Identification of Residues Essential for the Active Conformation of the ß-Adrenergic Receptor by Site-Directed Mutagenesis

Many hormones and neurotransmitters mediate their actions by stimulating one of a class of receptors which are functionally coupled to guanine nucleotide binding regulatory proteins (G-proteins). Activation of the G-protein by the agonist-occupied form of the receptor causes the release of GDP from the G-protein, allowing GTP to bind. The GTP-bound form of the G-protein is the active form, which dissociates from the receptor, leading to the activation or inhibition of specific effector enzymes and modulating levels of intracellular second messengers. The elucidation of the primary sequences of several G-protein coupled receptors by molecular cloning has shown them to share characteristic structural elements, presumably related to their common mechanism of action (see Dixon, et al, 1989, for review). All of these receptors whose sequences are known contain 7 stretches of conserved hydrophobic amino acids, thought to form transmembrane α- helices, which connect 8 more divergent hydrophilic regions, postulated to form alternating extracellular and intracellular loops. In our laboratory, we have used genetic anaiysis of-the s-adrenergic receptor (sAR), which is linked to GS to stimulate adenylyl cyclase, to identify structural domains and specific amino acid residues required for receptor function. A model for the structure of the sAR is shown in Figure 1. From these studies and from biochemical and genetic analysis of other G-protein coupled receptors, a model for receptor activity is emerging, which may serve to identify potential sites of pharmacological intervention within these signal transduction systems.