Truncation of the AT1A receptor's carboxyl-terminal 45 amino acids inhibits its endocytosis but does not prevent rapid desensitization, suggesting a distinct desensitization mechanism compared to other G-protein-coupled receptors.
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 (AT1A) receptor devoid of the carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL314 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 (approximately 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of 125Iangiotensin II from TL314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the level of which were comparable with the full-length AT1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT1A 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 AT1A receptor promotes sequestration. Thus, truncation of the AT1A 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 TL314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.
Thomas et al. (Sun,) studied this question.
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