Reciprocal mutations of highly conserved residues in transmembrane helices 2 and 7 of the alpha(2A)-adrenoceptor restore agonist activation of G(i1)alpha.
Fusion proteins were constructed between the alpha(2A)-adrenoceptor and the alpha-subunit of the G-protein G(i1). Mutation of the highly conserved Asp(79) in transmembrane (TM) helix 2 of the receptor to Asn reduced the capacity of agonists to activate G(i1)alpha by 95% without altering [3H]antagonist or agonist ligand-binding affinity. A reciprocal mutation in TM helix 7 (Asn(422)Asp) was without effect on signalling effectiveness. Combination of these two mutations overcame the effect of the Asp(79)Asp mutation. By examining alterations in this helix 2-helix 7 microdomain, we further demonstrate the utility of receptor-G-protein fusion proteins to quantitate mutational effects on receptor-G-protein interactions and information transfer.
Ward
Richard J
RJ
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Milligan
Graeme
G
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D019556:COS Cells; D017124:Conserved Sequence; D004837:Epinephrine / Q000378:metabolism / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007546:Isoquinolines / Q000378:metabolism; D009154:Mutation; D009287:Naphthyridines / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D007546:Isoquinolines; D009287:Naphthyridines; C104875:RS 79948-197; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001216:Asparagine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D004837:Epinephrine