An arrestin homolog of blowfly photoreceptors stimulates visual-pigment phosphorylation by activating a membrane-associated protein kinase.
An arrestin homolog (Arr2, 49-kDa protein) of blowfly (Calliphora erythrocephala) retinae undergoes light-dependent reversible binding to the photoreceptor membrane. In order to characterize this arrestin homolog and to study its function in a well-defined experimental system, we developed a purification scheme which used microvillar photoreceptor membranes as an affinity binding matrix. Additional purification steps included ammonium sulfate precipitation, gel filtration and binding to heparin-agarose. The molecular mass of purified Arr2, as judged by SDS/PAGE, is in the range 45-49 kDa. The isoelectric point, as judged by gel isoelectric focussing, is 8.7. Arr2 is specific to the retina, where it is subject to phosphorylation at multiple sites. Binding of purified Arr2 to isolated photoreceptor membranes efficiently activates the light-induced phosphorylation of visual pigment. Since the assay system used is deficient in rhodopsin phosphatase activity, the arrestin-stimulated phosphate incorporation into rhodopsin results solely from the activation of a protein kinase. Phosphorylation experiments with highly purified membrane preparations indicate that rhodopsin kinase is tightly associated with the rhabdomeric membrane or the microvillar cytoskeleton. Rhodopsin kinase is released from the membrane or inactivated upon treatment with urea. It is concluded that this arrestin is a regulator protein that controls visual-pigment phosphorylation by affecting the interaction of metarhodopsin and rhodopsin (metarhodopsin) kinase.
Bentrop
J
J
Institut für Zoologie I, Universität Karlsruhe, Germany.
Plangger
A
A
Paulsen
R
R
D000818:Animals; D019390:Arrestins; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004175:Diptera; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051606:G-Protein-Coupled Receptor Kinase 1; D007525:Isoelectric Focusing; D008027:Light; D008871:Microvilli / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D019390:Arrestins; D005136:Eye Proteins; D010750:Phosphoproteins; D014508:Urea; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1