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.
Highlights
Phosphorylation of serine and threonine residues in the carboxyl-terminal region ofmany G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways
In TL3 14 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 lA receptor
Hydropathy analysis of the deduced amino acid sequences predicts that the topology of both AT l and ATz receptors is typical of seven-transmembrane guanyl nucleotide-binding protein (Gprotein) coupled receptors; only AT1 receptors appear to efficiently couple G-proteins [7, 8]
Summary
Materials-Cell culture media. fetal calf serum, antibiotics, Geneticin (G418 su lfate). All Binding Studies-AIl binding studies on cultures of transfected CHO-Kl cells in 35-mm dishes were performed essentially as described previously (2 1); the AIl receptor binding buffer contained 50 mM TrisHCI, pH 7.5, 120 mM NaCI , 4 mM KCI, 5 mM MgC12, 1 mM CaCI2, 10 ,..gIm l bacitracin, and 2 mg/ml n-glucose. Tran s fe ct ion s run in para lle l with th e fu ll-len g th AT 1A r ecept or D NA in th esame vec to r co ns is te n tly prod uced clones e x p ress in g hi gh lev el s of 11:lf> IIA II bi ndin g (> 5 0 0 fmo l/m g of prot ein ), O ne clo neexp ress ing th e hi gh est le vel o f bind in g for th e t r u ncate d re cept or, d esi gn a t ed '1'1," 1,1.
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