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. 2002:343:578-600.
doi: 10.1016/s0076-6879(02)43159-x.

Rhodopsin and its kinase

Izabela Sokal  1 , Alexander Pulvermüller, Janina Buczyłko, Klaus-Peter Hofmann, Krzysztof Palczewski
Affiliations

Rhodopsin and its kinase

Izabela Sokal et al. Methods Enzymol. 2002.
No abstract available

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Figures

F<sc>ig</sc>. 1
Fig. 1
(A) Inactivation of the signaling state of Rho (Rho* or R*) by phosphorylation and binding of Arr. R represents the dark state, inactive form of Rho, Gt, transducin; and p44 is a short splice version of Arr. (B) Phylogenetic tree of GRKs. The tree was built with a bootstrap analysis of neighbor-joining distance using PAUPSearch in GCG (University of Wisconsin-Genetics Computer Group). The accession numbers are GRK1, Q15835; GRK2, X61157; GRK3, P35626; GRK4, P32298; GRK5, P34947; GRK6, P43250; ground squirrel GRK7, AF063016.
F<sc>ig.</sc> 2
Fig. 2
Assay RK activity. Time (A) and dose (B) dependence of RK activity. RK was partially purified from SF9 cells.
F<sc>ig.</sc> 3
Fig. 3
Molecular rules of opsin activation: phosphorylation of opsin in the presence of all-trans-retinal analogs of different lengths and their derivatives with blocked aldehyde groups. (A) Phosphorylation of opsin is carried out in the presence of all-trans-retinal, all-trans-C17 aldehyde, all-trans-C15 aldehyde, trans-C12 aldehyde, or their oximes generated with NH2OH, NH2OCH3, or NH2OCH2CH3 (8 M excess over 30 μM opsin). The solid horizontal line represents opsin activity. (B) Activity expressed as percentage of maximum phosphorylation for each of the aldehydes (marked as a continuous line). The phosphorylation reaction is carried out in 30 mM BTP, pH 6.5, containing 3 mM MgCl2. Data are reproduced with permission from the American Society for Biochemistry and Molecular Biology, Inc. [J. Buczyłko, J. C. Saari, R. K. Crouch, and K. Palczewski, J. Biol. Chem. 271, 20621 (1996)]. (C) Structures of all-trans-retinal, all-trans-C17 aldehyde, all-trans-C15 aldehyde, trans-C12 aldehyde.
F<sc>ig.</sc> 4
Fig. 4
Domain structure of RK, purification, and immunoblotting. (A) The N-terminal domain (∼180 amino acids) may be involved in the interaction with Rho*. The ATP-specific, catalytic domain is found in the middle of the sequence. The C-terminal domain is the site of autophosphorylation on Ser and Thr residues within the sequence FSTVKGV and is also modified by isoprenylation/carboxymethylation. This autophosphorylation changes the affinity for heparin and is used to purify RK. Bound RK is autophosphorylated on the heparin-Sepharose column and eluted at lower salt than is required for the elution of unphosphorylated kinase (see text). (B) Schematic representation of the elution profile for autophosphorylated and dephosphorylated RK from heparin-Sepharose column. At a high salt concentration (∼150 mM NaCl), unphosphorylated RK is bound to the resin and is eluted specifically with ATP/Mg2+ as a result of autophosphorylation. (C) Mobility shift of RK dephosphorylated by PrP2A (lane a) and autophosphorylated in the presence of ATP (lane b). Multiple forms of autophosphorylated RK are visualized by Western blotting of isolated bovine ROS (lane c) probed with monoclonal antibody G8.
F<sc>ig.</sc> 5
Fig. 5
Proteolysis of Rho and isolation of the C-terminal phosphorylated peptide. (A) SDS–PAGE of standards (lane a in kDa), 33P-labeled Rho (lane b), and Rho digested with endoproteinase Asp-N (lane c). (Rho)2 and (329G-Rho)2 represent dimers formed during sample preparation. The proteolytic-insoluble nonradioactive 329G-Rho fragment is separated from the soluble C-terminal 33P peptide by pelleting membranes. (B) The C-terminal peptide is initially purified on Ga3+-IMAC and finally on a C18 column using HPLC.
F<sc>ig.</sc> 6
Fig. 6
Predicated pattern of ions formed from the C-terminal fragment of Rho during mass spectrometrical analysis. The b series and y series of ions formed from (A, B) DDDEASTTVSKTETSQVAPA peptide (C) from unphosphorylated monophosphorylated peptides at Ser-334, or (D) from unphosphorylated monophosphorylated peptides Ser-338.
F<sc>ig.</sc> 7
Fig. 7
(A) Flash-induced light-scattering binding signals measured on suspensions of washed disk membranes reconstituted with purified RK. Binding of the proteins is studied using disk membranes containing either unphosphorylated (Rho, upper trace) or (pre)phosphorylated Rho (P-Rho, middle trace), respectively. In the lower trace, autophosphorylated RK (P-RK) is used with Rho. All signals were corrected for control signals measured without added kinase. Measuring conditions: 3 μM Rho and 1 μM RK. The flash illuminated 35% of Rho, the total volume is 300 μl, and the cuvette path length is 10 mm. (B) Inhibition of Gt activation by RK. Dissociation signals induced by flash of intensity R*/R = 9 × 10−3 in the presence of Gt and GTP. From the lower to the upper trace the amount of RK is increased. Measuring conditions: 3 μM Rho (washed membranes), 0.4 μM purified Gt, and 1 mM GTP. (C) Inhibition of Gt-dependent Meta II stabilization by RK. Upper trace, Meta II formation in the presence of 1 μM Gt; middle trace, suppression of Meta II formed with 1 μM Gt by the addition of 1 μM RK; lower trace, control with Rho alone. Measuring conditions: 2 μM Rho in 100 mM BTP, pH 8.0, containing 90 μM GDP; 4°; flash excitation, 20%; cuvette path length, 2 mm.
F<sc>ig.</sc> 8
Fig. 8
Model of RK binding to membranes. (A) Binding of RK to Rho*. The model illustrates the direct binding of RK to light R*. For simplification, this model does not distinguish between the different Meta states of Rho*. (B) Activation of Gt and its inhibition by RK. In the presence of GTP, at the preformed complex between R* and Gt, GTP binds to the Gtα subunit followed by the dissociation of Gtα from both R* and Gtβγ. This model shows that activation of Gt by R* is accompanied in vitro by a rapid release of the Gt subunits from the disk surface and that this release can be inhibited by the direct binding of RK to R*. It is important to note that only the fraction of Gt present on the membrane at the time of the flash is monitored in the dissociation signal. (C) Gt-dependent MII stabilization and its inhibition by RK. As illustrated in this model, binding of Gt stabilizes only metarhodopsin II (Meta II, λmax = 380 nm) at the cost of other, noninteractive and spectrophotometrically different forms of Rho*, e.g., metarhodopsin I (Meta I, λmax = 480 nm). RK can bind to Meta I and Meta II. When Gt and RK are present simultaneously, RK inhibits the complex formation between R* and Gt, resulting in the suppression of the extra-Meta II signal (see Fig. 7C).
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