Characterization of the GRK2 binding site of Galphaq

Abstract

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins. The G(q) subfamily of Galpha subunits couples GPCR activation to the enzymatic activity of phospholipase C-beta (PLC-beta). Regulators of G protein signaling (RGS) proteins bind to activated Galpha subunits, including Galpha(q), and regulate Galpha signaling by acting as GTPase activating proteins (GAPs), increasing the rate of the intrinsic GTPase activity, or by acting as effector antagonists for Galpha subunits. GPCR kinases (GRKs) phosphorylate agonist-bound receptors in the first step of receptor desensitization. The amino termini of all GRKs contain an RGS homology (RH) domain, and binding of the GRK2 RH domain to Galpha(q) attenuates PLC-beta activity. The RH domain of GRK2 interacts with Galpha(q/11) through a novel Galpha binding surface termed the "C" site. Here, molecular modeling of the Galpha(q).GRK2 complex and site-directed mutagenesis of Galpha(q) were used to identify residues in Galpha(q) that interact with GRK2. The model identifies Pro(185) in Switch I of Galpha(q) as being at the crux of the interface, and mutation of this residue to lysine disrupts Galpha(q) binding to the GRK2-RH domain. Switch III also appears to play a role in GRK2 binding because the mutations Galpha(q)-V240A, Galpha(q)-D243A, both residues within Switch III, and Galpha(q)-Q152A, a residue that structurally supports Switch III, are defective in binding GRK2. Furthermore, GRK2-mediated inhibition of Galpha(q)-Q152A-R183C-stimulated inositol phosphate release is reduced in comparison to Galpha(q)-R183C. Interestingly, the model also predicts that residues in the helical domain of Galpha(q) interact with GRK2. In fact, the mutants Galpha(q)-K77A, Galpha(q)-L78D, Galpha(q)-Q81A, and Galpha(q)-R92A have reduced binding to the GRK2-RH domain. Finally, although the mutant Galpha(q)-T187K has greatly reduced binding to RGS2 and RGS4, it has little to no effect on binding to GRK2. Thus the RH domain A and C sites for Galpha(q) interaction rely on contacts with distinct regions and different Switch I residues in Galpha(q).

Figure 1

(A) Model of the Gα_q-GRK2 RH domain complex and their interacting surfaces. The model of Gα_q was homology modeled based on the AlF₄⁻ bound structure of Gα_i in complex with RGS4 (11) and then docked with the RH domain as described in Experimental Procedures. The switch regions and the αA helix of Gα_q (purple and yellow) are labeled, as are the α5 and α6 helices of the GRK2 RH domain. These structural elements constitute the principal interaction surfaces of each protein. The proposed plane of the plasma membrane runs along the top of the complex, as shown in the figure. The switch regions of Gα_q are delineated by V182 to Y192 (switch I), V204- T224 (switch II), and D236-R247 (switch III). (B) Model of Gα_q in complex with RGS4, based on the atomic structure of Gα_i-RGS4 (11). The RH domains of GRK2 and RGS4 both interact with the switch regions of the G protein, but the surface of the RH domain used in the contact is unique. In the RGS4 complex, the α5 helix faces out of the page, while in the Gα_q-GRK2 complex it forms the principal contact surface. Panels (C) and (D) represent views of Gα_q and GRK2, respectively, as if the complex shown in panel (A) were opened like a book. (C) The GRK2-interacting surface of Gα_q. The residues shown as ball-and-stick models with green carbons are those mutated and analyzed in this study. Thick circles indicate residues that had a dramatic effect upon mutation (as per Table 1), thin circles indicate an “intermediate” effect, and no circles indicate no effect, at least upon GRK2 binding and inhibition of IP₃ release. The residues listed in orange are those that each Gα_q residue is predicted to contact. The black sphere represents Mg²⁺. (D) The Gα_q-interacting surface of the GRK2 RH domain. The residues shown as balland- stick models with green carbons are those mutated and analyzed in this (L118 and E130) and our previous study (22). Thick circles indicate residues that had a dramatic effect upon mutation, thin circles indicate an “intermediate” effect, and no circles indicate no effect, at least upon Gα_q binding. The residues listed in purple are those that each GRK2 residue is predicted to contact. All panels were created using PyMOL (38). The coordinates of the model of the Gα_q-GRK2 RH domain complex are available in a pdb file as Supplementary Data.

