Role of G-protein availability in differential signaling by alpha 2-adrenoceptors

The impact of G-protein expression on the coupling specificity of the human alpha (2B)-adrenergic receptor (alpha (2B)-AR) was studied in Sf9 cells. T he alpha (2B)-AR was shown to activate both coexpressed G(s)- and G(l)-prot eins in a [S-35]GTP gammaS binding assay. Noradrenaline and the synthetic a gonist UK14,304 were equally potent and efficacious in stimulating Gi activ ation. At the effector level (adenylyl cyclase), both ligands stimulated cA MP production. In the presence of forskolin, the effects of the agonists we re more complex. Noradrenaline stimulated cAMP production, while UK14,304 s howed a biphasic concentration-response curve with inhibition of stimulated cAMP production at low agonist concentrations and further stimulation at h igh agonist concentrations. G(s) coexpression. caused a monophasic stimulat ory response with both ligands. Coexpression with G(i) resulted in a biphas ic concentration-response curve for noradrenaline and a monophasic inhibiti on with UK14,304. Experiments with a panel of agonists demonstrated that th e more efficacious an agonist is in stimulating cAMP production, the weaker is its ability to couple to inhibition of cAMP accumulation via exogenous G(i). To be able to explain the mechanistic consequences of dual G-protein coupling described above, we developed a mathematical model based on the hy pothesis that an agonist induces different conformations of the receptor ha ving different affinity for different G-proteins. The model reproduced the profiles seen in the concentration-response curves with G(s) and G(i) coexp ression. The model predicts that the affinity of the receptor conformation for G-proteins as well as the availability of G-proteins will determine the ultimate response of the receptor. (C) 2001 Elsevier Science Inc. All righ ts reserved.