Mutational analysis of ligand binding activity of beta 2 adrenergic receptor expressed in Escherichia coli.

Abstract

A novel in situ screening procedure was used to identify neutral mutations in the human beta 2 adrenergic receptor (beta 2AR). The coding region of the human beta 2AR gene was subcloned under transcriptional control of an inducible T7 promoter and used to transform Escherichia coli. Colonies expressing the beta 2AR bound the radiolabeled antagonist [125I]iodocyanopindolol and could be identified by autoradiography after transfer to nitrocellulose filters. The region of the beta 2AR between residues 76 and 83, in the second transmembrane helix, was mutagenized by a saturation mutagenesis technique, so that virtually all of the beta 2AR genes contained at least one mutation. Colonies retaining ligand binding activity were isolated using the in situ screen. Sequence analysis of the active mutant receptor genes allowed the identification of individual amino acid side chains which are essential for either ligand binding or structural integrity of the beta 2AR receptor.