SMART MODE:

NORMAL GENOMIC

• Tomaru T et al.
• Isolation and characterization of a transcriptional cofactor and its novel isoform that bind the deoxyribonucleic acid-binding domain of peroxisome proliferator-activated receptor-gamma.
• Endocrinology. 2006; 147: 377-88
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• Using the DNA-binding domain (DBD) and hinge region of human peroxisome proliferator-activated receptor (PPAR)-gamma as bait in yeast two-hybrid screen, we isolated partial cDNA identical with that of the C terminal of KIAA1769. KIAA1769 encodes a 2080-amino acid protein (molecular mass, 231 kDa) that was recently identified to interact with PPARalpha and termed PPARalpha-interacting cofactor 285 (here referred to as PPARgamma-DBD-interacting protein 1 (PDIP1)-alpha). PDIP1 mRNA was expressed in 3T3-L1 adipocytes and THP-1 macrophages. We also identified the expression of the N terminal extended form of PDIP1alpha (referred to as PDIP1beta) consisting of 2649 amino acids (295 kDa) in human cultured cell lines by RT-PCR, and 5' rapid amplification of cDNA ends. Ribonuclease protection assay revealed that PDIP1beta mRNA was expressed more abundantly than PDIP1alpha mRNA. The C-terminal region of PDIP1 directly binds DBD of PPARgamma, and multiple LXXLL motifs in PDIP1 were not required for the interaction. PDIP1alpha and -beta similarly enhanced PPARgamma-mediated transactivation in transfection assays and short interfering RNA targeting PDIP1 mRNA significantly reduced transactivation by PPARgamma. No potent intrinsic activation domain was identified in either PDIP1 isoforms in mammalian one-hybrid assays, and mutation of all LXXLL motifs did not affect enhancement of PPARgamma-mediated transactivation. PDIP1alpha and -beta similarly augmented transactivation by PPARalpha, PPARdelta, thyroid hormone receptor (TR)-alpha1, TRbeta1, and retinoid X receptor-alpha. PDIP1alpha also enhanced estrogen receptoralpha- and androgen receptor-mediated transactivation, whereas PDIP1beta did not. PDIP1alpha showed receptor-specific synergism with activation function-2-interacting coactivators in PPARgamma- and TRbeta1-mediated transactivation. Together, PDIP1 might function as a transcriptional cofactor for a broad range of nuclear receptors, possibly in collaboration with specific activation function-2 interacting coactivators.

• Pulakat L et al.
• Ligand-dependent complex formation between the Angiotensin II receptor subtype AT2 and Na+/H+ exchanger NHE6 in mammalian cells.
• Peptides. 2005; 26: 863-73
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• Involvement of Angiotensin II (Ang II) in the regulation of sodium levels by modulating the Na+/H+ exchangers is demonstrated in many tissues. Screening of a mouse 17-day fetus cDNA library with the Angiotensin II receptor AT2 as the bait in yeast two-hybrid assay led us to identify an AT2-interacting mouse fetus peptide that shared 98% amino acid identity with the corresponding region of the human NHE6. NCBI Blast search showed that the clone 6430520C02 (GenBank Accession # AK032326) of the mouse genome project carried the complete sequence of this new mouse NHE6 isoform. The human and mouse NHE6 peptides share 97% overall homology. Further analysis showed that the region spanning the third intracellular loop and C-terminal cytoplasmic tail of the AT2 directly interacted with a 182 amino acid region that spans the predicted 5th intracellular loop and the initial part of the C-terminus of the mouse NHE6 in yeast two-hybrid assay. This 182-amino acid region that interacted with the AT2 also shares 98% homology with the corresponding region of rat NHE6 and therefore is highly conserved across species. We detected widespread expression of this NHE6 isoform in several rat tissues including 10-day fetus, 17-day fetus, and 30-day post-natal tissues of heart, brain, kidney and muscle. Moreover, the AT2 co-immunoiprecipitated with a hemagglutinin tagged NHE6 when expressed in human cell line MCF-7, and activated by AngII. This ligand-dependent complex formation between the AT2 and NHE6 suggests that the hormone Ang II may act as a regulator of NHE6, and Ang II-mediated direct protein-protein interaction between AT2 and NHE6 could be a mechanism for modulating the functions of the ubiquitously expressed NHE6 in different tissues.

