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. 2009 May 26;48(20):4285-93.
doi: 10.1021/bi900151g.

GRK2 Activation by Receptors: Role of the Kinase Large Lobe and Carboxyl-Terminal Tail

Free PMC article

GRK2 Activation by Receptors: Role of the Kinase Large Lobe and Carboxyl-Terminal Tail

Rachel Sterne-Marr et al. Biochemistry. .
Free PMC article


G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gbetagamma as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl-terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Residues in the GRK2 large lobe whose side chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of 20, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate two different GPCRS: rhodopsin, and the beta(2)-adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of nonreceptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had a 12-fold lower k(cat), but no changes in K(M), suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated beta(2)AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.


Figure 1
Figure 1. GRK2 Structure
Full-length GRK2 is oriented to display its predicted membrane-proximal surface, regions of which are expected to interact with the cytoplasmic loops and tails of an activated GPCR (6). In GRK2, the RH domain contacts both the kinase domain (green) and the PH domain (brown). In the RH domain, α-helices 1–9 are purple while α-helices 10 and 11, which are unique to the RH domains of GRKs, are magenta. The regions that interact with Gαq are colored cyan, that interact with Gβγ are yellow, and that interact with phosphatidylinositol bisphosphate (PIP2) are orange. The N-terminal 28 amino acids, a large portion of the kinase extension (476–495), a loop of the PH domain, and the C-terminal 21 residues are not structured in GRK2 crystals. The termini of ordered regions are indicated by Ser29, Gly475, Ile496, Gly569, Trp576, and Pro668 (arrows).
Figure 2
Figure 2. Kinase Domain Residues Targeted for Mutagenesis
The GRK2 kinase domain was modeled in a closed conformation based on an activated structure of PKA (PDB 1L3R). Two regions of the kinase domain were targeted for mutagenesis: the large lobe and the C-tail. To indicate the expected position of the phosphoacceptor binding site, the PKA inhibitor PKI (space-filling atoms in gray) was mapped onto the GRK2 structure. Nearby residues that could interact with other regions of the receptor (His280, Tyr281, Gln285, Ser284, His394, Lys395, and Lys397) were substituted with alanine. The AST of GRK2 has also been implicated in receptor interaction (20) but residues Glu476-Leu499 have been unstructured in all crystals of GRK2 reported thus far. However, a nearly fully ordered AST loop (black backbone) was observed in GRK1 (8). This structure was mapped onto GRK2 to provide an estimate of the position of amino acids in the AST. Pro473, Glu476, Val477, Asp481, Phe483, Asp484, Ile485, Phe488, Glu490, Gly495, and Leu499 in the AST were selected for site-directed mutagenesis. Substituted residues that showed diminished capacity to phosphorylate receptor and at least some of the non-receptor substrates have carbon atoms colored magenta, whereas those that did not have significant effects on rhodopsin phosphorylation have carbons colored yellow. (Inset) A space-filling model of GRK2 at the plasma membrane using the Fig. 1 coloring scheme (RH domain is purple and magenta, and the PH domain is colored brown). We speculated that the cleft between the lipid bilayer and the kinase large lobe could serve to accommodate the intracellular loops and carboxyl tail of a GPCR. Mutated residues in the kinase domain are shown as yellow spheres.
Figure 3
Figure 3. Expression and Phosphorylation of Light-activated Rhodopsin by GRK2 Kinase-Domain Mutants
A. WT and GRK2 mutant cDNAs were transiently transfected into COS-1 cells, and the amount of GRK2 in the HSS fraction of the cells was quantified by immunoblotting and densitometry. B. Kinase activity of WT and mutant GRK2 (~10 nM) in cell lysates was assessed using 15 µM light-activated rhodopsin and 200 µM γ-32P-ATP (0.2 dpm/fmol) as substrates. Coomassie-stained rhodopsin bands separated by SDS-PAGE were excised and the phosphate transferred was quantified by liquid scintillation counting. Error bars indicate SEM. One-way ANOVA was used to compare statistical significance of differences relative to WT. *, p<0.05; **, p<0.01.
Figure 4
Figure 4. Phosphorylation of GPCR and Non-receptor Substrates by GRK2 Kinase-Domain Mutants
Four mutants (Y281A, P473E, V477D, and I485A) were over-expressed in COS-1 or Sf9 insect cells and purified. Kinase assays were carried out with rhodopsin (A), β2AR in phosphatidylcholine vesicles (B), tubulin (C), α-synuclein (D), RESA peptide (E), and RASASA peptide (F) as described in Experimental Procedures. Error bars indicate SEM. One-way ANOVA was used to compare statistical significance of differences relative to WT. *, p<0.05; **, p<0.01.
Figure 5
Figure 5. Michaelis-Menten Kinetics
Kinase reactions were carried out with rhodopsin (2.5–20 µM), 100 nM WT GRK2, GRK2-Y281A, GRK2-P473E, and GRK2-I485A or 400 nM GRK2-V477D, and 1 mM [γ-32P] ATP (~0.2 dpm/fmol) for 2 min in the light. Rhodopsin was separated by electrophoresis, stained, and excised to quantitatively determine phosphate transferred. The data are derived from three experiments carried out in duplicate and error bars represent SEM. Initial rate data was fit to Michaelis-Menten rate equation and values for KM and kcat are given in Table 1.
Figure 6
Figure 6. V477D Mutant is Deficient in β2AR-mediated Activation
Phosphorylation of 1 mM RASASA peptide by 100–200 nM GRK2 or 200 nM V477D was assessed in the absence or presence of PI/DDM mixed micelles (PI/DDM), 80 nM isoproterenol-treated β2AR in PI/DDM mixed micelles (β2AR/ISO), or 80 nM alprenolol-treated β2AR in PI/DDM mixed micelles (β2AR/ALP). Two-way ANOVA was used to test statistical significance. Error bars indicate SEM. *, p<0.05; **, p<0.01; ***, p<0.001.

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