The role of C-terminal sequences of the α1BAR in its internalization and down-regulation were assessed. Wild-type and mutated hamster α1BARs were stably expressed in CHO cells. [3H]Prazosin binding and inositol phosphate (IP) formation were measured. Internalization was assayed as a loss of [3H]prazosin binding to intact cells on ice and as a shift of receptors to the light vesicle fraction on sucrose gradients after 30 min exposure of cells to 10 μM epinephrine. Down-regulation was assessed by loss of [3H]prazosin binding to membranes from cells exposed to 10 μM epinephrine for 24 hr. The construct truncated after Leu363 could not be detected in binding assays. The receptor truncated after Gln366 had normal binding and IP formation but failed to internalize or down-regulate. Receptors truncated after Gly380, Lys402, Ser425, Ala449 and Pro477 all exhibited normal binding, IP formation, and internalization. Although receptors truncated after Gly380 and Lys402 internalized normally, they did not down-regulate. Receptors truncated after Ser425 and Ala449 down-regulated normally, but truncation after Pro477 markedly inhibited down-regulation. These results identify multiple C-terminal domains of the α1BAR that are important for down-regulation, some of which are distinct from those required for internalization.
|Original language||English (US)|
|State||Published - Mar 20 1998|
ASJC Scopus subject areas
- Molecular Biology