Identification of distinct carboxyl-terminal domains mediating internalization and down-regulation of the hamster alpha(1B) adrenergic receptor

The roles of the carboxyl-terminal tail of the alpha(1B)-adrenergic recepto r in its expression, function, and regulation were investigated by site-dir ected mutagenesis. The receptor construct truncated after residue 363 seeme d not to be properly expressed. In contrast, the receptor truncated after r esidue 366 and all of the longer receptor constructs were properly expresse d and exhibited agonist and antagonist binding and activation of phosphoino sitide hydrolysis similar to the wild-type receptor. Agonist-induced seques tration of receptors within the plasma membrane, endocytosis into intracell ular vesicles, and eventual down-regulation were all absent in the receptor truncated after residue 366. A series of sequential truncations and a dele tion mutation identified a critical role for residues 403 to 425, which inc lude the previously identified sites for G protein-coupled receptor kinase phosphorylation, in agonist-induced internalization of the receptor. Simila r studies identified a critical role for residues 367 to 380 in agonist-ind uced downregulation. Individual point mutations converting either cysteine 367 or serine 369 to alanine selectively eliminated down-regulation, thus i dentifying two specific amino acid residues required for down-regulation. I mportantly, several of the mutated receptors that failed to show rapid agon ist-induced internalization nonetheless exhibited normal agonist-induced do wn-regulation. In addition to identifying specific regions and individual r esidues of the alpha(1B)-adrenergic receptor involved in internalization an d down-regulation, these studies provide mutated receptors that internalize but do not down-regulate, that downregulate without internalization, and t hat are defective in both internalization and down-regulation, all of which should be useful tools for further studies of the specific cellular compar tments and molecular mechanisms involved in receptor internalization and do wn-regulation.