CONTRIBUTION OF LIGAND STRUCTURE TO ACTIVATION OF ALPHA(2)-ADRENERGICRECEPTOR SUBTYPE COUPLING TO G(S)

Recently, we have demonstrated that alpha(2)-adrenergic receptors (alp ha(2)AR) functionally couple not only to G(i) but also to G(s). This a lpha(2)AR-G(s) coupling was subtype selective, in that the degree of a lpha(2)AR-G(s) (but not -G(i)) coupling was different between alpha(2) AR subtypes. It is not known whether the determinants of this subtype selectivity are found within the ligand-binding region of the receptor or within the intracellular G protein-coupling domains of the individ ual subtypes. We therefore expressed the three cloned human alpha(2)AR (alpha(2)C10, alpha(2)C4, and alpha(2)C2) in Chinese hamster ovary ce lls and studied the contribution of the ligand-binding domain to funct ional G(i) versus G(s) coupling, by determining the ability of various agonists (catecholamines, imidazolines, and azepines) to elicit alpha (2)AR-mediated inhibition and stimulation of adenylyl cyclase activity . Isolation of G(i) and G(s) responses was accomplished by incubating cells with cholera or pertussis toxin, respectively. Although each com pound was found to be a full agonist for alpha(2)AR-G(i) coupling, the efficacy of these agonists to elicit alpha(2)AR-G(s) coupling was mar kedly different, not only among drugs but also among the three alpha(2 )AR subtypes. The most notable differences occurred with the imidazoli ne agonists. Specifically, oxymetazoline stimulated adenylyl cyclase a ctivity 210 +/- 17% for alpha(2)C2 and 22 +/- 2.6% for alpha(2)C10 and displayed no stimulation for alpha(2)C4. UK-14304 stimulated adenylyl cyclase activity 240 +/- 16% for alpha(2)C10, 160 +/- 14% for alpha(2 )C4, and 86 +/- 9% for alpha(2)C2. Overall, the rank order of efficacy of these agonists to elicit stimulation of adenylyl cyclase activity by alpha(2)C10 was epinephrine = norephinephrine = UK-14304 > BHT-933 > BHT-920 > oxymetazoline. For alpha(2)C4 the rank was epinephrine = n orepinephrine = UK-14304, with oxymetazoline, BHT-920, and BHT-933 not eliciting any stimulation. For alpha(2)C2 the rank was epinephrine = norepinephrine > oxymetazoline > UK-14304 = BHT-920 > BHT-933. Thus, t he coupling of alpha(2)AR subtypes to G(s) occurs with endogenous cate cholamines as well as multiple synthetic agonists, and the degree of G (s) coupling is highly dependent on the structure of the agonist. Also , compounds that act as full agonists for G(i) coupling are not necess arily full agonists for G(s) coupling.