Functional reconstitution of the alpha 2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles.
We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the alpha 2-adrenergic receptor (alpha 2AR) which has been partially purified (approximately 500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte Ni and Ns, bovine retinal transducin and the recently discovered bovine brain No. Addition of the physiologic ligand, epinephrine, to vesicles containing the alpha 2AR and Ni results in stimulation of the GTPase activity in Ni. This stimulation of GTPase activity by epinephrine is prevented in the presence of the alpha-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the alpha 2AR and Ni has been established. The maximum turnover number for the alpha 2AR-mediated epinephrine-stimulated GTPase activity in Ni is similar to the maximal turnover numbers obtained for the beta-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in Ns and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of Pi released per min per mol of nucleotide regulatory protein). Functional similarities between the alpha 2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of Ni and No while being much less effective in promoting the activation of Ns. However, there are differences between the alpha 2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the alpha 2AR is much less effective in promoting this activation (i.e. approximately 20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin approximately equal to Ni approximately equal to No much greater than Ns; while for alpha 2AR, Ni approximately equal to No greater than transducin greater than or equal to Ns.
D000262:Adenylyl Cyclases / Q000032:analysis; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000032:analysis; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000502:physiology; D006801:Humans; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000032:analysis / Q000502:physiology; D009638:Norepinephrine / Q000494:pharmacology; D010743:Phospholipids; D011942:Receptors, Adrenergic, alpha / Q000032:analysis* / Q000502:physiology; D012243:Rhodopsin / Q000032:analysis / Q000502:physiology; D015293:Transducin; D014316:Tritium; D015016:Yohimbine / Q000378:metabolism
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D011942:Receptors, Adrenergic, alpha; D014316:Tritium; D015016:Yohimbine; D012243:Rhodopsin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015293:Transducin; D000262:Adenylyl Cyclases; D009638:Norepinephrine