• Shivakumar BR, Wang Z, Hammond TG, Harris RC
• EP24.15 interacts with the angiotensin II type I receptor and bradykinin B2 receptor.
• Cell Biochem Funct. 2005; 23: 195-204
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• The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 receptor (AT1) is known to interact with several classes of intracellular proteins that may modulate receptor function. Employing yeast two-hybrid screening of a human embryonic kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the AT1 receptor as a bait, we have isolated EP24.15 (EC 3.4.24.15, thimet oligopeptidase) as a potentially interacting protein. EP24.15 is widely distributed and is known to degrade bioactive peptides such as angiotensin I and II and bradykinin. In addition, EP24.15 was previously identified as a putative soluble angiotensin II binding protein. Two-hybrid screening also determined that EP24.15 can interact with the B2 bradykinin receptor. Transient expression of EP24.15 in a porcine kidney epithelial cell line stably expressing full length AT1 and full length B2 followed by affinity chromatography and co-immunoprecipitation confirmed EP24.15 association with both AT1 and B2 receptors. EP24.15 was also co-immunoprecipitated with AT1 and B2 in rat kidney brush border membranes (BBM) and basolateral membranes (BLM). Both AT1 and B2 undergo ligand-induced endocytosis. Analysis of endosomal fractions following immunoprecipitation with AT1 or B2 antibodies detected strong association of EP24.15 with the receptors in both light and heavy endosomal populations. Therefore, the present study indicates that EP24.15 associates with AT1 and B2 receptors both at the plasma membrane and after receptor internalization and suggests a possible mechanism for endosomal disposition of ligand that may facilitate receptor recycling.

• Imamura Y, Katahira T, Kitamura D
• Identification and characterization of a novel BASH N terminus-associated protein, BNAS2.
• J Biol Chem. 2004; 279: 26425-32
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• A B cell-specific adaptor protein, BASH (also known as BLNK or SLP-65), is crucial for B cell receptor (BCR) signaling. BASH binds to various signaling intermediates, such as Btk, PLCgamma2, Vav, and Grb2, through its well defined motifs. Although functional significance of such interactions has been documented, BASH-mediated signal transduction mechanism is not fully understood. Using the yeast two-hybrid system, we have identified a novel protein that binds to a conserved N-terminal domain of BASH, which we named BNAS2 (BASH N terminus associated protein 2). From its deduced amino acid sequence, BNAS2 is presumed to contain four transmembrane domains, which are included in a central MARVEL domain, and to localize to endoplasmic reticulum. BNAS2 was co-precipitated with BASH as well as Btk and ERK2 from a lysate of mouse B cell line. In the transfected cells, the exogenous BNAS2 was localized in a mesh-like structure in the cytoplasm resembling that of endoplasmic reticulum (ER) and nuclear membrane. BASH was co-localized with BNAS2 in a manner dependent on its N-terminal domain. RT-PCR analysis indicated that BNAS2 mRNA is expressed ubiquitously except for plasma cells. In chicken B cell line DT40, overexpression of BNAS2 resulted in an enhancement of BCR ligation-mediated transcriptional activation of Elk1, but not of NF-kappaB, in a manner dependent on the dose of BNAS2. Thus BNAS2 may serve as a scaffold for signaling proteins such as BASH, Btk, and ERK at the ER and nuclear membrane and may facilitate ERK activation by signaling from cell-surface receptors.

