Site-Directed Mutagenesis of Human β -adrenergic Receptors: Substitution of Aspartic Acid-130 by Asparagine Produces a Receptor with High-Affinity Agonist Binding that is Uncoupled from Adenylate Cyclase

By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human β -adrenergic receptor (β 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 β AR([Asn130]β AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]β AR. The mutant [Asn130]β AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type β AR), consistent with a single class of high-affinity binding sites. The mutant β AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the β 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]β ARs had no effect on intracellular levels of cAMP, indicating that the mutant β 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 β 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 β AR.