STABLE EXPRESSION OF A TRUNCATED AT(1A) RECEPTOR IN CHO-KI CELLS - THE CARBOXYL-TERMINAL REGION DIRECTS AGONIST-INDUCED INTERNALIZATION BUT NOT RECEPTOR SIGNALING OR DESENSITIZATION

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 carboxyl-terminal 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 II receptors with a dissociation constant of 1.08 nM and a receptor density of 560 fmol/mg of protein (similar to 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of [I-125] 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.