Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene (original) (raw)
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Regulatory Peptides, 2004
Homology modeling of the structure of the AT1 receptor, based on the high resolution rhodopsin crystal structure, indicated that it is unlikely that the binding of AngII to AT1 involves simultaneously all the receptor's residues reported in the literature to participate in this process. Site-directed mutagenesis using Ala substitution of charged residues Lys20, Arg23, Glu91 and Arg93 was performed to evaluate the participation of their side-chains in ligand binding and in triggering the cell's response. A comparative analysis by competition binding and functional assays using angiotensin II and the analog [Sar1]-angiotensin II suggests an important role for Arg23 of AT1 receptor in binding of the natural agonist. It is discussed whether some receptor's residues participate directly in the binding with AngII or whether they are part of a regulatory site.
Journal of Biological Chemistry, 1999
The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT 1 ) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT 1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT 1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT 1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT 1a receptor but not with those of angiotensin II type 2 (AT 2 ), m 3 muscarinic acetylcholine, bradykinin B 2 , endothelin B, and  2 -adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT 1a receptor association was confirmed by affinity chromatography, by specific coimmunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT 1a receptormediated activation of phospholipase C without affecting m 3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT 1a receptor and affects AT 1a receptor signaling.
Molecular Endocrinology, 1998
The molecular mechanism of angiotensin II type I receptor (AT 1 ) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr 332 Lys 333 Met 334 Ser 335 Thr 336 Leu 337 Ser 338 ) within the carboxyl terminus of the AT 1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT 1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT 1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT 1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys 325 , which removed the last 34 amino acids, almost completely inhibited Ang IIstimulated 32 P incorporation into the AT 1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitopetagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr 332 , Ser 335 , Thr 336 , and Ser 338 , previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar 1 Ile 8 -Ang II, which paradoxically causes internalization of wild-type AT 1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endo-cytosis, the internalization kinetics of wild-type AT 1A receptors and receptors mutated within the Thr 332 -Ser 338 region were compared. The four putative phosphorylation sites (Thr 332 , Ser 335 , Thr 336 , and Ser 338 ) were substituted with either neutral
Endocrinology, 2003
Asn111, localized in the third transmembrane domain of the AT1 receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller (Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT1, recognized 125I-Ang II with high affinities comparable to that of the wild-type AT1 receptor. In phospholipase C assays, the four mutants encompassed the entire spectrum of functional states, ranging from constitutive activity (without agonist) for N111A-AT1 and N111G-AT1 to a significant loss of activity (upon maximal stimulation) for N111Q-AT1 and a major loss of activity for N111W-AT1. In Ca2+ mobilization studies, N111W-AT1 produced a weak Ca2+ transient and, unexpectedly, N111G-AT1 also produced a Ca2+ transient that was much weaker than that of th...
Endocrinology, 2003
The pleiotropic actions of angiotensin II are mediated by the primarily G q protein-coupled type 1 angiotensin (AT 1) receptor. In this study a mutational analysis of the function of the conserved DRYXXV/IXXPL domain in the second intracellular loop of the rat AT 1A receptor was performed in COS7 cells. Alanine substitution studies showed that single replacement of the highly conserved Asp 125 and Arg 126 , but not Tyr 127 , moderately impaired angiotensin II-induced inositol phosphate signaling. However, concomitant substitution of both Asp 125 and Arg 126 caused marked reduction of both inositol phosphate signaling and receptor internalization. Alanine scanning of the adjacent residues showed that substitution of Ile 130 , His 132 , and Pro 133 reduced agonist-induced inositol phosphate signal generation, whereas mutations of Met 134 also impaired receptor internalization. Expression of the D125A mutant AT 1A receptor in COS7 cells endowed the receptor with moderate constitutive activity, as indicated by its enhanced basal Elk1 promoter activity and inositol phosphate response to partial agonists. Angiotensin II-induced stimulation of the Elk1 promoter showed parallel impairment with inositol phosphate signal generation in receptors containing mutations in this region of the AT 1A receptor. These data confirm that Ca 2؉ signal generation is required for the nuclear effects of angiotensin II-induced ERK activation. They are also consistent with the role of the conserved DRY sequence of the AT 1A receptor in receptor activation, and of Asp 125 in constraining the receptor in its inactive conformation. Furthermore, in the cytoplasmic helical extension of the third helix, an apolar surface that includes Ile 130 and Met 134 appears to have a direct role in G protein coupling.
