Simultaneous coupling of α₂-adrenergic receptors to two G-proteins with opposing effects: Subtype-selective coupling of α₂C10, α₂C4, and α₂C2 adrenergic receptors to G(i) and G(s)

Coupling of the three α₂-adrenergic receptor (α₂AR) subtypes to G(i) and G(s) was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the α₂AR agonist UK-14304, α₂C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, α₂C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of α₂C10 to both G(i) and G(s). To isolate functional G(s) and G(i) coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only α₂C10-mediated inhibition (~50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only α₂C10-mediated stimulation (~60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl- terminal portion of G(5α) blocked this functional α₂AR·G(s) coupling to the same extent as that found for β₂AR·G(s) coupling. In addition to functional G(s) coupling, we also verified direct, agonist-dependent, physical coupling of α₂AR to G(sα). In agonist-treated membranes, an agonist-receptor-G(sα) complex was immunoprecipitated with a specific α₂C10 antibody, and the G(s) component identified by both western blots using G(sα) antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the α₂AR subtypes, we investigated whether G-protein coupling was subtype- selective, using UK-14304 and cells with the same α₂AR expression levels (~5 pmol/mg). Coupling to G(i) was equivalent for α₂C10, α₂C4, and α₂C2: 53.4 ± 8.8% versus 54.9 ± 1.0% versus 47.6 ± 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to G(s) were found between the three α₂AR subtypes: stimulation of adenylyl cyclase was 57.9 ± 6.3% versus 30.7 ± 1.1% versus 21.8 ± 1.7% for α₂C10, α₂C4, and α₂C2, respectively. Thus, α₂AR have the potential to couple physically and functionally to both G(i) and G(s); for G(i) coupling we found a rank order of α₂C10 = α₂C4 = α₂C2, while for G(s) coupling, α₂C10 > α₂C4 > α₂C2.