Enzyme complementation between two deletion mutants of ß-galactosidase (ß-gal; and ) can be used to study protein-protein interactions (Mohler et al., 1996; Graham et al., 2001). Following ligand stimulation, ß-arrestin 2 is recruited to the ß2-adrenoceptor (ß₂AR) (Barak et al., 1997). The two inactive ß-gal mutants (a and ) have been attached to the ß2AR and ß-arrestin 2 to follow the interaction between these two proteins (Yan et al., 2002). Here we have used ß-gal comp-lementation to assess the pharmacology of this interaction.

A C2C12 cell line stably expressing both the ß2-AR-ß-gal- and ß-arrestin-ß-gal- fusion (Yan et al., 2002) was used in the present study. Experiments were performed in serum free DMEM media. Antagonists were added 30 min prior to the addition of agonists at 37ºC. Incubations were then continued for 1-hour at 37ºC with agonist and ß-gal activity was then measured using the Gal-Screen® system according to the manufacturer instructions (Applied Biosystems).

Isoprenaline, adrenaline and noradrenaline stimulated an inc-rease in b-gal activity (ß₂-AR/ß-arrestin 2 complementation) that was 2.13 ± 0.10 (pEC₅₀ = 5.93 ± 0.08; n=8), 2.33 ± 0.06 (pEC₅₀ = 5.74 ± 0.13; n=9) and 2.20 ± 0.07 (pEC₅₀ = 5.46 ± 0.04; n=8) fold over basal levels respectively. In contrast, salbutamol and terbutaline produced maximal responses that were 39.69 ± 2.21 (pEC₅₀ = 6.32 ± 0.10;n=9) and 63.9 ± 6.37 (pEC₅₀ = 5.55 ± 0.07; n=8) percent of the maximal response to isoprenaline (0.1mM) measured in the same experiment.

The ß-adrenoreceptor antagonist propranolol produced parallel shifts in the concentration-response curve to isoprenaline consistent with competitive antagonism (pA₂ = 8.97 ± 0.14; n=6). Timolol and the ß₂-selective antagonist ICI 118551 shifted the isoprenaline concentration-response curve to the right, but also reduced the maximal responses. This was consistent with a hemi-equilibrium between slowly dissociating antagonists and a response with a low receptor reserve (Christopoulous et al., 1999; Baker et al., 2003; Hopkinson et al., 2000). Estimation of pA₂ values according to the method of Christopoulous et al., (1999) produced pA₂ values of 8.70±0.16 (n=5) and 9.66±0.16 (n=6) for ICI 118551 and timolol respectively.

These data indicate that assays based on ß-gal complementation provide a sensitive and quantitative approach for the study of the molecular pharmacology of agonist-stimulated interactions between the ß2-AR and ß-arrestin 2.

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AAC holds a BBSRC research studentship. We thank Applied Biosystems for provision of the InteraX^TM system ß2-AR-ß-gal- and ß-arrestin-ß-gal- cell line.