Pairs of residues targeted for disulfide bridge formation. (A) Cα–Cα and Cβ–Cβ interatomic distances for chosen pairs. The Cα–Cα and Cβ–Cβ distances of typical disulfide bonds are <6.5 and 3.4–4.2 Å, respectively. (B and C) Locations of CC mutants on the GRK6 structure. The Cβ–Cβ distances are indicated. The N5C/D476C (B) and L13C/I472C (C) pairs were designed to cross-link αN with the C-tail, whereas the V11C/Y189C, L12C/Y189C, and L12C/A202C pairs (C) were designed to cross-link αN with the kinase N-lobe. The color scheme is the same as in Figure 1.

Effects of Asp-N-cleaved activated Rho (Asp-NRho*) on peptide C phosphorylation by GRK1₅₃₅H₆. Both the wt protein (■) and the cross-linked T8C/N480C mutant (□) show activation by Asp-N Rho* in a concentration-dependent fashion, while the ΔN19 mutant did not show activation by Asp-NRho* (●). Data were fitted to eq 1 from four independent experiments. Y_min, Y_max, and K_D,app values are (79 ± 5) × 10⁻⁶ s⁻¹, (780 ± 80) × 10⁻⁶ s⁻¹, and 12 ± 5 µM for wt and (370 ± 40) × 10⁻⁶ s⁻¹, (1900 ± 170) × 10⁻⁶ s⁻¹, and 15 ± 6 µM for the cross-linked T8C/N480C mutant, respectively.

Phosphorylation of GPCRs by GRKs, Using GRK1 and Rho as an Example

Key interactions between αN and the kinase domain observed in GRK6 are conserved in GRK1. (A) Overall structure of GRK6 in the closed conformation (PDB entry 3NYN). The RH domain and C-terminal region, the kinase domain, αN, and the C-tail are colored gray, yellow, green, and purple, respectively. The nucleoside analogue sangivamycin bound in the active site is shown as a sphere model. (B) Hydrogen bonds formed among Arg190 of the kinase N-lobe, Asn9 of αN, and the C-tail of GRK6. The structure of GRK1 in a complex with ATP (PDB entry 3C4Z, light blue) was superimposed with GRK6 using the N-lobe. The AST loop (residues 472–477) of GRK1 adopts a conformation similar to that in GRK6. Phe273 of αD on the kinase large lobe of GRK6 interacts with the backbone of the C-tail, as does the analogous Tyr274 of GRK1. (C) van der Waals interactions formed between αN and the kinase domain of GRK6, including Leu12 and Leu13 of αN, Ile472 of the C-tail, and Met211 and Tyr189 of the kinases mall lobe. Corresponding positions in GRK1, including Val476, Leu212, and Phe190, are colored light blue. (D) Residues of GRKs involved in key interactions between αN and the kinase domain, and between the small lobe and the C-tail, are highly conserved. Conserved contact residues in GRK6 are colored red, and residues previously identified as being important for kinase activity in GRK1 and GRK6 are labeled with purple and blue asterisks, respectively. Note that bovine GRK1 is used in this study.

GRK1₅₃₅H₆ T8C/N480C forms a disulfide bond between positions 8 and 480 under oxidizing conditions. (A) K₃Fe(CN)₆-treated GRK1₅₃₅H₆ T8C/N480C, but not wt, ΔN19, or other CC mutants, displays a delayed migration on nonreducing 10% SDS–PAGE that can be reversed by DTT: lane 1, before K₃Fe(CN)₆ treatment; lane 2, after K₃Fe(CN)₆ treatment; lane 3, after K₃Fe(CN)₆ treatment and then incubation with DTT. (B) GRK1₅₃₅H₆ T8C/N480C is monomeric after K₃Fe(CN)₆ oxidation. The elution profile from size-exclusion chromatography (Superdex 200, 25 mL) is shown as a solid line, and that of molecular mass standards is shown as a dotted line with numbers indicating molecular masses in kilodaltons. Nonreducing 10% SDS–PAGE analyzed fractions from the column are shown in the inset. Fractions of monomers show an upward gel shift, as seen in panel A. (C) Overlays of HPLC runs of peptidic fragments from oxidized GRK1₅₃₅H₆ T8C/N480C in the absence (black) or presence (red and blue) of DTT. The inset shows the degree of reduction via SDS–PAGE by application of DTT before tandem mass spectrometry analysis. (D) Identification of the Cys8–Cys480 disulfide bond by tandem mass spectrometry. The specific disulfide bond was verified on the basis of the MS² spectra of the doubly charged ion at m/z 920.39. Ions used to identify fragments of chain A (EC⁸VVANSAF) are colored blue, whereas ions used to identify chain B (AKC⁴⁸⁰IQDVGA) are colored red. Product ions generated due to neutral losses are labeled using primes (loss of water) and asterisks (loss of ammonia). Further statistics for the confirmation of the disulfide-linked peptide by mass spectroscopy are provided in Table S1 of the Supporting Information.

Effects of GRK1₅₃₅H₆ variants on transducin (G_t) activation by Rho* and Meta II decay. Data for reactions without GRK1, with wt GRK1, with cross-linked T8C/N480C, and with ΔN19 are colored black, blue, purple, and green, respectively. Rate constants were calculated from three independent experiments, and data from a representative reaction are shown. (A) GRK1 inhibits activation of G_t by Rho*. The activation of G_t was monitored by the increase in its intrinsic tryptophan fluorescence because of nucleotide exchange catalyzed by Meta II (λ_excitation = 300 nm, and λ_emission = 345 nm). GTPγS (5 µM) was added to initiate the reaction after data had been recorded for 300 s. (B) GRK1 promotes Meta II decay. Meta II decay was measured by the increase of the intrinsic tryptophan fluorescence of Rho due to the release of the retinal chromophore (λ_excitation = 295 nm, and λ_emission = 330 nm).

Reaction Diagrams for Phosphorylation of GPCRs and Peptides by GRKs