Enhanced stability of wild-type and constitutively active α 2A -adrenoceptors by ligands with agonist, silent and inverse agonist properties

Abstract

The hypothesis that prolonged treatment of a constitutively active receptor with inverse agonists may lead to increased receptor density was tested for the alpha(2)-adrenoceptor (AR) inverse agonist (+)-RX 811059 at both the wild-type (WT) and Thr(373)Lys alpha(2A) ARs in CHO-K1 cells by monitoring [(3)H]RX 821002 and [(35)S]GTPgammaS binding responses. One-hundred micromolar KCl instead of NaCl in the [(35)S]GTPgammaS membrane binding assay favoured the detection of a high-magnitude constitutive alpha(2A) AR activity. Under this condition, (+)-RX 811059 was an inverse agonist [ E(max) (% vs. basal): Thr(373)Lys alpha(2A) AR (-52+/-2) > WT alpha(2A) AR (-31+/-6)] while atipamezole was a silent neutral antagonist for both WT and Thr(373)Lys alpha(2A) ARs. The B(max) value of [(3)H]RX 821002 binding sites to membranes of transfected CHO-K1 cells was <90% for the Thr(373)Lys alpha(2A) AR compared with the WT alpha(2A) AR (9.1+/-1.4 pmol/mg protein); K(d) values were similar (1.16+/-0.19 nM and 1.51+/-0.15 nM, respectively). Forty-eight-hours' pre-treatment of cells with either 0.1 microM (+)-RX 811059, 1 microM atipamezole or 1 microM of the efficacious agonist d-medetomidine increased the amount of [(3)H]RX 821002 binding sites of both WT (52%-59%) and mutant (306%-447%) Thr(373)Lys alpha(2A) ARs. The same alpha(2) AR ligands also prevented the loss of [(3)H]RX 821002 binding sites as induced by incubation of transfected CHO-K1 cellular membranes at 37 degrees C for 4 h (WT alpha(2A) AR) and 2 h (Thr(373)Lys alpha(2A) AR); 0.1 microM (+)-RX 811059 and 1 microM atipamezole caused an increase compared with the control amount of [(3)H]RX 821002 binding sites to the Thr(373)Lys alpha(2A) AR by 73% and 50%, respectively. In conclusion, no relationship was found between inverse agonism and alpha(2A) AR up-regulation. It is suggested that this is due to structural stabilisation of the alpha(2A) AR, irrespective of the nature of the ligand.

A Single Point Mutation (N514Y) in the Human M3Muscarinic Acetylcholine Receptor Reveals Differences in the Properties of Antagonists: Evidence for Differential Inverse Agonism

“…The N514Y M 3 (but not the WT-M 3 ) receptor expression level could be significantly increased by chronic (24 h) incubation in the presence of an inverse agonist. Ligand-mediated receptor up-regulation has been reported previously for constitutively active mutant receptors (Smit et al, 1996;Stevens et al, 2000;Li et al, 2001;Pauwels and Tardif, 2002), although the precise molecular mechanisms involved are still debated (Parnot et al, 2002). For some CAM-GPCR, the degree of up-regulation is positively correlated with both the degree of constitutive activity in the system and the ability of a ligand to decrease basal activity.…”

“…If a CAM-GPCR functions in a similar way to its cognate agonist-induced WT counterpart, it would follow that only inverse agonists would cause up-regulation by virtue of stabilizing the inactive conformation (Parnot et al, 2002). However, other studies (Gether et al, 1997;Pauwels and Tardif, 2002) have shown that the creation of a CAM can diminish stabilizing constraints within the receptor, leading to an inherently unstable receptor that is more susceptible to destabilization and/or proteolytic degradation (Stevens et al, 2000). In this case, the expression level of the mutant is increased by any ligand [(inverse) agonist/antagonist] regardless of its efficacy.…”

Inverse Agonist Activity of Selected Ligands of the Cysteinyl-Leukotriene Receptor 1

“…‫,ءء‬ p Ͻ 0.01; ‫,ء‬ p Ͻ 0.05. under our conditions because our treatment with inverse agonists was relatively short. The increased cell surface expression could be due to the stabilization of the receptor, either intracellularly to increase receptor maturation and expression (Petaja-Repo et al, 2002) or by stabilizing the receptor on the cell surface (Wilson and Limbird, 2000;Pauwels and Tardif, 2002). Because there is no indication that the antagonists we used in our experiments are cell-permeable, we would favor the hypothesis that the inverse agonists possibly stabilize constitutively active receptor on the cell surface and possibly interfere with the constitutive internalization process.…”

Role of the second and third extracellular loops of the histamine H4 receptor in receptor activation

“…Some structurally labile receptors could be stabilized by expression in the presence of ligands (Gether et al 1997;Pauwels and Tardif 2002;Roth et al 2008). Also, the agonist HA and the inverse agonist THIO were able to increase the B max value of [ 3 H]HA binding to wild-type hH 4 R ).…”