Role of G protein-coupled receptor kinases (GRKs) in β2 -adrenoceptor-mediated glucose uptake

Abstract

Truncation of the C-terminal tail of the β₂ -AR, transfection of βARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the β₂ -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant β₂ -ARs were generated and receptor affinity for [³ H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by β₂ -AR agonists, cAMP accumulation, GLUT4 translocation, [³ H]-2-deoxyglucose uptake, and β₂ -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between β₂ -AR and β-arrestin2 or between β₂ -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to β₂ -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to β₂ -AR agonists occurred in CHO-GLUT4myc cells expressing β₂ -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type β₂ -AR. However, β₂ -ARs lacking phosphorylation sites failed to recruit β-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the β₂ -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

Keywords: GRK2; glucose uptake; β2 adrenoceptor.

FIGURE 1

Saturation of (A–D) and competition for (E–H) [³H]‐CGP12177A binding in CHO‐GLUT4myc cells stably expressing wild‐type or mutant β₂‐adrenoceptors (AR). Binding (fmol/mg protein) determined in CHO‐GLUT4myc cells expressing (A) β₂‐AR wild‐type, (B) β₂‐AR (−)GRKcom, (C) β₂‐AR (−)GRK/PKA, or (D) β₂‐AR DALL by incubation with [³H]‐CGP12177A for 2 h. Competition for [³H]‐CGP12177A(~2 nM) binding was determined by incubating with isoprenaline or BRL37344 for 2 h (E–H). Non‐specific binding was determined with (−)‐propranolol (10 μM). Points show mean ± standard deviation (SD) of 5 independent experiments performed in quadruplicate (saturation) or triplicate (competition).

FIGURE 2

Concentration‐dependent cAMP accumulation (A–D), glucose uptake (E–H), and GLUT4 translocation (I–L) in response to isoprenaline or BRL37344 in CHO‐GLUT4myc cells stably expressing β₂‐adrenoceptor (AR) variants. cAMP was measured in CHO‐GLUT4myc cells expressing either (A) β₂‐AR wild‐type, (B) β₂‐AR (−)GRKcom, (C) β₂‐AR (−)GRK/PKA, or (D) β₂‐AR DALL treated with isoprenaline or BRL37344 for 30 min in the presence of 0.5 mM IBMX. Results are expressed as % of cAMP produced by isoprenaline (10 μM). 2‐deoxy‐[³H]‐glucose uptake was measured in (E) β₂‐AR wild‐type, (F) β₂‐AR (−)GRKcom, (G) β₂‐AR (−)GRK/PKA, or (H) β₂‐AR DALL following stimulation with isoprenaline, BRL37344 or insulin (10 μM) for 2 h. GLUT4 translocation was measured after 2 h stimulation with isoprenaline, BRL37344, or insulin (1 μM) in (I) β₂‐AR wild‐type, (J) β₂‐AR (−)GRKcom, (K) β₂‐AR (−)GRK/PKA, or (L) β₂‐AR DALL. Data was quantified by automated multiwave scoring. Values are mean ± standard deviation (SD) of 5–6 independent experiments performed in duplicate. Data are normalized to values in vehicle‐treated cells at 2 h.

FIGURE 3

Time course of SNAP‐β₂‐AR internalization in response to isoprenaline or BRL37344. Time course of β₂‐AR internalization following stimulation with 10 μM isoprenaline or 10 μM BRL37344 in (A) SNAP‐β₂‐AR wild‐type, (B) SNAP‐β₂‐AR (−)GRKcom, (C) SNAP‐β₂‐AR (−)GRK/PKA, or (D) SNAP‐β₂‐AR DALL cells. Data were quantified using an automated granularity algorithm. Values are mean ± standard deviation (SD) in 5 independent experiments performed in duplicate. Data are normalized to values in vehicle‐treated cells at 60 min.

FIGURE 4

Interaction between the β₂‐adrenoceptor and β‐arrestin2 in HEK293 cells examined by bioluminescent resonance energy transfer (BRET). HEK293A cells were transfected with bicistronic vectors encoding β‐arrestin2‐venus with Rluc8 tagged β₂‐AR wild‐type compared to (A) β₂‐AR (−)GRKcom, (B) β₂‐AR (−)GRK/PKA, or (C) β₂‐AR DALL. About 48 h after transfection, cells were assayed in a 100 μL final volume with 5 μM coelenterazine‐H and either isoprenaline or BRL37344 (1 μM) after serum starving for 4 h. Results are mean ± standard deviation (SD), n = 5.

FIGURE 5

GRK isoform mRNA expression in L6 cells and effect of siRNA knockdown on GRK2 mRNA expression. (A) RT‐PCR of GRK isoforms in L6 cells in 3 independent experiments. (B) Knockdown of GRK2 mRNA following transfection of GRK2 siRNA. Relative GRK2 gene expression was determined by qRT‐PCR. Results are mean ± standard deviation (SD), n = 3.

FIGURE 6

Effects of GRK2 inhibition on β‐arrestin2 recruitment in HEK cells and glucose uptake in rat L6 skeletal muscle cells. (A) BRET assay was performed to investigate the effect of GRK2 inhibition on β‐arrestin2 recruitment using a bicistronic vector expressing the β₂‐AR and β‐arrestin2 in HEK293A cells. The GRK inhibitors paroxetine and CMPD101 (both 10 μM) reduced BRET between the β₂‐AR and β‐arrestin2 following isoprenaline (100 nM). BRL37344 (100 nM) did not cause β‐arrestin2 recruitment. In L6 cells, paroxetine and CMPD101 (10 μM) also reduced glucose uptake in response to isoprenaline (100 nM; B,C) and BRL37344 (10 μM; D,E). Concentration‐dependent increases in glucose uptake to isoprenaline (2 h) were observed in L6 myoblasts transfected with siRNA (GRK2 scrambled) but completely inhibited in myoblasts following transfection with GRK2 siRNA (F). Data are normalized to values from vehicle‐treated cells at 2 h. All data are mean ± standard deviation (SD) in 5 independent experiments performed in duplicate.

FIGURE 7

Interaction between the β₂‐adrenoceptor and GRK2 in HEK293 cells examined by bioluminescent resonance energy transfer (BRET). HEK293A cells were transfected with vectors encoding GRK2‐venus and either Rluc8 tagged β₂‐AR wild‐type, β₂‐AR (−)GRKcom, β₂‐AR (−)GRK/PKA, or β₂‐AR DALL. About 48 h after transfection, cells were assayed in a 100 μL final volume with 5 μM coelenterazine‐H and ligands [1 μM for isoprenaline] after serum starving for 4 h. Results are mean ± standard deviation (SD), n = 9.