title: 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.
abstract: 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 [125I]angiotensin 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.
authors: Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
authors: W G
authors: WG
authors: Thomas
authors: T J
authors: TJ
authors: Thekkumkara
authors: T J
authors: TJ
authors: Motel
authors: K M
authors: KM
authors: Baker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA; D019204:GTP-Binding Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D011945:Receptors, Angiotensin; D004247:DNA; D019204:GTP-Binding Proteins