Figure 2

Interaction of GST-GRK2-(45–178) with Gα_q point mutants activated by AlF₄⁻, the Q209L or the R183C mutation. (A) HEK-293 cells were transfected with EE tagged versions of Gα_q point mutants and Gβ and Gγ constructs. Cells were lysed and binding to GST-GRK2-(45–178) in the presence (+) or the absence (−) of AlF₄⁻ was determined as described in Experimental Procedures. The (+) and (−) lanes represent 40% of the Gα_q or Gα_q mutant pulled down from the 125 μl of lysate. In these experiments we detect little to no binding of GST-GRK2-(45–178) to Gα_q or Gα_q point mutants in the absence of AlF₄⁻. Underneath the representative western blot the percent of each Gα_q mutant pulled down by GST-GRK2-(45–178) in the presence of AlF₄⁻ is compared to the control, which is the percent of Gα_q pulled down by GST-GRK2 (45–178) in the presence of AlF₄⁻, and is represented graphically as the percent of control + S.D. (B) HEK-293 cells were transfected with EE tagged versions of Gα_q-Q209L point mutants and Gβ and Gγ constructs. Cells were lysed and binding of the QL mutants to GST-GRK2-(45–178) was determined as described in Experimental Procedures. Results are plotted as described in A. (C) HEK-293 cells were transfected with EE tagged versions of Gα_q-R183C point mutants and Gβ and Gγ constructs. Cells were lysed and binding of the RC mutants to GST-GRK2-(45–178) was determined as described in Experimental Procedures. The lanes labeled “P” represent 40% of the Gα_q or Gα_q mutant that was present in the pulldown from 200 μl of lysate. The lanes in A, B and C labeled “L” represent 4% of total Gα_q or Gα_q mutant available in the lysate for pull-down. Results are plotted as described in A. The (⋆) indicates that the amount of the marked Gα_q mutant pulled-down is significantly different (p < 0.05) by one-way ANOVA followed by a Dunnett post-test, than the amount of Gα_q pulled-down by GST-GRK2. The (⋆⋆) indicates that statistical analysis could not be performed on the binding of GST-GRK2-RH to the K77A-QL, P185K-QL or P185K-RC mutants because there was no detectable pull-down. The data are averages from three to six independent experiments.

Figure 3

Effect of the Q152A point mutation in Gα_q-RC on the ability of GRK2 to inhibit inositol phosphate production. (A) HEK-293 cells were transfected with 0.1 μg of the constitutively active Gα_q-R183C or Gα_q-Q152A-RC and 0.2 μg of myc, His-tagged Gβ and 0.1 μg of Gγ and increasing amounts of GRK2-K220R and empty vector up to a total of 1.0 μg of DNA. 24 hrs after transfection the cells were labeled with 2 μCi/ml myo-[³H]inositol and 16 hours later inositol phosphate production was determined, as described in Experimental Procedures. The results shown are averages from five independent experiments each done in triplicate and displayed as percent control ± S.D. The control is the inositol phosphate production stimulated by Gα_q-R183C or Gα_q- Q152A-RC in the absence of any co-expressed GRK2-K220R. A (⋆) denotes a statistically significant difference (p < 0.05) by two-way ANOVA followed by a Bonferroni post-test, between the indicated Gα_q-Q152A-RC bar and the Gα_q-RC bar transfected with the same amount of GRK2-K220R. A (#) indicates a statistically significant difference (p < 0.05) by one-way ANOVA followed by a Dunnett post-test, between the indicated bar and the control, either Gα_q-RC or Gα_q-Q152A-RC in the absence of cotransfected GRK2-K220R. (B) Western blots of total cellular lysates from a representative inositol phosphate experiment from (A) probed with the EE monoclonal antibody showing that increasing GRK2-K220R expression does not effect expression of Gα_q-RC or Gα_q-Q152A-RC. The bottom panel of Figure 4 B shows the level of GRK2- K220R overexpression. The bands corresponding to 10, 25 and 100 ng of GRK2-K220R transfected can be seen after very short exposures; however, the GRK2 band corresponding to 5 ng of cDNA transfected is barely visible, even after long exposures, suggesting that comparatively low levels of GRK2 expression can significantly inhibit Gα_q signaling.

