Measurement of agonist-induced guanine nucleotide turnover by the G-protein Gi1alpha when constrained within an alpha2A-adrenoceptor-Gi1alpha fusion protein.
A fusion protein was generated between the porcine alpha2A-adrenoceptor and a pertussis-toxin-insensitive (Cys351-->Gly) variant of the alpha subunit of Gi1alpha by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the alpha2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro- 1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. Vmax estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0+/-0.2 min-1 for Gi1alpha when the alpha2A-adrenoceptor was maximally stimulated by UK14304 with a Km for GTP of 0.37+/-0.07 microM. Co-expression of excess beta1gamma2 along with the alpha2A-adrenoceptor-Gi1alpha fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1+/-0.2-fold) without alteration in agonist EC50. These studies demonstrate the functionality of the fusion construct, its capacity to interact with betagamma complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.
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Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Carr
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Milligan
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D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000068438:Brimonidine Tartrate; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013552:Swine; D014162:Transfection
D000316:Adrenergic alpha-Agonists; D017931:DNA Primers; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D000068438:Brimonidine Tartrate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go