Phe(303) in TMVI of the alpha(1B)-adrenergic receptor is a key residue coupling TM helical movements to G-protein activation.
We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor, a highly conserved residue in G-protein-coupled receptors (GPCRs), is critically involved in receptor-activation and G-protein-coupling [Chen, S. H., Lin, F., Xu, M., Hwa, J., and Graham, R. M. (2000) EMBO J. 19, 4265-4271]. Here, we show that saturation mutagenesis of Phe(303) results in a series of mutants with different levels of constitutive activity for inositol phosphate (IP) signaling. Mutants F303G and F303N showed neither basal nor agonist-stimulated IP turnover, whereas F303A displayed increased basal activity but an attenuated maximal response to (-)-epinephrine-stimulation. F303L, on the other hand, showed all features of a typical constitutively active GPCR with markedly increased basal activity and increased potency and efficacy of agonist-stimulated IP signaling. All mutants displayed higher agonist-binding affinities than the wild-type receptor, and by thermal stability studies, those able to signal showed increased susceptibility to inactivation with an order of sensitivity (F303L > F303A > WT) directly related to their degree of constitutive activity. Using the substituted cysteine accessibility method (SCAM) and equilibrium binding studies, we further show that the F303A and F303L mutants result in TM helical movements that differ in accordance with their degree of constitutive activity. These findings, therefore, confirm and extend our previous data implicating Phe(303) as a key residue coupling TM helical movements to G-protein-activation.
Chen
Songhai
S
Molecular Cardiology Unit, Victor Chang Cardiac Research Institute, St. Vincent's Hospital, School of Medicine, Sydney, NSW 2010, Australia.
Lin
Fang
F
Xu
Ming
M
Graham
Robert M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry*; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013696:Temperature; D013997:Time Factors; D014162:Transfection
C547716:ADRA1B protein, human; D008024:Ligands; D010716:Phosphatidylinositols; D018340:Receptors, Adrenergic, alpha-1; D010649:Phenylalanine; D019204:GTP-Binding Proteins; D003545:Cysteine; D004837:Epinephrine