Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases

Abstract

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Figure 1

Agonist-dependent association of GRK2 with the plasma membrane is inhibited by the GRK2 carboxyl terminus (GRK2ct). Cos-7 cells transiently overexpressing GRK2 (CN, ISO) or GRK2 and the GRK2ct (GRK2ct/ISO) were incubated either in medium alone (CN) or medium containing 10 μM isoproterenol (ISO) for 5 min. Cells were harvested and plasma membranes were subjected to Western blot analysis using an anti-GRK2 monoclonal antibody. The intensity of the immunoreactive band corresponding to GRK2 was measured by laser densitometry. Data represent the mean ± SEM. of three independent experiments expressed as fold increase over basal. The basal value represents that amount of membrane-associated GRK2 observed in the absence of agonist (ISO) treatment.

Figure 2

Gβγ binds to GRK2 but not GRK5 in intact cells. (A) Cos-7 cells were transfected with pcDNA-1 vector (NT), GRK2 (GRK2), Gβ₁ and Gγ₂ (βγ), or GRK2, Gβ₁, and Gγ₂ (GRK2 + βγ). Cells were solubilized in 1.0% CHAPS-HEDN, and GRK2 was immunoprecipitated using an anti-GRK2 monoclonal antibody. Immunoprecipitated proteins were subsequently subjected to Western blot analysis using an anti-Gβ antibody. Purified bovine brain Gβγ was included on the Western blot as a positive control (Control). (B) Immunoprecipitation of GRK5 from cells transfected with empty vector (NT), GRK5 (GRK5), Gβ₁ and Gγ₂ (βγ), or GRK5, Gβ₁, and Gγ₂ (GRK5 + βγ). The immunoprecipitations and Gβ Western blot analysis were performed as described for A with the exception that an anti-GRK5 antibody was used for immunoprecipitation. The lower blots in A and B show the GRK2, Gβ, and GRK5 contents of the Cos-7 cell lysates. The Western blots shown are representative of three independent experiments.

Figure 3

(Upper) Time course of agonist-stimulated GRK2-Gβγ complex formation. Cos-7 cells transfected with GRK2, Gβ₁, and Gγ₂ were incubated overnight in serum-free media prior to LPA treatment. Following agonist treatment and at the indicated times cells were lysed and GRK2 was immunoprecipitated. The Gβ content of GRK2 immunoprecipitates was determined by Western blot analysis. Blots were quantified by laser densitometry. The data at each time point represent the mean ± SEM. of three separate determinations. The data are presented as fold increase in Gβ content relative to an unstimulated cell control. (Inset) Representative Western blot showing Gβ immunoreactivity. (Lower) Blots showing Gβ and GRK2 immunoreactivity present in equivalent protein aliquots of cell lysate.

Figure 4

Agonist-specific formation of GRK/Gβγ complexes. Cos-7 cells transfected with either GRK2 (A) or GRK3 (B) were starved overnight in serum-free medium prior to exposure to lysophosphatidic acid (LPA; 10 μM), isoproterenol (ISO; 10 μM), or the thrombin receptor agonist SFLLRN (100 μM). GRK2 or GRK3/Gβγ complex formation was assessed by coimmunoprecipitation. The data represent mean ± SEM values from four independent experiments and are expressed as fold increase over unstimulated (control) cells. (Inset) Immunoblot of a representative experiment.

Figure 5

Gβγ binding specificity of GRK2 and GRK3 carboxyl termini. (A) Recombinant Gβ₁, Gβ₂, or Gβ₃ subunits from Sf9 cells were subjected to Western blot analysis using Gβ-specific antibodies. (B) Purified GST, GST-GRK2ct, or GST-GRK3ct proteins were incubated with purified bovine brain Gβγ in the presence of glutathione-Sepharose beads. Protein–bead complexes were washed and analyzed for Gβ isoforms by Western blotting using the specific Gβ₁, Gβ₂, or Gβ₃ antibodies. (C) GST-GRK2ct or GST-GRK3ct proteins were incubated with purified recombinant Gβ₁γ₅ or Gβ₃γ₅ and analyzed for complex formation as previously described. Blots were developed using Gβ_common antibodies. A Western blot representative of at least three independent experiments is shown.