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

Publisher Website
Google Scholar
Abstract
1. The effects of substitution of the Ser200 and Ser204 residues with alanine on the signalling properties of the cloned human alpha2A-adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2. The Ser-->Ala200 or the Ser-->Ala204 mutant forms of the alpha2A-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 microM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser-->Ala204 mutant form of the alpha2A-adrenoceptor but showed no stimulation in cells expressing the Ser-->Ala200 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 Ser200 and Ser204 residues of the alpha2A-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 Ser204 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.