• Chen D, Zhao M, Mundy GR
• Bone morphogenetic proteins.
• Growth Factors. 2004; 22: 233-41
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• Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smad1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and related genes have shown that BMP signaling plays critical roles in heart, neural and cartilage development. BMPs also play an important role in postnatal bone formation. BMP activities are regulated at different molecular levels. Preclinical and clinical studies have shown that BMP-2 can be utilized in various therapeutic interventions such as bone defects, non-union fractures, spinal fusion, osteoporosis and root canal surgery. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue. BMPs are members of the TGFbeta superfamily. The activity of BMPs was first identified in the 1960s (Urist, M.R. (1965) "Bone formation by autoinduction", Science 150, 893-899), but the proteins responsible for bone induction remained unknown until the purification and sequence of bovine BMP-3 (osteogenin) and cloning of human BMP-2 and 4 in the late 1980s (Wozney, J.M. et al. (1988) "Novel regulators of bone formation: molecular clones and activities", Science 242, 1528-1534; Luyten, F.P. et al. (1989) "Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation", J. Biol. Chem. 264, 13377-13380; Wozney, J.M. (1992) "The bone morphogenetic protein family and osteogenesis", Mol. Reprod. Dev. 32, 160-167). To date, around 20 BMP family members have been identified and characterized. BMPs signal through serine/threonine kinase receptors, composed of type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, type IA and IB BMP receptors (BMPR-IA or ALK-3 and BMPR-IB or ALK-6) and type IA activin receptor (ActR-IA or ALK-2) (Koenig, B.B. et al. (1994) "Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells", Mol. Cell. Biol. 14, 5961-5974; ten Dijke, P. et al. (1994) "Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4", J. Biol. Chem. 269, 16985-16988; Macias-Silva, M. et al. (1998) "Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2", J. Biol. Chem. 273, 25628-25636). Three type II receptors for BMPs have also been identified and they are type II BMP receptor (BMPR-II) and type II and IIB activin receptors (ActR-II and ActR-IIB) (Yamashita, H. et al. (1995) "Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects", J. Cell. Biol. 130, 217-226; Rosenzweig, B.L. et al. (1995) "Cloning and characterization of a human type II receptor for bone morphogenetic proteins", Proc. Natl Acad. Sci. USA 92, 7632-7636; Kawabata, M. et al. (1995) "Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-beta receptor", J. Biol. Chem. 270, 5625-5630). Whereas BMPR-IA, IB and II are specific to BMPs, ActR-IA, II and IIB are also signaling receptors for activins. These receptors are expressed differentially in various tissues. Type I and II BMP receptors are both indispensable for signal transduction. After ligand binding they form a heterotetrameric-activated receptor complex consisting of two pairs of a type I and II receptor complex (Moustakas, A. and C.H. Heldi (2002) "From mono- to oligo-Smads: the heart of the matter in TGFbeta signal transduction" Genes Dev. 16, 67-871). The type I BMP receptor substrates include a protein family, the Smad proteins, that play a central role in relaying the BMP signal from the receptor to target genes in the nucleus. Smad1, 5 and 8 are phosphorylated by BMP receptors in a ligand-dependent manner (Hoodless, P.A. et al. (1996) "MADR1, a MAD-related protein that functions in BMP2 signaling pathways", Cell 85, 489-500; Chen Y. et al. (1997) "Smad8 mediates the signaling of the receptor serine kinase", Proc. Natl Acad. Sci. USA 94, 12938-12943; Nishimura R. et al. (1998) "Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12", J. Biol. Chem. 273, 1872-1879). After release from the receptor, the phosphorylated Smad proteins associate with the related protein Smad4, which acts as a shared partner. This complex translocates into the nucleus and participates in gene transcription with other transcription factors (). A significant advancement about the understanding of in vivo functions of BMP ligands, receptors and signaling molecules has been achieved in recent years.

• Wilcox A, Katsanakis KD, Bheda F, Pillay TS
• Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex.
• J Biol Chem. 2004; 279: 38881-8
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• The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.

• Guo DF et al.
• A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells.
• J Biol Chem. 2004; 279: 21109-20
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• Angiotensin II stimulates cellular hypertrophy in cultured vascular smooth muscle and renal proximal tubular cells. This effect is believed to be one of earliest morphological changes of heart and renal failure. However, the precise molecular mechanism involved in angiotensin II-induced hypertrophy is poorly understood. In the present study we report the isolation of a novel angiotensin II type 1 receptor-associated protein. It encodes a 531-amino acid protein. Its mRNA is detected in all human tissues examined but highly expressed in the human kidney, pancreas, heart, and human embryonic kidney cells as well as rat vascular smooth muscle and renal proximal tubular cells. Protein synthesis and relative cell size analyzed by flow cytometry studies indicate that overexpression of the novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in cultured rat vascular smooth muscle and renal proximal tubular cells. In contrast, the hypertrophic effects was reversed in renal proximal tubular cell lines expressing the novel gene in the antisense orientation and its dominant negative mutant, which lacks the last 101 amino acids in its carboxyl-terminal tail. The hypertrophic effects are at least in part mediated via protein kinase B activation or cyclin-dependent kinase inhibitor, p27(kip1) protein expression level in vascular smooth muscle, and renal proximal tubular cells. Moreover, angiotensin II could not stimulate cellular hypertrophy in renal proximal tubular cells expressing the novel gene in the antisense orientation and its mutant. These findings may provide new molecular mechanisms to understand hypertrophic agents such as angiotensin II-induced cellular hypertrophy.

• Seta K, Sadoshima J
• Phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor mediates angiotensin II-induced trans-activation of the epidermal growth factor receptor.
• J Biol Chem. 2003; 278: 9019-26
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• Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR.

