Document Detail

Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase.
MedLine Citation:
PMID:  2840663     Owner:  NLM     Status:  MEDLINE    
By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR.
C M Fraser; F Z Chung; C D Wang; J C Venter
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  85     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  1988 Aug 
Date Detail:
Created Date:  1988-09-07     Completed Date:  1988-09-07     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  5478-82     Citation Subset:  IM    
Section of Receptor Biochemistry and Molecular Biology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD 20892.
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MeSH Terms
Adenylate Cyclase / metabolism*
Asparagine / genetics*,  metabolism
Aspartic Acid / genetics*,  metabolism
Base Sequence
Binding, Competitive
Catecholamines / metabolism
Cell Line
Gene Expression Regulation
Guanine Nucleotides / pharmacology
Molecular Sequence Data
Receptors, Adrenergic, beta / genetics*,  metabolism
Sequence Homology, Nucleic Acid
Reg. No./Substance:
0/Catecholamines; 0/Guanine Nucleotides; 0/Receptors, Adrenergic, beta; 56-84-8/Aspartic Acid; 7006-34-0/Asparagine; EC Cyclase

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