Site-directed mutagenesis of alpha 2A-adrenergic receptors: identification of amino acids involved in ligand binding and receptor activation by agonists.

Molecular cloning of the alpha 2A-adrenergic receptor has shown that this receptor is a member of the gene superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors. The alpha 2A-adrenergic receptor expressed in Chinese hamster ovary (CHO) cells attenuates and potentiates forskolin-stimulated cAMP production through independent signaling pathways. To examine the role of three conserved aspartic acid and two conserved serine residues in alpha 2A-adrenergic receptor function, we substituted asparagine for aspartic acid or alanine for serine and characterized the mutant receptors in stably transfected CHO cells. Asn113 mutant alpha 2-adrenergic receptors display no [3H] yohimbine specific binding, at concentrations up to 1000 nM. In transfected cells expressing the Asn113 mutant receptor, agonists, at concentrations up to 0.1 mM, produce small decreases (approximately 10% of wild-type values) in forskolin-stimulated cAMP and potentiate forskolin-stimulated cAMP concentrations in a dose-dependent manner, with EC50 values approximately 500-fold higher than those for the wild-type receptor. These findings suggest that Asp113 may be involved in high affinity binding of agonists and antagonists, as has been previously reported for beta 2-adrenergic and m1 muscarinic acetylcholine receptors. Asn79 mutant alpha 2-adrenergic receptors display high affinity agonist binding that is insensitive to guanine nucleotides, suggesting an altered receptor-G protein coupling. Furthermore, agonist binding to Asn79 mutant receptors elicits no change in forskolin-stimulated cAMP concentrations, similar to earlier findings that the corresponding residue in beta 2-adrenergic and muscarinic receptors is required for effector stimulation. Asp130 appears to influence receptor-G protein coupling. Mutation of this residue eliminates high affinity, guanine nucleotide-sensitive, agonist binding and produces a rightward shift in the dose-response curves for agonist-mediated inhibition of forskolin-stimulated cAMP production, compared with the wild-type receptor. Moreover, agonist potentiation of forskolin-stimulated cAMP levels is abolished if Asp130 is replaced by Asn, supporting the hypothesis that inhibition and potentiation of forskolin-stimulated cAMP production in CHO cells proceed through distinct signaling pathways. Characterization of Ala204 mutant alpha 2A-adrenergic receptors suggests a possible role for Ser204 in hydrogen bond interactions with the para-hydroxyl group of the phenyl ring of the catecholamines, as has been previously described for the corresponding serine in beta 2-adrenergic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)