Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

Abstract

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.

Figure 1

Gα_s mediates the first phase of the stimulation of ERK MAPK by β₂-AR. (A) Top: different concentrations of isoproterenol increased the kinase activity of ERK MAPK in MEF cells. Whole-cell lysates were prepared from MEF cells. Activated ERK MAPK proteins were immunoprecipitated from cell lysates by a monoclonal antibody against phospho-p44/42 ERK MAPK (crosslinked to agarose beads). The ERK MAPK activity was measured by the phosphorylation of substrate GST-Elk-1, which was detected by Western blotting with an anti-phospho-Elk-1 antibody. Bottom: Western blot with anti-ERK MAPK antibody showing that similar amounts of cell lysates were used in each lane. (B) The data in (A) were quantified and the stimulation of MAPK was shown in comparison to the basal (without isoproterenol treatment). The data in the presence of ICI-118551 are marked with x. Data represent mean±s.d. of three experiments. (C, D) Dose–response of ERK activation by isoproterenol in CHO cells stably expressing human β₂-AR. (E) Dose–response of cAMP production by isoproterenol stimulation in MEF cells. (F) No stimulation of ACs by isoproterenol in Gα_s^−/− cells. (G) Increase of cAMP by forskolin in Gα_s^−/− cells. (H, I) Different concentrations of isoproterenol increased the kinase activity of ERK MAPK in Gα_s^−/− cells. The data point in the presence of 10 μM PP2 is marked with x. Data represent mean±s.d. of three experiments.

Figure 2

The second phase of the stimulation of ERK MAPK by β₂-AR does not require G proteins and receptor internalization. (A) No stimulation of ACs by isoproterenol in β₁-AR^−/−/β₂-AR^−/− cells. (B) Increase of cAMP by forskolin in β₁-AR^−/−/β₂-AR^−/− cells. (C) No stimulation of ERK MAPK by isoproterenol in β₁-AR^−/−/β₂-AR^−/− cells. Stimulation of ERK MAPK in MEF cells was used as positive control (lanes 1 and 2). (D, E) PTX pre-treatment did not alter the dose–response curve of ERK MAPK stimulation by isoproterenol in Gα_s^−/− cells. Data represent mean±s.d. of three experiments. (F) PTX pre-treatment blocked the ERK MAPK stimulation by Gi-coupled m2 mAChR in Gα_s^−/− cells. (G) Expression of RGS4 and the RGS domain of p115 Rho GEF had no effect on ERK MAPK stimulation by isoproterenol in Gα_s^−/− cells. (H, I) Different concentrations of isoproterenol increased the kinase activity of ERK MAPK in β-arrestin 2^−/− cells. (J, K) Different concentrations of isoproterenol increased the kinase activity of ERK MAPK in β-arrestin 1^−/−/β-arrestin 2^−/− cells. Data represent mean±s.d. of three experiments.

Figure 3

Src is required for the second phase of the stimulation of ERK MAPK by β₂-AR. (A, B) Effect of different concentrations of isoproterenol on the kinase activity of ERK MAPK in SYF cells. (C, D) Effect of different concentrations of isoproterenol on the kinase activity of ERK MAPK in SYF/c-Src cells. Data represent mean±s.d. of three experiments. (E) Dose–response of cAMP production by isoproterenol stimulation in SYF cells.

Figure 4

Direct interaction between Src proteins and β₂-AR. (A) Stimulation of c-Src kinase activity by 10 μM isoproterenol in MEF, Gα_s^−/−, and β-arrestin 1^−/−2^−/− cells. Data represent mean±s.d. of three experiments. (B) In vitro binding assays of purified unphosphorylated Src with the purified unphosphorylated C-terminal tail of β₂-AR (as GST fusion protein). GST alone was used as a negative control. (C) Purified phosphorylated Src was assayed for interaction with the C-terminal tail of β₂-AR in the presence or absence of ATP. (D) Co-immunoprecipitation of endogenous β₂-AR with endogenous c-Src from HEK-293 cells in the presence or absence of isoproterenol. (E) Phosphorylation of the purified C-terminal tail of β₂-AR by purified Src. Data are representative of three experiments.

Figure 5

Direct activation of Src by β₂-AR. (A) Coomassie blue staining of purified human β₂-AR from Sf9 cells, purified c-Src from Sf9 cells, purified Gα_s from E. coli, and purified Gβ₁γ₂ from Sf9 cells (Gγ₂ protein was off the gel). (B) Purified β₂-AR directly activated purified c-Src. Top panel: Western blot with anti-phospho-tyrosine antibody to show the autophosphorylation of Src (pSrc) and the phosphorylation of β₂-AR by Src (pβ₂-AR) (the reaction was for ∼30 s) (10% SDS–PAGE). ALP: alprenolol; ISO: isoproterenol. Bottom panel: the same filter was probed with anti-Src antibody to show that similar amounts of Src were used in each reaction. (C) Quantification of data in (B). Error bars show mean±s.d., ^*P<0.001 (Student's t-test). (D) Purified β₂-AR directly activated purified c-Src. GST-CDB3 fusion protein was used as exogenous substrate for Src. (E) Quantification of data in (D). Error bars show mean±s.d., ^*P<0.001 (Student's t-test). (F) Acceleration of GTPγS binding to Gs (α_s+βγ) by β₂-AR. Gα_s, Gβγ, and β₂-AR together with alprenolol (▪) or isoproterenol (•) were incubated on ice for 10 min. After incubation at 30°C for 5 min, [³⁵S]GTPγS was added. At various time points, aliquots were removed and ³⁵S was counted to measure GTPγS loading. (G) β₂-AR increased the autophosphorylation of Src (as well as the phosphorylation of β₂-AR) after incubations for 30 s, 1 min, and 2 min (7% SDS–PAGE). After 4 min incubation, there was no difference between the phosphorylation with or without ISO. Bottom panel: a shorter ECL exposure of the same filter shown above. Data are representative of three to five experiments.

Figure 6

A β₂-AR-mutant defective in c-Src binding abolishes the second phase of the ERK response. (A) Sequence alignment of the relevant regions of the β₂-AR and turkey β-AR (the sequences of the remaining C-terminal tails are not shown). (B) In vitro GST-pull-down assay with purified GST fusion proteins and purified c-Src. (C) cAMP assays with β₁^−/−/β₂^−/− cells and β₁^−/−/β₂^−/− cells expressing the β₂-AR-mutants. (D, E) Different concentrations of isoproterenol increased the kinase activity of ERK MAPK in β₁^−/−/β₂^−/− cells expressing the β₂-AR-mutant-2. (F) Diagram of G-protein-dependent and -independent pathways initiated from β₂-AR. At low concentrations of isoproterenol, β₂-AR signals through Gα_s to activate ACs to produce cAMP. At high concentrations of isoproterenol, β₂-AR could directly activate c-Src leading to the activation of ERK MAPK. Also, at high concentrations of isoproterenol, β₂-AR initiates its own internalization. Both Src and β-arrestin 2 are required for receptor internalization. There is quite an amount of crosstalk in these pathways (indicated by open arrows) such as direct activation of Src by Gα_s, by PKA phosphorylation, or by β-arrestin recruitment. Src could also phosphorylate Gα_s and enhance the activity of Gα_s. Src has also been reported to phosphorylate and activate GRKs. This extensive crosstalk might underline the shift of EC₅₀ values in Gα_s^−/− and SYF cells compared to wild-type MEF cells. cAMP and PKA could directly act on target proteins to induce immediate responses. The ERK MAPK pathway could work through gene expression to induce a long-lasting effect.