G protein-coupled receptor kinase 6 (GRK6) selectively regulates endogenous secretin receptor responsiveness in NG108-15 cells

Abstract

1. To determine the role of G protein-coupled receptor kinases (GRKs) in the regulation of endogenous secretin receptor responsiveness, we have transiently overexpressed both wild-type (WT) and dominant negative mutant (DNM) GRKs in NG108-15 mouse neuroblastoma x rat glioma hybrid cells and investigated the effects of this on agonist-stimulated adenylyl cyclase activity. 2. Overexpression of WT GRK6 selectively inhibited secretin-stimulated cyclic AMP accumulation (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.1+/-2.0 and 6.2+/- 0.8 in control and WT GRK overexpressing cells, respectively) without affecting cyclic AMP responses mediated by the adenosine A(2) receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) or the prostanoid-IP receptor agonist iloprost, or the direct activator of adenylyl cyclase, forskolin. On the other hand DNM GRK6 (Lys(215)Arg) overexpression produced the opposite effect--a selective increase in the secretin-stimulated cyclic AMP response was observed in cells overexpressing DNM GRK6 compared to plasmid-transfected cells (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.6+/-2.7 and 29.6+/-5.6 for control and DNM GRK6-overexpressing cells, respectively). 3. Overexpression of WT GRK5 likewise inhibited the secretin-stimulated cyclic AMP response, however, this effect was not as selective as with GRK6, since adenosine A(2) receptor responsiveness was also suppressed by GRK5 overexpression. Unlike DNM GRK6, overexpression of DNM GRK5 failed to modulate secretin or A(2) adenosine receptor signalling suggesting that endogenous GRK5 is unlikely to regulate desensitization of these receptors in NG108-15 cells. 4. Overexpression of WT GRK2 did not affect secretin-stimulated cyclic AMP accumulation. Instead, GRK2 overexpression selectively inhibited A(2) adenosine receptor responsiveness, confirming our previous findings. 5. Together these results suggest a selective role of endogenous GRK6 in regulating secretin receptor responsiveness in NG108-15 cells. In addition, these data indicate that GRKs exert a surprising degree of selectivity in the regulation of natively expressed GPCR responses.

Figure 1

GRK overexpression in NG108-15 cells. Cells were transfected with 5 μg DNA containing empty vector, wild-type (WT) GRK or DNM GRK constructs. Whole cell lysates were subjected to SDS–PAGE followed by Western transfer and immunoblotting with GRK-specific primary antibodies as detailed in the Methods. (A) GRK6 overexpression in NG108-15 cells. Lane 1, lysate from NG108-15 cells stably expressing WT GRK6. In lanes 2–4 lysate preparations were from transiently transfected cells. Lane 2, lysate from cells transiently expressing WT GRK6; Lane 3, lysate from cells transiently expressing DNM GRK6; Lane 4, lysate from cells transiently expressing the vector pcDNA₃; Lane 5, lysate from non-transfected NG108-15 cells. (B) GRK5 overexpression in NG108-15 cells. Lane 1, 8 ng of purified GRK5; Lane 2, lysate from cells transiently expressing WT GRK5; Lane 3, lysate from cells transiently expressing DNM GRK5; Lane 4, lysate from cells transiently expressing the vector pcDNA₃. (C) GRK2 overexpression in NG108-15 cells. Lane 1, 8 ng of purified GRK2; Lane 2, lysate from cells transiently expressing WT GRK2; Lane 3, lysate from cells transiently expressing the vector pEGFP-C2; Lane 4, lysate from non-transfected NG108-15.

Figure 2

WT GRK6 overexpression selectively inhibits secretin-stimulated cyclic AMP accumulation. (A) WT GRK6-overexpressing and plasmid-transfected control cells were incubated with secretin (100 nM) for the times indicated. Secretin-stimulated cyclic AMP accumulation was significantly decreased in cells overexpressing WT GRK6 versus control (P=0.008, two-way ANOVA; n=5). (B) WT GRK6-overexpressing and plasmid-transfected control cells were incubated with either secretin (100 nM), iloprost (1 μM), NECA (10 μM) or forskolin (10 μM) for 15 min. Cyclic AMP accumulation is expressed as fold-stimulation over basal and data are mean±s.e.mean of values from at least five independent experiments with each point performed in quadruplicate (P=0.002 compared to respective control, paired t-test)

Figure 3

Effect of WT GRK6 or DNM GRK6 overexpression on concentration-effect curves for secretin-stimulated cyclic AMP accumulation. Cells transiently expressing pcDNA₃ (control), WT GRK6 or DNM GRK6 were incubated with secretin at the concentrations indicated for 15 min. Values for cyclic AMP accumulation have been expressed as a per cent of the maximum level of cyclic AMP obtained in control cells. Data points represent the mean±s.e.mean of values from three independent experiments with each point performed in quadruplicate.

