Abstract

The roles of the carboxyl-terminal tail of the α_1B-adrenergic receptor in its expression, function, and regulation were investigated by site-directed mutagenesis. The receptor construct truncated after residue 363 seemed not to be properly expressed. In contrast, the receptor truncated after residue 366 and all of the longer receptor constructs were properly expressed and exhibited agonist and antagonist binding and activation of phosphoinositide hydrolysis similar to the wild-type receptor. Agonist-induced sequestration of receptors within the plasma membrane, endocytosis into intracellular vesicles, and eventual down-regulation were all absent in the receptor truncated after residue 366. A series of sequential truncations and a deletion mutation identified a critical role for residues 403 to 425, which include the previously identified sites for G protein-coupled receptor kinase phosphorylation, in agonist-induced internalization of the receptor. Similar studies identified a critical role for residues 367 to 380 in agonist-induced down-regulation. Individual point mutations converting either cysteine 367 or serine 369 to alanine selectively eliminated down-regulation, thus identifying two specific amino acid residues required for down-regulation. Importantly, several of the mutated receptors that failed to show rapid agonist-induced internalization nonetheless exhibited normal agonist-induced down-regulation. In addition to identifying specific regions and individual residues of the α_1B-adrenergic receptor involved in internalization and down-regulation, these studies provide mutated receptors that internalize but do not down-regulate, that down-regulate without internalization, and that are defective in both internalization and down-regulation, all of which should be useful tools for further studies of the specific cellular compartments and molecular mechanisms involved in receptor internalization and down-regulation.