A Novel Rhodopsin Kinase in Octopus Photoreceptor Possesses a Pleckstrin Homology Domain and Is Activated by G Protein βγ-Subunits*
- From the Department of Life Science, Himeji Institute of Technology, Harima Science Garden City, Akoh-gun, Hyogo 678-1279, Japan
Abstract
G protein-coupled receptor kinases (GRKs) play an important role in stimulus-dependent receptor phosphorylation and desensitization of the receptors. Mammalian rhodopsin kinase (RK) and β-adrenergic receptor kinase (βARK) are the most studied members among known GRKs. In this work, we purified RK from octopus photoreceptors for the first time from invertebrate tissues. The molecular mass of the purified enzyme was 80 kDa as estimated by SDS-polyacrylamide gel electrophoresis, and this was 17 kDa larger than that of the vertebrate enzymes. Unlike vertebrate RK, octopus RK (ORK) was directly activated by βγ-subunits of a photoreceptor G protein. We examined the effects of various known activators and inhibitors of GRKs on the activity of the purified ORK and found that their effects were different from those on either bovine RK or βARK. To analyze the primary structure of the enzyme, we cloned the cDNA encoding ORK from an octopus retinal cDNA library. The deduced amino acid sequence of the cDNA was highly homologous to βARK over the entire molecule, including a pleckstrin homology domain located in the C-terminal region, and homology to RK was significantly lower. Furthermore, Western blot analysis of various octopus tissues with an antibody against the purified ORK showed that ORK is expressed solely in the retina, which confirmed the identity of the enzyme as rhodopsin kinase. Thus, ORK appears to represent a unique subgroup in the GRK family, which is distinguished from vertebrate RK.
Footnotes
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↵* This work was supported in part by Research Grants 0740819 and 08257219 (to M. T.) and 9780611 (to S. K.) from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ, GenBank™/EBI Data Bank with accession number(s)AB009875.
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↵‡ To whom correspondence should be addressed. Tel.: 81-7915-8-0196; Fax: 81-7915-8-0197; E-mail: mtsuda{at}sci.himeji-tech.ac.jp.
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↵1 The abbreviations used are: GRKs, G protein-coupled receptor kinases; RK, rhodopsin kinase; βARK, β-adrenergic receptor kinase; ORK, octopus rhodopsin kinase; PH, pleckstrin homology; GTPγS, guanosine 5′-(3-O-thio)triphosphate; APMSF, 4-(amidinophenyl)methanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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↵2 S. Kikkawa, unpublished data.
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- Received October 6, 1997.
- Revision received January 6, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
