Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation

S Chen; F Lin; Ming Xu; John Hwa; R M Graham

doi:10.1093/emboj/19.16.4265

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Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation

Journal article

Open access

Peer reviewed

Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation

S Chen, F Lin, Ming Xu, John Hwa and R M Graham

The EMBO journal, Vol.19(16), pp.4265-4271

08/15/2000

DOI: 10.1093/emboj/19.16.4265

PMID: 10944109

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Abstract

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.

Transfection

Ligands

Adrenergic alpha-1 Receptor Agonists

Molecular Sequence Data

Immunoblotting

Receptors, Adrenergic, alpha-1 - chemistry

Dose-Response Relationship, Drug

Genes, Dominant

Glycine - chemistry

Phenylalanine - chemistry

Conserved Sequence

Phosphatidylinositols - metabolism

Inositol Phosphates - metabolism

Cyclic AMP - metabolism

Amino Acid Sequence

Mutagenesis, Site-Directed

Signal Transduction - genetics

Precipitin Tests

Hydrolysis

Sequence Homology, Amino Acid

Point Mutation

Animals

Receptors, Adrenergic, alpha-1 - genetics

Protein Binding

Adrenergic alpha-Agonists - pharmacology

COS Cells

DNA, Complementary - metabolism

Epinephrine - pharmacology

Asparagine - chemistry

GTP-Binding Proteins - metabolism

Details

Title: Subtitle: Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation
Creators: S Chen - Molecular Cardiology Unit, Victor Chang Cardiac Research Institute, St Vincent's Hospital, Sydney 2010, Australia
F Lin
Ming Xu
John Hwa
R M Graham
Resource Type: Journal article
Publication Details: The EMBO journal, Vol.19(16), pp.4265-4271
DOI: 10.1093/emboj/19.16.4265
PMID: 10944109
NLM abbreviation: EMBO J
ISSN: 0261-4189
eISSN: 1460-2075
Publisher: England
Language: English
Date published: 08/15/2000
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: 9984025430102771

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