• Guo DF, Chenier I, Tardif V, Orlov SN, Inagami T
• Type 1 angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membrane.
• Biochem Biophys Res Commun. 2003; 310: 1254-65
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• The carboxyl terminus of the type 1 angiotensin II receptor (AT(1)) plays an important role in receptor phosphorylation, desensitization, and internalization. The yeast two-hybrid system was employed to isolate proteins associated with the carboxyl terminal region of the AT(1A) receptor. In the present study, we report the isolation of a novel protein, ARAP1, which promotes recycling of AT(1A) to the plasma membrane in HEK-293 cells. ARAP1 cDNA encodes a 493-amino-acid protein and its mRNA is ubiquitously expressed in rat tissues. A complex of ARAP1 and AT(1A) was observed by immunoprecipitation and Western blotting in HEK-293 cells. In the presence of ARAP1, recycled AT(1A) showed a significant Ca(2+) release response to a second stimulation by Ang II 30 min after the first treatment. Immunocytochemical analysis revealed co-localization of recycled AT(1A) and ARAP1 in the plasma membrane 45 min after the initial exposure to Ang II. Taken together, these results indicate a role for ARAP1 in the recycling of the AT(1) receptor to the plasma membrane with presumable concomitant recovery of receptor signal functions.

• Soond SM, Terry JL, Colbert JD, Riches DW
• TRUSS, a novel tumor necrosis factor receptor 1 scaffolding protein that mediates activation of the transcription factor NF-kappaB.
• Mol Cell Biol. 2003; 23: 8334-44
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• We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Co-immunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-alpha. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-kappaB and increased NF-kappaB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS(1-723)) was found to inhibit NF-kappaB activation by TNF-alpha. Co-precipitation and co-immunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK.

• Lopez-Ilasaca M, Liu X, Tamura K, Dzau VJ
• The angiotensin II type I receptor-associated protein, ATRAP, is a transmembrane protein and a modulator of angiotensin II signaling.
• Mol Biol Cell. 2003; 14: 5038-50
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• Our group identified angiotensin II type 1 (AT1) receptor-associated protein (ATRAP) in a yeast two-hybrid screen for proteins that bind to the carboxyl-terminal cytoplasmic domain of the AT1. In this work, we characterize ATRAP as a transmembrane protein localized in intracellular trafficking vesicles and plasma membrane that functions as a modulator of angiotensin II-induced signal transduction. ATRAP contains three hydrophobic domains at the amino-terminal end of the protein, encompassing the amino acid residues 14-36, 55-77, and 88-108 and a hydrophilic cytoplasmic carboxyl-terminal tail from residues 109-161. Endogenous and transfected ATRAP cDNA shows a particulate distribution; electron microscopy reveals the presence of ATRAP in prominent perinuclear vesicular membranes; and colocalization analysis by immunofluorescence shows that ATRAP colocalizes in an intracellular vesicular compartment corresponding to endoplasmic reticulum, Golgi, and endocytic vesicles. Real-time tracking of ATRAP vesicles shows constitutive translocation toward the plasma membrane. Using epitope-tagged forms of ATRAP at either the amino or carboxyl end of the molecule, we determined the orientation of the amino end as being outside the cell. Mutant forms of ATRAP lacking the carboxyl end are unable to bind to the AT1 receptor, leading to the formation of prominent perinuclear vesicle clusters. Functional analysis of the effects of ATRAP on angiotensin II-induced AT1 receptor signaling reveals a moderate decrease in the generation of inositol lipids, a marked decrease in the angiotensin II-stimulated transcriptional activity of the c-fos promoter luciferase reporter gene, and a decrease in cell proliferation.

• Yu J et al.
• Global chimeric exchanges within the intracellular face of the bradykinin B2 receptor with corresponding angiotensin II type Ia receptor regions: generation of fully functional hybrids showing characteristic signaling of the AT1a receptor.
• J Cell Biochem. 2002; 85: 809-19
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• The intracellular (IC) face of the G-protein coupled receptors (GPCR), bradykinin (BK) B2 and angiotensin (AT) 1a, is similar in sequence homology and in size. Both receptors are known to link to Galphai and Galphaq but differ markedly in a number of physiologic actions, particularly with respect to their hemodynamic action. We made single as well as multiple, global replacements within the IC of BKB2R with the corresponding regions of the AT1aR. When stably transfected into Rat-1 cells, these hybrid receptors all bound BK with high affinity. Single replacement of the intracellular loop 2 (IC2) or the distal 34 residues of the C-terminus (dCt) with the corresponding regions of AT1aR resulted in chimera, which turned over phosphotidylinositol (PI) and released arachidonic acid (ARA) as WT BKB2R. In contrast, incorporation of the AT1aR IC3 in a single replacement abolished signal transduction. However, the simultaneous exchange of IC2 and IC3 of BKB2R with AT1aR resulted in a receptor responding to BK with PI turnover and ARA release approximately 4-fold greater than WT BKB2R. Likewise, the simultaneous replacement of IC2 and dCt resulted in a 2.8- and 1.6-fold increase in PI turnover and ARA release, respectively. In contrast, the dual replacement of IC3 and dCt could not overcome the deleterious effects of the IC3 replacement, resulting in very low PI activation and ARA release. Replacement of all three IC domains (IC2, IC3, and dCt) resulted in PI closer to that of AT1aR than BKB2R. The uptake of the receptor chimeras was similar to that of WT BKB2R with the exception of the IC3/dCt dual mutant, which exhibited very poor internalization (18% at 60'). When transfected into Rat-1 cells, the AT1aR markedly increased the expression of connective tissue growth factor (CTGF) mRNA, while BK slightly decreased it. The dual IC2/dCt and triple IC2/IC3/dCt hybrids both upregulated CTGF mRNA in response to BK. These results show that the IC face of the BKB2R can be exchanged with that of AT1aR, producing hybrid receptors, which take on the functional characteristics of AT1aR. The characterization of the chimera with stepwise replacement of the IC domains should allow for assignment of specific roles to the individual loops and C-terminus in the signaling and internalization of the BKB2R and facilitate the generation of a receptor with BKB2R binding and AT1aR function.

