Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation.
alpha(1)-adrenergic receptors (alpha(1)-ARs) are members of the G-protein-coupled receptor (GPCR) superfamily and activate inositol phosphate (IP) turnover. We show that glycine and asparagine mutations of Phe303 in transmembrane segment VI (TMVI) of the alpha(1B)-AR, a highly conserved residue in GPCRs, although increasing agonist affinity, abolish agonist-activated IP signalling. Co-expression of the Phe303 mutants also inhibited (-)epinephrine-stimulated IP signalling by wild-type alpha(1B)-AR and other G(q)-coupled receptors, as well as IP signalling mediated by AlF(4)(-) stimulation of both wild-type G(q alpha) and a constitutively active mutant. The inability of the Phe303 mutants to signal is due to induction of a receptor conformation that dissociates G-protein binding from activation. As a result, the Phe303 mutants sequester G(q alpha) and stoichiometrically inhibit Gq signalling in a dominant-negative manner. We further show that both the enhanced basal and agonist-stimulated IP-signalling activity of the constitutively active alpha(1B)-AR mutants, C128F and A293E, are inhibited in the double mutants, C128F/F303G and A293E/F303G. Phe303, therefore, appears to be critically involved in coupling TMVI alpha-helical movement, a key step in receptor activation, to activation of the cognate G-protein.
Chen
S
S
Molecular Cardiology Unit, Victor Chang Cardiac Research Institute, St Vincent's Hospital, Sydney 2010, Australia.
Lin
F
F
Xu
M
M
Hwa
J
J
Graham
R M
RM
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D019556:COS Cells; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D005799:Genes, Dominant; D005998:Glycine / Q000737:chemistry; D006868:Hydrolysis; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D010716:Phosphatidylinositols / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D018076:DNA, Complementary; D007295:Inositol Phosphates; D008024:Ligands; D010716:Phosphatidylinositols; D018340:Receptors, Adrenergic, alpha-1; D010649:Phenylalanine; D001216:Asparagine; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D005998:Glycine; D004837:Epinephrine