Identification of the D₂‐_‐Dopamine Receptor Binding Subunit in Several Mammalian Tissues and Species by Photoaffinity Labeling

Abstract

Abstract Photoaffinity labeling of the D 2 ‐ ‐ dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D 2 ‐ ‐ dopaminergic antagonist probe [ 125 I] N ‐( p ‐azidophenethyl)spiperone ([ 125 I]N 3 ‐ ‐ NAPS). In tissues containing D 2 ‐ ‐ receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of M r = 94,000. Specificity of the labeling is typically D 2 ‐ ‐ dopaminergic in character. The covalent labeling is blocked by (+)‐butaclamol but not by the inactive (‐) isomer. Agonists block incorporation with the order of potency: N‐n‐ propylnorapomorphine > apomorphine > dopamine. The D 2 ‐ ‐ selective antagonist spiperrone blocks labeling of the M r = 94,000 peptide whereas the D 1 ‐ ‐ selective antagonist SCH‐23390 is ineffective. Thus, these results indicate that the ligand binding subunit of the D 2 ‐ ‐ dopamine receptor resides on a M r = 94,000 peptide in these various tissues from several species. Under conditions where proteolysis is not stringently controlled, peptides of lower M r (32–38,000) are labeled at the expense of the M r = 94,000 peptide. The most efficient protease inhibitor tested in these systems was EDTA, suggesting that the generation of these lower M r receptor fragments might be the result of a metal‐dependent proteolysis in the membrane preparations. In the rat neurointermediate lobe, a tissue containing D 2 ‐ ‐ receptors. [ 125 I]N 3 ‐ ‐ NAPS specifically labels a major peptide of M r ± 120,000 in addition to the M r = 94,000 peptide. This peptide may represent an unprocessed or differently processed form of the receptor or a pharmacologically similar but biochemically distinct form of the receptor in this tissue. Alternatively, the M r = 94,000 peptide labeled in all the other tissues, even under the conditions used, may already represent a fragment of this high‐M r peptide.