Translocation of arrestin induced by human A(3) adenosine receptor ligands in an engineered cell line: comparison with G protein-dependent pathways - PubMed (original) (raw)

Comparative Study

Translocation of arrestin induced by human A(3) adenosine receptor ligands in an engineered cell line: comparison with G protein-dependent pathways

Zhan-Guo Gao et al. Pharmacol Res. 2008 Apr.

Abstract

Structurally diverse ligands were studied in A(3) adenosine receptor (AR)-mediated beta-arrestin translocation in engineered CHO cells. The agonist potency and efficacy were similar, although not identical, to their G protein signaling. However, differences have also been found. MRS542, MRS1760, and other adenosine derivatives, A(3)AR antagonists in cyclic AMP assays, were partial agonists in beta-arrestin translocation, indicating possible biased agonism. The xanthine 7-riboside DBXRM, a full agonist, was only partially efficacious in beta-arrestin translocation. DBXRM was shown to induce a lesser extent of desensitization compared with IB-MECA. In kinetic studies, MRS3558, a potent and selective A(3)AR agonist, induced beta-arrestin translocation significantly faster than IB-MECA and Cl-IB-MECA. Non-nucleoside antagonists showed similar inhibitory potencies as previously reported. PTX pretreatment completely abolished ERK1/2 activation, but not arrestin translocation. Thus, lead candidates for biased agonists at the A(3)AR have been identified with this arrestin-translocation assay, which promises to be an effective tool for ligand screening.

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Figures

Figure 1

Figure 1

Concentration-response curves in the β-arrestin translocation assay for known synthetic AR pharmalogical probes (CPA 2, CGS21680 3, NECA 1, and Cl-IB-MECA 4) and the native nucleotide ligands (ADP 5, ATP 6) of various P2Y receptor subtypes. Results are expressed mean ± SEM from three separate experiments performed in duplicate.

Figure 2

Figure 2

Selected structures of ligands that were tested in the β-arrestin translocation assay using the CHO-ADORA3 Pathhunter cell line. A. Adenosine derivatives containing the natural riboside-like 5′-CH2OH group. B. NECA-like 5′-CONH-alkyl derivatives and ring-constrained (N)-methanocarba derivatives. C. Diverse structures that were shown previously to interact with the human A3AR.

Figure 2

Figure 2

Selected structures of ligands that were tested in the β-arrestin translocation assay using the CHO-ADORA3 Pathhunter cell line. A. Adenosine derivatives containing the natural riboside-like 5′-CH2OH group. B. NECA-like 5′-CONH-alkyl derivatives and ring-constrained (N)-methanocarba derivatives. C. Diverse structures that were shown previously to interact with the human A3AR.

Figure 2

Figure 2

Selected structures of ligands that were tested in the β-arrestin translocation assay using the CHO-ADORA3 Pathhunter cell line. A. Adenosine derivatives containing the natural riboside-like 5′-CH2OH group. B. NECA-like 5′-CONH-alkyl derivatives and ring-constrained (N)-methanocarba derivatives. C. Diverse structures that were shown previously to interact with the human A3AR.

Figure 3

Figure 3

Concentration-response curves for structurally diverse A3AR modulators in the β-arrestin translocation assay in the CHO-ADORA3 Pathhunter cell line. Results are expressed mean ± SEM from three separate experiments performed in duplicate.

Figure 4

Figure 4

Kinetic analysis of four A3AR agonists in the β-arrestin translocation assay in the CHO-ADORA3 Pathhunter cell line. Results are expressed mean ± SEM from three separate experiments performed in duplicate.

Figure 5

Figure 5

Two nonnucleoside A3AR antagonists, the triazoloquinazoline derivative MRS1220 114 (A,C) and the pyridine derivative MRS1523 108 (B,D), characterized pharmacologically in the β-arrestin translocation assay in the CHO-ADORA3 Pathhunter cell line. Concentration response curves for the agonist Cl-IB-MECA 4 (A,B) and Schild analysis [19] of the antagonism was carried out (C,D). The Schild slopes were calculated to be 0.73 and 1.40 for C and D, respectively.

Figure 6

Figure 6

Effect of PTX pretreatment (200 ng/ml) on β-arrestin translocation induced by IB-MECA. PTX was incubated with cells for 24 h before the measurement. Results are expressed as mean ± SEM from three experiments.

Figure 7

Figure 7

Studies of A3AR-mediated phosphorylation of ERK1/2 in the CHO-ADORA3 Pathhunter cell line. (A) Effects of pertussis toxin (PTX) pretreatment (24 h) on MRS3558 (1 μM)-induced activation of ERK1/2, and (B) pretreatment (24 h) with nucleosides IB-MECA and DBXRM desensitize the activation of ERK1/2. In each experiment, the maximal level of ERK1/2 activation was set to 100% and that observed in unstimulated to 0%. Results are expressed as mean ± SEM (n=3).

Figure 8

Figure 8

Comparison of the potency and efficacy of Cl-IB-MECA 4 at various A3AR-mediated signaling pathways. NECA was used a full agonist in all assays. The efficacy of NECA in each assay was expressed as 100%. Results are expressed as mean ± SEM of 3–5 separate experiments performed in duplicate or triplicate. The potency and Emax values from arrestin and cyclic AMP assays were listed in Table 1. The EC50 (nM) and Emax (compared with NECA) values of Cl-IB-MECA in the calcium assay are 47 ± 9 nM and 57± 6%, respectively.

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