Detection of receptor ligands by monitoring selective stabilization of aRenillaluciferase‐tagged, constitutively active mutant, G‐protein‐coupled receptor

Abstract

1 The wild-type b 2 -adrenoceptor and a constitutively active mutant of this receptor were Cterminally tagged with luciferase from the sea pansy Renilla reniformis. 2 C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant b 2 -adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. 3 Treatment of cells expressing constitutively active mutant b 2 -adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. 4 The pEC 50 for ligand-induced luciferase upregulation and ligand a nity to bind the receptor were highly correlated. 5 Similar upregulation could be observed following sustained treatment with agonist ligands. 6 These e ects were only observed at a constitutively active mutant of the b 2 -adrenoceptor. Coexpression of the wild-type b 2 -adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. 7 Co-expression of the constitutively active mutant b 2 -adrenoceptor-Renilla luciferase and an equivalent mutant of the a 1b -adrenoceptor C-terminally tagged with green¯uorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. 8 This approach o ers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. 9 As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors.

Generation and Analysis of Constitutively Active and Physically Destabilized Mutants of the Human β1-Adrenoceptor

“…7B). Furthermore, as we noted previously for the CAM␤ 2 -adrenoceptor (Ramsay et al, 2001), ligand-induced up-regulation was not restricted to antagonist/inverse agonist ligands. Up-regulation of both the CAMK␤ 1 -adrenoceptor-G s ␣ and the Arg 156 Ala␤ 1 -adrenoceptor-G s ␣ constructs was produced to similar extents by sustained exposure to isoprenaline (Fig.…”

“…The basis for the differences between these two ␤-adrenoceptors is unclear but may relate to their differential targeting in cells because it seems that the ␤ 2 -adrenoceptor is more selectively targeted to detergent-insensitive membrane domains that also allow marked concentration of heterotrimeric G proteins (Rybin et al, 2000). Although constitutively active mutants of the ␤ 2 -adrenoceptor that have a markedly higher capacity for signaling in the absence of agonist have been widely studied (Samama et al, 1993;MacEwan and Milligan, 1996;Gether et al, 1997;Ramsay et al, 2001), much less information is available on the ␤ 1 -adrenoceptor. In many regards, this is surprising given the quantitative importance of this receptor in cardiac function.…”

“…There was, however, ligand specificity for up-regulation because neither of these constructs was up-regulated by exposure to the markedly ␤ 2 -adrenoceptor-selective blocker ICI118551 (10M) (Fig. 7C), which does cause up-regulation of the CAM␤ 2 -adrenoceptor (McLean et al, 1999;Ramsay et al, 2001). …”

“…Consequently, a CAM␤ 2 -adrenoceptor-Renilla reniformis luciferase construct (Ramsay et al, 2001) was digested with XhoI and XbaI to eliminate R. reniformis luciferase to ligate the digested G s ␣ fragment from the PCR in-frame with the CAM␤ 2 -adrenoceptor.…”

“…An interesting feature of this receptor is that it is physically destabilized compared with the wild-type ␤ 2 -adrenoceptor (Gether et al, 1997), and this can be suppressed by the binding of ligands. Thus in the face of ongoing synthesis, the addition of ␤ 2 -adrenoceptor ligands to cells expressing the CAM␤ 2 -adrenoceptor results in upregulation of the polypeptide (Pei et al, 1994;MacEwan and Milligan, 1996;McLean et al, 1999;Ramsay et al, 2001). This has been demonstrated to provide a useful means to identify, without any a prioiri knowledge, ligands that interact with the receptor .…”