The functional significance of conserved polar amino acids within the putative transmembrane region of the fi-adrenergic receptor (PiAR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding PAR 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 BAR 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 .BAR, 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 BAR. The recent cloning of the genes encoding the hamster (2) and human 32AR (3), the avian PAR (4), and the porcine Ml muscarinic cholinergic receptor (MAR) (5) and the deduction of the primary sequences of these proteins suggest a structural basis for the mechanistic similarities among the Gprotein-linked receptors. These hormone receptors share sequence homology with each other and with the visual opsins, which transduce their signals through the activation of the G-protein transducin (6). Most of the conservation in sequence among these proteins is found within seven hydrophobic domains of approximately 20-25 residues in length. Based on the model proposed for the opsins (7), these hydrophobic regions of the receptors, which are linked by more divergent hydrophilic regions of various lengths, would alternately traverse the membrane with the N terminus of the PAR exposed externally and the C terminus exposed intracellularly (2). 4384The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

No abstract

On the basis of the homology between the amino acid sequences of the beta‐adrenergic receptor (beta AR) and the opsin proteins we have proposed that the ligand binding domain lies within the seven transmembrane hydrophobic regions of the protein, which are connected by hydrophilic regions alternatively exposed extracellularly and intracellularly. We have systematically examined the importance of each of these regions by making a sequential series of deletions in the gene for the hamster beta AR which encompass most of the protein coding region. The ability of the corresponding mutant receptors to be expressed, localized to the cell membrane, and bind beta‐adrenergic ligands has been analyzed, using transient expression in COS‐7 cells. The hydrophobic regions and the hydrophilic segments immediately adjacent to the membrane cannot be removed without affecting the processing and membrane localization of the beta AR. However, most of the hydrophilic regions appear to be dispensable for ligand binding. In addition, we observed that substitution of the conserved cysteine residues at positions 106 and 184 dramatically altered the ligand binding characteristics of the beta AR, suggesting the occurrence of a disulfide bond between these two residues in the native protein. These data are discussed in terms of the tertiary structure of the beta AR.