; D047392:beta-Cyclodextrins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D003902:Detergents; D005136:Eye Proteins; D008055:Lipids; D008074:Lipoproteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D051603:Recoverin; D017830:Octoxynol; D002784:Cholesterol; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D010727:Phosphoric Diester Hydrolases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D006152:Cyclic GMP; D002118:Calcium