Figure 4

DNM GRK6 overexpression selectively enhances secretin-stimulated cyclic AMP accumulation. (A) DNM GRK6-overexpressing and plasmid-transfected control cells were incubated with secretin (100 nM) for the times indicated. Secretin-stimulated cyclic AMP accumulation was significantly increased in cells overexpressing DNM GRK6 versus control (P=0.03, two-way ANOVA, n=5). (B) DNM GRK6-overexpressing and plasmid-transfected control cells were challenged with either secretin (100 nM), iloprost (1 μM), NECA (10 μM) or forskolin (10 μM) for 15 min. Cyclic AMP accumulation is expressed as fold-stimulation over basal and data are mean±s.e.mean of values from at least five independent experiments with each point performed in quadruplicate (P<0.05 compared to respective control, paired t-test).

Figure 5

WT GRK5 overexpression inhibits secretin-stimulated cyclic AMP accumulation. (A) WT GRK5-overexpressing and plasmid-transfected control cells were challenged with secretin (100 nM) for the times indicated. Secretin-stimulated cyclic AMP accumulation was significantly reduced in cells overexpressing WT GRK5 versus control (P=0.0038, two-way ANOVA, n=4). (B) WT GRK5-overexpressing and plasmid-transfected control cells were challenged with either secretin (100 nM), iloprost (1 μM), NECA (10 μM) or forskolin (10 μM) for 15 min. Cyclic AMP accumulation is expressed as fold-stimulation over basal and data are mean±s.e.mean of values from at least five independent experiments with each point performed in quadruplicate. Both secretin and NECA-stimulated cyclic AMP accumulation were reduced by WT GRK5 overexpression as compared to control values (P=0.0338 and P=0.0101, respectively, paired t-test).

Figure 6

Effect of DNM GRK5 overexpression on agonist-stimulated cyclic AMP accumulation. (A) DNM GRK5-overexpressing and plasmid-transfected control cells were challenged with secretin (100 nM) for the times indicated. Secretin-stimulated cyclic AMP accumulation was not significantly affected by DNM GRK5 overexpression compared to control (P>0.05, two-way ANOVA, n=3). (B) DNM GRK5-overexpressing and plasmid-transfected (control) cells were challenged with either secretin (100 nM), iloprost (1 μM), NECA (10 μM) or forskolin (10 μM) for 15 min. There was no difference in agonist-stimulated cyclic AMP accumulation between DNM GRK5-overexpressing and control cells (n=4). Data are mean±s.e.mean of values from at least three independent experiments with each point performed in quadruplicate.

Figure 7

WT GRK2 overexpression selectively inhibits NECA-stimulated cyclic AMP accumulation. (A) WT GRK2-overexpressing and plasmid-transfected control cells were challenged with NECA (10 μM) for the times indicated. NECA-stimulated cyclic AMP accumulation was decreased in cells overexpressing WT GRK2 versus control (P=0.0012, two-way ANOVA, n=5). (B) WT GRK2-overexpressing and plasmid-transfected control cells were challenged with either secretin (100 nM), iloprost (1 μM), NECA (10 μM) or forskolin (10 μM) for 15 min. Data are mean±s.e.mean of values from five independent experiments with each point performed in quadruplicate. GRK2 overexpression selectively inhibited NECA-stimulated cyclic AMP accumulation compared to control (P=0.02, paired t-test).

Figure 8

WT β-arrestin-1 overexpression inhibits secretin and NECA-stimulated cyclic AMP accumulation. (A) β-arrestin-1 overexpression in NG108-15 cells. Cells were transfected with WT β-arrestin-1-GFP. Whole cell lysates were subjected to SDS–PAGE followed by Western transfer and immunoblotting with an anti-GFP antibody as detailed in the Methods. A strong band of the expected molecular weight (∼70 kDa) was observed following β-arrestin-1-GFP transfection. β-arrestin-1-overexpressing and plasmid-transfected control cells were incubated with (B) secretin (100 nM) or (C) NECA (10 μM) for the times indicated. Both secretin and NECA-stimulated cyclic AMP accumulation was significantly decreased in cells overexpressing β-arrestin-1 versus control (P=0.0239 and P=0.0258, respectively; two-way ANOVA). (D) Overexpression of β-arrestin-1 did not affect iloprost or forskolin-stimulated cyclic AMP accumulation at a 15 min time-point of agonist challenge. Cyclic AMP accumulation is expressed as fold stimulation over basal and data are mean±s.e.mean of values from four independent experiments with each point performed in quadruplicate.