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

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 [³H]-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, [³H]-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.