The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

Abstract

Previously, Lefkowitz and coworkers (8) cloned and sequenced cDNAs for the first member of this proposed receptor kinase family, the f3-adrenergic receptor kinase (j3ARK) (8). This kinase phosphorylates and regulates the function of 8-adrenergic (and possibly other G-proteincoupled) receptors by catalyzing phosphorylation of the receptor on a serine/threonine-rich cluster found at its carboxyl terminus (9). Functionally, a very analogous enzyme is rhodopsin kinase (RK). Discovered almost two decades ago (4-6), this enzyme is known to phosphorylate rhodopsin, thereby initiating its deactivation. The biochemical and functional properties of RK are such as to suggest that it might be closely related to DARK (10). Accordingly we set out to clone cDNAs for RK so as to elucidate its primary structure and thereby clarify structural, evolutionary, and functional relationships with PARK, as well as to understand the nature and diversity of the proposed family of receptor kinase molecules.tt MATERIALS AND METHODSProtein Purification and Peptide Sequencing. RK was purified from bovine retina as described (11). The enzyme was extracted from photobleached rod outer segment membranes with 60 mM KC1. The kinase was further purified with a DEAE-cellulose step, and pooled activity was then applied to a hydroxyapatite column. RK was eluted in a buffer of 20 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane, 1 mM Mg(OAc)2, and 95 mM KC1. Fractions (1 ml) were concentrated to 300 A.l under reduced vacuum (Speed-Vac, Savant) in individual Eppendorf tubes. To each of the 10 tubes was added 700 p.l of CNBr in formic acid (99%) to give a final CNBr concentration of 10 mM; the digestion was allowed to proceed for 24 hr in the dark at room temperature. Each sample was dried under reduced pressure and rehydrated with 500 ,ul of 90%o water/10%o acetonitrile/0.1% trifluoroacetic acid. The 10 samples were individually applied to an Aquapore C4 reverse-phase column (Applied Biosystems; 2.1 x 30 mm) at a flow rate of 200 p.l/min; the column was washed with 1.5 ml of 0.1% trifluoroacetic acid in water between each sample application. Once the last sample was loaded, a gradient of 0-70% acetonitrile was developed. Fractions were collected at 1-min intervals. A control digest, consisting of 1 ml of buffer handled exactly as the kinasecontaining samples and subjected to CNBr treatment, was chromatographed under identical conditions. Peaks unique to the RK-containing digest were submitted for gas-phase peptide sequencing (R. Randall, Howard Hughes Medical Institute Biopolymer Lab, Durham, NC).cDNA Library Screening and Polymerase Chain Reaction (PCR). Screening of a bovine Okayama-Berg cDNA library (12) with the peptide-derived oligodeoxynucleotide 5'-Abbreviations: RK, rhodopsin kinase; fARK, fi-adrenergic receptor kinase; RACE, rapid amplification of cDNA ends.

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

“…synaptic membranes), their effective concentrations in these compartments could be even higher (74,75,78,97). The behavior of membrane-tethered GRK2 mutants is reminiscent of strict dependence on receptor activation of visual GRK1-and GRK7-dependent phosphorylation of rhodopsin and cone opsins, even though both visual GRKs are constitutively membrane-associated via C-terminal prenylation (50,80). Although GRK1 was shown to phosphorylate many molecules of inactive rhodopsin upon activation of each rhodopsin molecule by light (19,20), this phenomenon could be explained within the framework of the general model that GRK1 activation requires its physical interaction with an active GPCR (8).…”

Interaction between the Conserved Region in the C-terminal Domain of GRK2 and Rhodopsin Is Necessary for GRK2 to Catalyze Receptor Phosphorylation

“…The C-terminal domain of GRKs is involved in membrane translocation of the enzymes (1). Much attention so far has been paid to the distinctive structure in the C-terminal domains of respective GRKs such as the CAAX motif in GRK1 that directs farnesylation (30,31), ␤␥ binding regions in GRK2 and GRK3 (5)(6)(7)(8), and the palmitoylation sites in GRK4 (32) and GRK6 (33). However, little heed was given to the region within the C-terminal domain that is rather conserved among the six GRK members (filled with slanted line in Fig.…”

A Novel Rhodopsin Kinase in Octopus Photoreceptor Possesses a Pleckstrin Homology Domain and Is Activated by G Protein βγ-Subunits

“…Polycations activate RK (16), but not ␤ARK (17). The sequence similarity between the two kinases is not high, and the differences are most evident in the C-terminal regions (7). RK lacks ϳ120 C-terminal residues present in the corresponding region of ␤ARK.…”

“…Both of these kinases have been purified to homogeneity, and their specificities and activities have been examined in reconstituted systems (5,6). In addition to RK (7) and two kinds of ␤ARK (8,9), at least three other members of the GRK family (GRK4 -6) have been cloned in mammals (10 -12), and several related genes have also been cloned from other organisms such as Drosophila (13) and Caenorhabditis elegans (14).…”