A polymorphism of G-protein coupled receptor kinase5 alters agonist-promoted desensitization of beta2-adrenergic receptors

Abstract

Beta-agonist treatment of asthma displays substantial interindividual variation, which has prompted polymorphism discovery and characterization of beta2-adrenergic (beta2AR) signaling genes. beta2AR function undergoes desensitization during persistent agonist exposure because of receptor phosphorylation by G-protein coupled receptor kinases (GRKs). GRK5 was found to be highly expressed in airway smooth muscle, the tissue target for beta-agonists. The coding region is polymorphic at codon 41, where Gln can be substituted by Leu (minor allele), but almost exclusively in those of African descent. In transfected cells, GRK5-Leu41 evoked a greater degree of agonist-promoted desensitization of adenylyl cyclase compared with GRK5-Gln41. Consistent with this functional effect, agonist-promoted beta2AR phosphorylation was greater in cells expressing GRK5-Leu41, as was the rate of agonist-promoted receptor internalization. In studies with mutated beta2AR lacking PKA-phosphorylation sites, this phenotype was confirmed as being GRK-specific. So, GRK5-Leu41 represents a gain-of-function polymorphism that evokes enhanced loss-of-function of beta2AR during persistent agonist exposure, and thus may contribute to beta-agonist variability in asthma treatment of African-Americans.

Fig. 1

Desensitization and phosphorylation phenotypes of GRK5-Gln41 and GRK5-Leu41. In (a) and (b), CHW-1102 cells stably expressing β₂AR were transfected to express the two GRK5 variants. Cells were exposed to vehicle or vehicle plus isoproterenol, washed, membranes prepared, and membrane adenylyl cyclase activities determined. Cells expressing GRK5-Leu41 displayed greater desensitization than those expressing the Gln41 form. Results are from 4 experiments. In (c), COS-7 cells were co-transfected with FLAG-tagged β₂AR and the two GRK5 variants, loaded with ³²P-orthophosphate, and agonist-promoted phosphorylation of β₂AR determined (see ref [7] for methods). Shown is a representative result from 3 experiments. See text for mean results. In (d) and (e), CHO cells were co-transfected with a mutated β₂AR lacking PKA phosphorylation sites (see text) and the GRK5 variants. Experiments were carried out as in (a) and (b), and showed greater desensitization of the modified β₂AR in cells expressing GRK5-Leu41 compared to GRK5-Gln41. Results are from 7 experiments. Iso, isoproterenol

Fig. 2

Expression of GRK5 in various cell lines. Western blots were performed using GRK5-and GRK2-specific antibodies, and a control antibody for GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA) at titers of 1:1000, with 25 µg of protein from whole-cell lysates for each sample. In (a) results are shown for the indicated cell lines that endogenously express β₂AR. Human airway smooth muscle (HASM) had the greatest expression of GRK5 compared to human airway epithelial cells (BEAS2B) and other cell lines. In contrast, this marked difference was not observed with GRK2. In (b), endogenous GRK expression is shown from the three model cell lines used in the current study for transfections. Results are from a representative experiment.

Fig. 3

Agonist-promoted β₂AR internalization phenotypes of GRK5-Gln41 and GRK5-Leu41. Cells expressing β₂AR and the indicated GRK5 variant were exposed to 10 µM isoproterenol for the times shown, and the extent of internalized receptors in intact cells determined by the binding of [³H]-CGP12177 (which binds only to cell-surface β₂AR, see text and ref [10]). GRK5-Leu41 cells had a higher rate of β₂AR internalization compared to that of GRK5-Gln41 cells (t1/2 = 3.8 ± 0.20 vs. 7.2 ± 0.33 min, P < 0.001). Results are mean ± SE from 4 experiments.