Premium
The effect of site‐directed mutagenesis of two transmembrane serine residues on agonist‐specific coupling of a cloned human α 2A ‐adrenoceptor to adenylyl cyclase
Author(s) -
Rudling Jane E,
Kennedy Karen,
Evans Peter D
Publication year - 1999
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0702614
Subject(s) - adenylyl cyclase , agonist , chinese hamster ovary cell , receptor , biology , octopamine (neurotransmitter) , pertussis toxin , stimulation , forskolin , mutant , g protein , endocrinology , biochemistry , medicine , gene , serotonin
The effects of substitution of the Ser 200 and Ser 204 residues with alanine on the signalling properties of the cloned human α 2A ‐adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. The Ser→Ala 200 or the Ser→Ala 204 mutant forms of the α 2A ‐adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (±)‐ para ‐octopamine and (±)‐ meta ‐octopamine, respectively, than cells expressing the wild‐type receptor. Binding studies indicate that the effects are not due to an increased agonist affinity for the mutant receptors and that they are likely to be due to agonist‐mediated conformational changes in receptor structure. After incubation with pertussis toxin, (±)‐ meta ‐octopamine (100 μ M and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser→Ala 204 mutant form of the α 2A ‐adrenoceptor but showed no stimulation in cells expressing the Ser→Ala 200 mutant receptor. Under these conditions (±)‐ para ‐octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild‐type receptor. The results emphasise the importance of the Ser 200 and Ser 204 residues of the α 2A ‐adrenoceptor in exerting an inhibitory influence on the ability of (±)‐ para ‐octopamine and (±)‐ meta ‐octopamine respectively, to induce a receptor‐agonist conformation capable of inhibiting forskolin‐stimulation of cyclic AMP levels. It is clear that Ser 204 also prevents meta ‐octopamine from generating a receptor‐agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist‐specific coupling of this receptor to different second messenger systems.British Journal of Pharmacology (1999) 127 , 877–886; doi: 10.1038/sj.bjp.0702614