Site-directed mutagenesis and continuous expression of human beta-adrenergic receptors. Identification of a conserved aspartate residue involved in agonist binding and receptor activation.

Using a new expression vector that allows stable and steroid inducible expression of the human beta 2-adrenergic receptor in mouse L cells, we have examined the functional significance of the highly conserved aspartate residue in the putative second transmembrane region of the receptor. Substitution of aspartate 79 with asparagine produced a mutant receptor that displays the expected affinity and stereoselectivity for antagonists but a 40-, 140-, and 240-fold reduction in its affinity for isoproterenol, epinephrine, and norepinephrine, respectively. This receptor mutant does not display guanine nucleotide-sensitive high affinity binding of agonists. Addition of saturating concentrations of isoproterenol to cell cultures expressing the mutant receptor produces a slight, albeit significant, increase in intracellular levels of cyclic AMP as compared to cells expressing wild type receptor. These observations demonstrate that substitution of aspartate with asparagine at residue 79 in the human beta-adrenergic receptor differentially affects the binding of catecholamines and produces a functional uncoupling of receptors and stimulatory guanine nucleotide regulatory proteins (Gs). These data are consistent with a role for aspartate 79 as a counterion to the amine in catecholamines and in agonist-induced activation of the beta-adrenergic receptor associated with high affinity ligand binding, Gs coupling, and adenylate cyclase stimulation.