Many cell surface receptors that mediate the actions of hormones and neurotransmitters are coupled to the activation or inhibition of intracellular enzymes or of ion channels by GTP-binding regulatory proteins (G proteins). The activated G proteins thereby transmit information about receptor occupancy to the interior of cells. We have, for many years, studied one of these G protein coupled receptors: the opiate receptor. The neuroblastoma x glioma hybrid cell line, NG108-15, is richly endowed with opiate receptors, and is a particularly good source of this protein since it expresses only the delta type of opiate receptor. In the past year we have isolated many peptides believed to be fragments of opiate receptors and determined their amino acid sequences. We have prepared oligonucleotides corresponding to these sequences and are in the process of screening a cDNA library prepared from NG108-15 cells with these and other probes. We hope to thereby isolate a clone of the opiate receptor cDNA, learn its complete amino acid sequence, elucidate its mechanism of action, and study the regulation of its synthesis and metabolism. It has become clear that the G protein coupled receptors are members of a closely related family of structures. By the combined use of specific antibodies and synthetic peptides we had earlier characterized a highly conserved receptor domain that activates G-proteins, and showed that no other intracellular domain fully shares these properties. We have in the past year initiated a study of the structural basis of agonist-antagonist discrimination in the related G protein-coupled alpha-2 adrenergic receptor. This protein is coupled to the same G proteins as is the delta opiate receptor and is pharmacologically related as well. We have expressed a cloned alpha-2 adrenergic receptor in both COS and CHO cells and measured receptor numbers by binding assays and receptor coupling by inhibition of adenylyl cyclase and stimulation of GTPase. We found that coupling to G proteins required the presence of an inordinately large number of receptors in the transfected cells, suggesting that a G protein may be limiting. We have therefore prepared COS cells which overexpress each of the two G proteins known to be responsible for opiate receptor and alpha-2 receptor actions. We expect that these cells will allow the more efficient coupling of the two receptors and may also find uses in expression cloning of this type of receptor. Efforts are now also underway to mutate the alpha-2 adrenergic receptor in ways designed to test the role of flexibility and motion in determining the efficacy of agonists.
