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.

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Abstract
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) tha 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 [sn130].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 and 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 guinine 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.