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 eryt hrocephala) retinae undergoes light-dependent reversible binding to th e photoreceptor membrane. In order to characterize this arrestin homol og 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 t o heparin-agarose. The molecular mass of purified Arr2, as judged by S DS/PAGE, is in the range 45-49 kDa. The isoelectric point, as judged b y gel isolelectric focussing, is 8.7. Arr2 is specific to the retina, where it is subject to phosphorylation at multiple sites. Binding of p urified 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 arrest in-stimulated phosphate incorporation into rhodopsin results solely fr om the activation of a protein kinase. Phosphorylation experiments wit h highly purified membrane preparations indicate that rhodopsin kinase is tightly associated with the rhabdomeric membrane or the microvilla r cytoskeleton. Rhodopsin kinase is released from the membrane or inac tivated upon treatment with urea. It is concluded that this arrestin i s a regulator protein that controls visual-pigment phosphorylation by affecting the interaction of metarhodopsin and rhodopsin (metarhodopsi n) kinase.