Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization

Abstract

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.

Figure 1

Effects of GRK mutants on G_s-coupled receptor-mediated cAMP accumulation in ASM. Primary ASM cultures were infected with retrovirus and selected with 250 μg/mL G418 to generate lines stably expressing GFP, GRK2CT-GFP (CT-GFP), GRK2K220R-GFP (KR-GFP), or GRK2NT-GFP (NT-GFP). Cells grown to near-confluence in 24 well plates were stimulated with 1 mM RO-20-1724 and 1 μM ISO (A), 1 μM PGE₂ (B), or 50 μM NECA (C) for 0–10 min, or 100 μM FSK for 10 min. cAMP was isolated and quantified by RIA as described in Experimental Procedures. Data represent the mean ± SE values from 6–9 experiments, with each experiment employing a different culture derived from a unique donor. *p < 0.05, 2-way ANOVA.

Figure 2

Effects of GRK2/3 knockdown on G_s-coupled receptor-mediated cAMP accumulation in ASM. (A) Immunoblot analysis of effects of transfection of control (CON) or GRK2/3 siRNA on GRK2/3 expression in ASM cultures. (B) Cultures transfected as in (A) were plated in 24 well plates and stimulated with 1 mM RO-20-1724 and 1 μM ISO (A), 1 μM PGE₂ (B), or 50 μM NECA (C) for 0–10 min, or 100 μM FSK for 10 min. cAMP was isolated and quantified by RIA as described in Experimental Procedures. Data represent the mean ± SE values from 6 experiments, using 6 sets of cultures each derived from a unique donor. *p < 0.05, 2-way ANOVA.

Figure 3

Characterization of EP receptor mRNA expression and EPR-mediated Ca²⁺ mobilization in ASM cells. (A) Reverse transcriptase PCR analysis of EP receptor subtype mRNA expression in ASM cultures. mRNA of each of the EPR subtypes as well as GAPDH was amplified for the indicated number of cycles, and products were purified and electrophoresed in a 2% agarose, EtBR-containing gel. (B–D) Primary cultures of ASM were plated on coverslips, loaded with FURA-2 AM, pretreated 5 min with vehicle, and then stimulated as indicated. Cells were subsequently washed with HBSS, then pretreated with vehicle or 1 μM SC19220, and stimulated again, with agonist-stimulated Ca²⁺ mobilization assessed as described in Experimental Procedures. B and C are representative traces depicting Ca²⁺ mobilization stimulated by 1 μM PGE₂ (B) or 1 μM sulprostone (C) in the presence/absence of 1 μM SC19220; (D) is the graphical representation of mean ± SE values of 3 (PGE₂) or 4 (sulprostone) independent experiments.

Figure 4

EPR-mediated p42/p44 phosphorylation and cAMP accumulation. (A–D) ASM cells plated in 12 well plates were pretreated for 12 h with vehicle or 50 ng/mL PTX then stimulated for 0–3 h with 1 μM PGE₂, 1 μM sulprostone, or 10 μM butaprost. Cell lysates were harvested, and phospho-p42/p44, VASP, and β-actin levels were assessed by immunoblotting. Data depicted are representative of experiments performed using 4 separate cultures. (E) ASM cells plated in 24 well plates were pretreated for 12 h with vehicle or 50 ng/mL PTX then stimulated for 10 min with vehicle, 1 μM PGE₂, or 100 μM FSK. (F) ASM cells were transfected with either scrambled (CON) or EP2R siRNA oligonucleotides, plated in 24 well plates, and stimulated 96 h later with vehicle, 1 μM PGE₂, 10 μM butaprost, or100 μM FSK. Knockdown of EP2R mRNA (69 + 15%) was assessed by real-time PCR using cells plated in parallel in 6 well plates. Neither PTX treatment nor EP2R siRNA affected FSK-stimulated cAMP accumulation, and data were normalized to this value determined for each group. Data represent the mean ± SE values from 6 (E) and 4 (F) experiments. *p < 0.05, Student’s t-test.

Figure 5

Effects of GRK inhibition on selective EP2 R signaling. Time-dependent, butaprost-stimulated cAMP accumulation in GFP- vs GRK2CT-GFP (A)- and GFP- vs GRK2K220R-GFP (B)-expressing cells, and in cells transfected with control siRNA vs GRK2/3 siRNA (C), assessed as per experiments described in Figure 1. (C) Data represent the mean ± SE values from 6 (A), 5 (C), or 4 (B) paired observations.

Figure 6

Effects of GRK mutant expression on ISO- and PGE₂-mediated inhibition of ASM growth. ASM cultures expressing GFP, CT-GFP, or KR-GFP were grown to near-confluence in 24 well plates then growth arrested in 0.1% BSA serum-free media for 24 h. Cell were then pretreated 10 min with vehicle, 1 μM ISO, 1 μM PGE₂, or 50 μM NECA, then stimulated with 10 nM EGF. [³H] thymidine incorporation was subsequently assessed as described in Experimental Procedures. Data represent the mean ± SE values from 6 paired observations from cultures of GFP vs CT-GFP cells and 4 paired observations from cultures of GFP vs KR-GFP cells.