Journal article
Phe303 in TMVI of the α1B-Adrenergic Receptor Is a Key Residue Coupling TM Helical Movements to G-protein Activation
Biochemistry (Easton), Vol.41(2), pp.588-596
01/15/2002
DOI: 10.1021/bi011868k
Abstract
We showed previously that Phe303 in transmembrane segment (TM) VI of the α1B-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 Phe303 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 Phe303 as a key residue coupling TM helical movements to G-protein-activation.
Details
- Title: Subtitle
- Phe303 in TMVI of the α1B-Adrenergic Receptor Is a Key Residue Coupling TM Helical Movements to G-protein Activation
- Creators
- Songhai ChenFang LinMing XuRobert M Graham
- Resource Type
- Journal article
- Publication Details
- Biochemistry (Easton), Vol.41(2), pp.588-596
- DOI
- 10.1021/bi011868k
- ISSN
- 0006-2960
- eISSN
- 1520-4995
- Publisher
- American Chemical Society
- Language
- English
- Date published
- 01/15/2002
- Academic Unit
- Anatomy and Cell Biology; Iowa Neuroscience Institute; Craniofacial Anomalies Research Center; Fraternal Order of Eagles Diabetes Research Center; Neuroscience and Pharmacology; Internal Medicine
- Record Identifier
- 9984070386302771
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