The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human α2A-adrenoceptor to adenylyl cyclase

1 The effects of substitution of the Ser(200) and Ser(204) residues with alanine on the signalling properties of the cloned human alpha(2A)-adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2 The Ser-->Ala(200) or the Ser-->Ala(204) mutant forms of the alpha(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. 3 After incubation with pertussis toxin, (+/-)-meta-octopamine (100 mu M and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser-->Ala(204) mutant form of the alpha(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. 4 The results emphasise the importance of the Ser(200) and Ser(204) residues of the alpha(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. 5 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.