Search

The Thr149Ala mutation in a putative protein kinase C phosphorylation site of the 5-HT1A receptor’s second intracellular loop has been shown to affect the closing of Ca2+ channels and Ca2+ mobilisation without interfering with the inhibitory cAMP pathway (Mol Pharmacol 52 (1997) 164). Here, the Ca2+ responses for a series of 5-HT1A agonists were compared between the wild-type (wt) and mutant Thr149Ala 5-HT1A receptor as part of a fusion protein containing a Gα15 protein. Neither the mutation nor the fusion process modified the [3H]WAY 100635-based ligand binding profile of the fusion proteins as compared to the wt 5-HT1A receptor protein. Whereas at the wt 5-HT1A receptor, 5-HT induced a Ca2+ response in CHO-K1 cells via endogenous Gi/o proteins, the Ca2+ response to 5-HT at the mutant Thr149Ala 5-HT1A receptor was fully dependent on either the co-expression or the fusion to a recombinant Gα15 protein. Buspirone, flesinoxan and 8-OH-DPAT produced a graded partial response (26 to 62%) at the wt 5-HT1A:Gα15 fusion protein; F 13640, 5-CT and F 14679 behaved as higher-efficacy agonists with maximal Ca2+ responses similar to 5-HT. The maximal Ca2+ responses at the mutant Thr149Ala 5-HT1A:Gα15 fusion protein were significantly attenuated for flesinoxan and 8-OH-DPAT (−45 and −36%, respectively); the response to the other 5-HT agonists was not significantly affected. A similar effect was observed upon treatment with phorbol 12-myristate 13-acetate at the Thr149Ala 5-HT1A:Gα15 fusion protein. In conclusion, the amplitude of the Ca2+ responses induced by partial, but not that to fuller 5-HT1A receptor agonists, is affected by the Thr149Ala mutation of the 5-HT1A:Gα15 fusion protein. [Copyright &y& Elsevier]

Variations in the measurement of ligand's intrinsic activity between receptor subtypes is a common consequence of unequal receptor:G protein density ratios. We have investigated ligand activation at the α2-adrenoceptor (α2-AR) subtypes under defined expression conditions of one receptor molecule for one Gα protein molecule using fusion proteins. Fusion between either a wt α2C AR or a mutant Thr381Lys α2C AR and a chimeric Gαq/i1 protein displayed robust, transient (-)-adrenaline-mediated Ca2+ responses with similar potencies (pEC50: 7.78 and 7.66) and kinetic properties. A comparison of the intrinsic activities of α2 AR agonists found d-medetomidine to be the only compound with an efficacy similar to that of (-)-adrenaline. The Ca2+ responses as mediated by UK 14304, oxymetazoline and clonidine became more potent and efficacious at the Thr381Lys α2C AR, whereas the response as mediated by talipexole displayed a higher potency with an unaltered maximal response. Whereas only small differences in ligand's intrinsic activities between the wt α2A, α2B and α2C AR fusion proteins were observed with most ligands, oxymetazoline was virtually silent at the α2A AR while active as a partial and apparently full agonist at the α2C AR and α2B AR, respectively. The mutant α2 AR subtypes could be differentiated using the apparent positive efficacy of ligands that used to be defined as antagonists. The following rank order of maximal responses was observed for the Thr381Lys α2C AR: idazoxan ≃ SKF 86466 > atipamezole >> dexefaroxan; Thr373Lys α2A AR: SKF 86466 > idazoxan = atipamezole > dexefaroxan; and Thr370Lys α2B AR: atipamezole > idazoxan ≃ dexefaroxan. RX 811059 (10 µM) was the only compound to be completely silent at both the wt and mutant α2 AR subtypes. In conclusion, silent α2 AR ligands are probably rare in these specified α2 AR systems. Most antagonists may actually possess partial agonist properties at the α2 AR subtypes, which are facilitated by the same mutation in the distal portion of their third intracellular loop. [ABSTRACT FROM AUTHOR]

A fusion protein was constructed between the recombinant human α2A-adrenoceptor and a mouse Gα15 protein to measure the efficacy of agonist-induced Ca2+ responses at a receptor:Gα15 protein stoichiometry of 1. Activation of this fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50: 7.28±0.04) increase in the intracellular Ca2+ concentration. The magnitude of the Ca2+ response was related to the amount of the fusion protein and the number of surface α2A-adrenoceptor binding sites as estimated by [3H]RX 821002 binding. Whereas UK 14304 was as efficacious as (-)-adrenaline, the following ligands displayed partial agonist properties [Emax in percentage vs. (-)-adrenaline: d-medetomidine (76±3) > BHT 920 (53±3) > clonidine (39±4) >> oxymetazoline (10±1)]. This ligand activation profile was not affected over a 30-fold range of expression of the fusion protein in contrast to the observed enhancement of the partial agonists' maximal responses by co-expression of the α2A-adrenoceptor with increasing amounts of the Gα15 protein. In conclusion, the fusion protein approach opens perspectives to quantify intrinsic activities of ligands under controlled experimental conditions of a fixed receptor:Gα15 protein ratio of 1. [ABSTRACT FROM AUTHOR]

Fusion proteins were constructed between either a wild-type or mutant Thr370Lys α2B-adrenoceptor (α2B AR) and a mouse Gα15 protein to analyze ligand-receptor interactions at a receptor/Gα15 protein density ratio of 1. Activation of the wild-type α2B AR-Gα15 fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50 = 7.37 ± 0.13) increase in the intracellular Ca2+ concentration, which could be antagonized by RX 811059 (pKB = 7.55 ± 0.15). Whereas d-medetomidine and oxymetazoline were as efficacious agonists as (-)-adrenaline, the following ligands displayed partial agonist properties: BRL 44408 < atipamezole < clonidine < UK 14304 < BHT 920. A comparison with the mutant Thr370Lys α2B AR-Gα15 fusion protein displayed similar Ca2+ kinetics and a ligand-mediated receptor activation profile characterized by higher potencies and greater maximal Ca2+ responses for the ligands being investigated, including the putative antagonists dexefaroxan and idazoxan. RX 811059 and RX 821002 remained silent. Similar conclusions could be made on enhancement of the ligands’ intrinsic activities by coexpression of the mutant Thr370Lys α2B AR with either a Gα15 or GαO Cys351Ile protein. The Thr370Lys α2B AR-Gα protein interactions may modify the tertiary structure of the mutant receptor in such a way that some putative α2 AR antagonists are capable of stabilizing an active receptor conformation, thereby generating positive efficacy. [ABSTRACT FROM AUTHOR]