Identification of Residues Required for Ligand Binding to the β -adrenergic Receptor

• Published in: Proceedings of the National Academy of Sciences - PNAS 1987-07, Vol.84 (13), p.4384-4388
• Main Authors: Strader, Catherine D., Sigal, Irving S., Register, R. Bruce, Candelore, Mari Rios, Rands, Elaine, Richard A. F. Dixon
• Format: Article
• Language: English
• Subjects: Adrenergic beta-Agonists - metabolism; Adrenergic beta-Antagonists - metabolism; Agonists; Amino Acid Sequence; Animals; beta -adrenergic; Binding sites; Biological and medical sciences; Cattle - genetics; Cell membranes; Cell receptors; Cell structures and functions; COS cells; Cricetinae; Eye Proteins - genetics; Fundamental and applied biological sciences. Psychology; Humans; Ligands; Molecular and cellular biology; Mutation; Opsins; P branes; Plasmids; Protein Binding; Proteins; Receptors; Receptors, Adrenergic, beta - genetics; Receptors, Adrenergic, beta - metabolism; Receptors, Muscarinic - genetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rod Opsins; Sequence Homology, Nucleic Acid; Species Specificity; Swine - genetics; Turkeys - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.13.4384

The functional significance of conserved polar amino acids within the putative transmembrane region of the β -adrenergic receptor (β AR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding β AR and expression of the mutant genes in COS-7 cells. Although a substitution of aspartate at position 113 with an asparagine residue did not affect expression or processing of the protein, the resulting mutant β AR did not show detectable binding toward the antagonist iodocyanopindolol. Replacement of the aspartate and asparagine residues at positions 79 and 318, respectively, had no effect on the affinity of the receptor toward antagonists but reduced the affinity of the receptor toward agonists by 1 order of magnitude. Furthermore, we observed that substitution of the proline at position 323 with a serine residue resulted in improper or incomplete processing of the β AR, presumably reflecting a role for this residue in the folding of the receptor. Together with our previous results from deletion mutagenesis studies, these observations indicate that the ligand binding site involves the transmembrane region of the β AR.