Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization
Thomas, W.G.; Thekkumkara, T.J.; Motel, T.J.; Baker, K.M.
Journal of Biological Chemistry 270(1): 207-213
1995
ISSN/ISBN: 0021-9258 PMID: 7814375 Accession: 009451519
Phosphorylation of serine and threonine residues in the carboxyl-terminal region of many G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways. In Chinese hamster ovary cells, we have stably expressed a truncated mutant of the angiotensin II (AT-1A) receptor devoid of the carboxylterminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL-314 to indicate truncation after leucine 314, expressed a single class of angiotensin H receptors with a dissociation constant of 1.08 nm and a receptor density of 560 fmol/mg of protein ( apprx 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of (125I)angiotensin II from TL-314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL-314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the levels of which were comparable with the full-length AT-1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT-1A receptors. Interestingly, angiotensin II-induced endocytosis of the truncated receptor was almost completely inhibited, suggesting that a recognition motif within the carboxyl-terminal 45 amino acids of the AT-1A receptor promotes sequestration. Thus, truncation of the AT-1A receptor after leucine 314 inhibits agonist-induced internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins, and activation of signal transduction pathways. The rapid desensitization and refractoriness of the angiotensin II-induced calcium transient in the TL-314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT-1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.