The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human alpha(2A)-adrenoceptor toadenylyl cyclase

1 The effects of substitution of the Ser(200) and Ser(204) residues with al anine on the signalling properties of the cloned human alpha(2A)-adrenocept or, stably expressed in Chinese hamster ovary (CHO) cell lines, have been i nvestigated using noradrenaline and the structural isomers of octopamine. 2 The Ser-->Ala(200) or the Ser-->Ala(204) mutant forms of the alpha(2A)-ad renoceptor, when expressed in cells in the absence of pertussis toxin pretr eatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (+/-)-para-octopamine and (+/-)-meta-octopamine, respect ively, than cells expressing the wild-type receptor. Binding studies indica te that the effects are not due to an increased agonist affinity for the mu tant receptors and that they are likely to be due to agonist-mediated confo rmational changes in receptor structure. 3 After incubation with pertussis toxin, (+/-)-meta-octopamine (100 mu M an d above) produced a stimulation of cyclic AMP levels in cells expressing th e Ser-->Ala(204) mutant form of the alpha(2A)-adrenoceptor but showed no st imulation in cells expressing the Ser-->Ala(200) mutant receptor. Under the se 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) residue s 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-sti mulation 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, emphasi sing the importance of this residue in the agonist-specific coupling of thi s receptor to different second messenger systems.