Mutating the dileucine motif of the human beta(2)-adrenoceptor reduces the high initial rate of receptor phosphorylation by GRK without affecting postendocytic sorting

Abstract

The internalization of beta(2)-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on beta(2)-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the beta(2)-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. beta(2)-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A beta(2)-adrenoceptors. The mutant beta(2)-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type beta(2)-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A beta(2)-adrenoceptors was reduced to approximately 25% that of wild type beta(2)-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A beta(2)-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type beta(2)-adrenoceptors. Therefore, although the role of L339,L340 in beta(2)-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient beta(2)-adrenoceptors endocytosis.

Fig. 1

The first-order rate constants for agonist-induced endocytosis are decreased in the L339A,L340A β₂-adrenoceptors. Two independent HEK293 cell lines stably expressing wild type β₂-adrenoceptors and two lines expressing L339A,L340A β₂-adrenoceptors were constructed as described in the Materials and methods. Cells were grown in 24-well clusters, treated for varying times with 10 µM isoproterenol, then chilled and incubated with 9 nM [³H] CGP12177 to detect surface receptor levels expressed as a fraction of surface receptors in untreated cells as described in the Materials and methods. (A). Receptors remaining on the surface were plotted as a function of time after agonist addition. The internalization curves were modeled using nonlinear regression to obtain first-order rate constants for endocytosis (k_e) and recycling (k_r) as described in Morrison et al. (1996). (B). The data points from the first 3 min of internalization were transformed and a linear regression was performed. The slopes of these lines approximate the endocytic rate constants. All rate values are shown in Table 2. * represents all points plotted on the graph that are significantly different between the wild type and L339A,L340A β₂-adrenoceptor cell lines (unpaired Student's t-test, P<0.0001). N = 9–13, data are presented as mean (symbol) ± S.E.M. (vertical bars). Some error bars are smaller than the symbols.

Fig. 2

β₂-adrenoceptors with the L339A,L340A mutation show an increase in the EC₅₀ for internalization with isoproterenol. Stably transfected cells expressing wild type or L339A,L340A β₂-adrenoceptors were treated with varying concentrations of isoproterenol for 20 min before being chilled and assayed for surface receptors by [³H] CGP12177 binding and receptor internalization calculated as fraction internalized after normalizing counts to those of surface receptor in untreated cells as described in Materials and methods. The EC₅₀ values are shown in Table 3. Data are presented as mean ± S.E.M. * represents points plotted on the graph that are significantly different between the wild type and L339A,L340A β₂-adrenoceptor cell lines (unpaired Student's t-test, P<0.05). N = 4–5, and the data were curve-fitted to a sigmoidal dose–response curve using nonlinear regression analysis.

Fig. 3

β₂-adrenoceptor recycling is not significantly affected by the L339A,L340A mutation. Stably transfected cell lines expressing wild type receptors or L339A,L340A mutant receptors were treated with 5 µM isoproterenol for 20 min, then washed at 4 °C to remove agonist before returning to 37 °C for various times up to 60 min. The return of receptors to the cell surface was assessed by binding to the hydrophilic radioligand [³H]CGP12177 and the fraction of surface receptors was calculated as fraction of surface receptors in untreated cells as described in Materials and methods. The other pair of cell lines produced similar results. * represents points plotted on the graph that are significantly different between the wild type and L339A,L340A β₂-adrenoceptor cell lines (unpaired Student's t-test, P<0.05). N = 5–6, data are presented as mean ± S.E.M.

Fig. 4

Downregulation of β₂-adrenoceptors is not directly affected by the L339A,L340A mutation. (A). Stably transfected cells expressing wild type or L339A,L340A β₂-adrenoceptors were treated with varying concentrations of isoproterenol for 24 h, then assessed for surface receptor levels using [³H]CGP12177 binding, and for total receptor levels using the same radioligand, but in the presence of 0.05% digitonin, as described in Materials and methods. Data are presented as a percentage of untreated cells. N = 5, similar results were obtained with another pair of clones. Data are presented as mean (symbols) ± S.E.M (vertical bars). (B). HEK 293 stable cell lines expressing wild type (upper panel) and L339A,L340A mutant (lower panel) β₂-adrenoceptors were grown on glass cover slips and treated with isoproterenol (10 µM) for 24 h or received no treatment in the presence of 100 µM leupeptin. Receptors were immunolabeled using Texas Red secondary antibody and LAMP-1 was labeled green using FITC secondary antibody and images were deconvolved as described in Materials and methods. Arrows highlight yellow (overlap of red and green) punctate labeling where receptors localize with LAMP-1 in lysosomes. Images are representative of 5–10 cells from 4–5 different experiments in both pairs of cell lines.

Fig. 5

Receptors with the L339A,L340A mutations show a reduced rate of agonist-induced phosphorylation of serines 355,356. Stably transfected HEK293 cells expressing wild type or L339A,L340A β₂-adrenoceptors were treated with 10 µM isoproterenol for the indicated times, then chilled and harvested to assess receptor phosphorylation by immunoblotting, as described in the Materials and methods. (A) Quantification of immunoblots: phosphoserine band density values were normalized per receptor band density values. Phosphorylation was calculated as a fraction of maximum phosphorylation of wild type receptors at 5 min. Data are presented as mean ± S.E.M., N = 4. * represents significantly different from wild type receptor cell lines (unpaired t-test, P<0.05). (B) A representative immunoblot of the phosphorylation time course quantified in panel A, using antibodies against the carboxyl terminus of β₂-adrenoceptor and its phosphoserines 355,356. Top panels, wild type β₂-adrenoceptors; bottom panels, L339A. L340A β₂-adrenoceptors.

Fig. 6

Receptors with the L339A,L340A mutations show an increased EC₅₀ for phosphorylation of serines 355,356. Stably transfected HEK293 cells expressing wild type or L339A,L340A β₂-adrenoceptors were treated with isoproterenol for 2 min, then chilled and harvested for immunoblotting to detect phosphorylation of serines 355,356 as described in the Materials and methods. (A) Quantification of immunoblots: phosphoserine density values were normalized per receptor density values of the same lane. Phosphorylation was calculated as a percentage of maximum phosphorylation for wild type receptors at 10 µM of agonist. The data were fitted to a sigmoidal dose–response curve using nonlinear regression analysis. N = 3–5, Data are presented as mean (symbols) ± S.E.M (vertical bars). The EC₅₀ values are shown in Table 3. (B) A representative immunoblot of the phosphorylation dose–response using antibodies against the carboxyl terminus of β₂-adrenoceptors and its phosphoserines 355,356. Top panels, wild type β₂-adrenoceptors; bottom panels, L339A. L340A β₂-adrenoceptors.