British Journal of Pharmacology, 2000
The role of different residues of the rat AT1A receptor in the interaction with the N- and C-terminal ends of angiotensin II (AngII) was studied by determining ligand binding and production of inositol phosphates (IP) in COS-7 cells transiently expressing the following AT1A mutants: T88H, Y92H, G196I, G196W and D278E.G196W and G196I retained significant binding and IP-production properties, indicating that bulky substituents in position 196 did not affect the interaction of AngII's C-terminal carboxyl with Lys199 located three residues below.Although the T88A mutation did not affect binding, the T88H mutant had greatly decreased affinity for AngII, suggesting that substitution of Thr88 by His might hinder binding through an indirect effect.The Y92H mutation caused loss of affinity for AngII that was much less pronounced than that reported for Y92A, indicating that His in that position can fulfil part of the requirements for binding.Replacing Asp278 by Glu caused a much smaller reduction in affinity than replacing it by Ala, indicating the importance of Asp's β-carboxyl group for AngII binding.Mutations in residues Thr88, Tyr92 and Asp278 greatly reduced affinity for AngII but not for Sar1 Leu8-AngII, suggesting unfavourable interactions between these residues and AngII's aspartic acid side-chain or N-terminal amino group, which might account for the proposed role of the N-terminal amino group of AngII in the agonist-induced desensitization (tachyphylaxis) of smooth muscles.The role of different residues of the rat AT1A receptor in the interaction with the N- and C-terminal ends of angiotensin II (AngII) was studied by determining ligand binding and production of inositol phosphates (IP) in COS-7 cells transiently expressing the following AT1A mutants: T88H, Y92H, G196I, G196W and D278E.G196W and G196I retained significant binding and IP-production properties, indicating that bulky substituents in position 196 did not affect the interaction of AngII's C-terminal carboxyl with Lys199 located three residues below.Although the T88A mutation did not affect binding, the T88H mutant had greatly decreased affinity for AngII, suggesting that substitution of Thr88 by His might hinder binding through an indirect effect.The Y92H mutation caused loss of affinity for AngII that was much less pronounced than that reported for Y92A, indicating that His in that position can fulfil part of the requirements for binding.Replacing Asp278 by Glu caused a much smaller reduction in affinity than replacing it by Ala, indicating the importance of Asp's β-carboxyl group for AngII binding.Mutations in residues Thr88, Tyr92 and Asp278 greatly reduced affinity for AngII but not for Sar1 Leu8-AngII, suggesting unfavourable interactions between these residues and AngII's aspartic acid side-chain or N-terminal amino group, which might account for the proposed role of the N-terminal amino group of AngII in the agonist-induced desensitization (tachyphylaxis) of smooth muscles.British Journal of Pharmacology (2000) 130, 1263–1268; doi:10.1038/sj.bjp.0703430
Biochemical and Biophysical Research Communications, 1999
Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [ 125 I-Sar 1-Ile 8 ]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125 I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [ 125 I-Sar 1-Ile 8 ]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands.
Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins
International Immunopharmacology, 2008
Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT 1 / Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT 1 receptor, a non-functional AT 1 receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT 1 receptor and Mas showed a binding profile similar to that observed for the wild-type AT 1 expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT 1 . Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/ K20A and Mas. In addition, immunofluorescence analysis localized the AT 1 receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT 1 receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.
Molecular Pharmacology, 1997
Two nonpeptide ligands that differ chemically by only a single methyl group but have agonistic (L-162,782) and antagonistic (L-162,389) properties in vivo were characterized on the cloned angiotensin AT 1 receptor. Both compounds bound with high affinity (K I ϭ 8 and 28 nM, respectively) to the AT 1 receptor expressed transiently in COS-7 cells as determined in radioligand competition assays. L-162,782 acted as a powerful partial agonist, stimulating phosphatidylinositol turnover with a bellshaped dose-response curve to 64% of the maximal level reached in response to angiotensin II. Surprisingly, L-162,389 also stimulated phosphatidylinositol turnover, albeit only to a small percentage of the angiotensin response. The prototype nonpeptide AT 1 agonist L-162,313 gave a response of ϳ50%. The apparent EC 50 values for all three compounds in stimulating phosphatidylinositol turnover were similar, ϳ30 nM, corresponding to their binding affinity. Each of the three compounds also acted as angiotensin antagonists, yet in this capacity the compounds differed markedly, with IC 50 values ranging from 1.05 ϫ 10 Ϫ7 M for L-162,389 to 6.5 ϫ 10 Ϫ6 for L-162,782. A series of point mutations in the transmembrane segments (TMs)