Figure 4

Interaction of GST-RGS2 with Gα_q point mutants activated by AlF₄⁻, the Q209L or the R183C mutation. (A) HEK-293 cells were transfected with EE tagged versions of Gα_q point mutants and Gβ and Gγ constructs. Cells were lysed and binding to GST-RGS2 in the presence (+) or the absence (−) of AlF₄⁻ was determined as described in Experimental Procedures. The (+) and (−) lanes represent 40% of the Gα_q or Gα_q mutant pulled down from the 125 μl of lysate. In these experiments we detect little to no binding of GST- RGS2 to Gα_q or Gα_q point mutants in the absence of AlF₄⁻. Underneath the representative western blot the percent of each Gα_q mutant pulled down by GST- RGS2 in the presence of AlF₄⁻ is compared to the control, which is the percent of Gα_q pulled down by GST-RGS2 in the presence of AlF₄⁻, and is represented graphically as the percent of control ± S.D. (B) HEK-293 cells were transfected with EE tagged versions of Gα_q-Q209L point mutants and Gβ and Gγ constructs. Cells were lysed and binding of the QL mutants to GST-RGS2 was determined as described in Experimental Procedures. Results are plotted as described in A. (C) HEK-293 cells were transfected with EE tagged versions of Gα_q-R183C point mutants and Gβ and Gγ constructs. Cells were lysed and binding of the RC mutants to GST-RGS2 was determined as described in Experimental Procedures. The lanes labeled “P” in B and C represent 40% of the Gα_q or Gα_q mutant that was present in the pull-down from 200 μl of lysate. The lanes in A, B and C labeled “L” represent 4% of total Gα_q or Gα_q mutant available in the lysate for pull-down. Results are plotted as described in A. The (⋆) indicates that the amount of the marked Gα_q mutant pulled-down is significantly different (p < 0.05) by one-way ANOVA followed by a Dunnett post-test, than the amount of Gα_q pulled-down by GST-RGS2. The (⋆⋆) indicates that statistical analysis could not be performed on the binding of GST-RGS2 to the K77A-QL, P185K-QL, P185K-RC, T187K or T187K-QL mutants because there was no detectable pull-down. The data are averages from three to six independent experiments.

Figure 5

RGS2 inhibition of inositol phosphate production stimulated by Gα_q-RC and Gα_q-RC mutants. (A) HEK-293 cells were transfected with 0.1 μg of the constitutively active Gα_q-R183C, Gα_q-Q81A/RC, Gα_q-R92A/RC, Gα_q-Q152A/RC, or Gα_q-RC/T187K and 0.2 μg of myc, His-tagged Gβ and 0.1 μg of Gγ and increasing amounts of RGS2 and empty vector up to a total of 1.0 μg of DNA. 24 hrs after transfection the cells were labeled with 2 μCi/ml myo-[³H]inositol and 16 hours later inositol phosphate production was determined, as described in Experimental Procedures. The results shown are averages from three independent experiments each done in triplicate and displayed as percent control + SD. The control is the inositol phosphate production stimulated by each mutant in the absence of any co-expressed RGS2. The statistical significance of the difference between the indicated bar and Gα_q-R183C, in the absence of any additional mutations, transfected with equal amounts of RGS2 is denoted by ⋆ (p < 0.05) by oneway ANOVA followed by a Dunnett post-test. A (#) indicates a statistically significant difference (p < 0.05) by one-way ANOVA followed by a Dunnett post-test, between the indicated bar and the control, either Gα_q-RC or a Gα_q-RC mutant in the absence of cotransfected RGS2. (B) Western blots of total cellular lysates from a representative inositol phosphate experiment from (A) probed with the EE monoclonal antibody showing that increasing RGS2 expression does not effect expression of Gα_q-RC or any of the mutants. We were not able to detect the level of RGS2 overexpression in these experiments; however the decrease in inositol phosphate production suggests that RGS2 expression was increased.

Figure 6

Further mapping of the Gα_q/11 binding site on the GRK2 RH domain. Upper Panel. Glutathione-agarose beads bearing GST fusion proteins, either WT GSTGRK2-(45–178) or GST-GRK2-(45–178) substituted as indicated, were incubated with bovine brain extract (as a source of Gα_q/11) in the presence (+) or absence (−) of aluminum fluoride (AlF₄⁻). Bound Gα_q/11 was visualized by immunoblotting. Lower Panel. Fusion proteins used in the GST-pull-down assay above were separated by SDS-PAGE and visualized by Coomassie staining.