• Li B, Cong F, Tan CP, Wang SX, Goff SP
• Aph2, a protein with a zf-DHHC motif, interacts with c-Abl and has pro-apoptotic activity.
• J Biol Chem. 2002; 277: 28870-6
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• c-Abl is a non-receptor tyrosine kinase implicated in DNA damage-induced cell death and in growth factor receptor signaling. To further understand the function and regulation of c-Abl, a yeast two-hybrid screen was performed to identify c-Abl-interacting proteins. Here we report the identification of Abl-philin 2 (Aph2), encoding a novel protein with a unique cysteine-rich motif (zf-DHHC) and a 53-amino acid stretch sharing homology with the creatine kinase family. The zf-DHHC domain is highly conserved from yeast to human. Two proteins containing this motif, Akr1p and Erf2p, have been characterized in Saccharomyces cerevisiae, both implicated in signaling pathways. Deletion analysis by two-hybrid assays revealed that the N-terminal portion of Aph2 interacts with the C terminus of c-Abl. Aph2 was demonstrated to interact with c-Abl by co-immunoprecipitation assays. Aph2 is expressed in most tissues tested and is localized in the cytoplasm, mainly in the endoplasmic reticulum (ER). The sequences required for ER location reside in the N terminus and the zf-DHHC motif of Aph2. It has been reported that a portion of c-Abl is localized in the ER. We demonstrate here that Aph2 and c-Abl are co-localized in the ER region. Overexpression of Aph2 leads to apoptosis as justified by TUNEL assays, and the induction of apoptosis requires the N terminus. Co-expression of c-Abl and Aph2 had a synergistic effect on apoptosis induction and led to a decreased expression of both proteins, suggesting either that these two proteins are mutually down-regulated or that cells expressing both c-Abl and Aph2 rapidly disappeared from the culture. These results suggest that Aph2 may be involved in ER stress-induced apoptosis in which c-Abl plays an important role.

• Yu J, Prado GN, Taylor L, Pal-Ghosh R, Polgar P
• Hybrid formation between the intracellular faces of the bradykinin B2 and angiotensin II AT1 receptors and signal transduction.
• Int Immunopharmacol. 2002; 2: 1807-22
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• Most frequently, the physiologic functions of the angiotensin II (Ang II) type 1 receptor (AT1R) and bradykinin B2 receptor (BKB2R) are antagonistic, particularly with respect to the regulation of vascular tone. Despite major differences in their physiologic actions, the receptors share sequence similarities. Both link to Galpha(i) and Galpha(q) and transduce very similar signal paths, not only those relating to the traditional G-protein associated second messengers, but also those involved in transactivation mechanisms involving receptor tyrosine kinases. With respect to these paths, some differences in signaling may be accounted for by cell type specificity. However, alternative signal cascades for these two receptors are becoming increasingly evident. One such is the recruitment of signaling molecules upon receptor translocation and internalization. The AT1R translocates into clathrin-coated pits and internalizes upon recruitment of beta-arrestin 2 which then recruits ASK1 and JNK3. The BKB2R translocates and internalizes mainly via caveolae. Another signaling divergence may be due to the direct activation of small G-proteins by both receptors. AT1R activates the RhoA, Rac1, Cdc42 while BKB2R couples only with Rac1 and Cdc42. Both receptors may serve as docking stations for intracellular proteins. One such example is the YIPP motif within the C-terminus of the ATIR which associates with the JAK/STAT pathway. Another potential alternative is the activation of tyrosine/serine kinase phosphatases by BK. This mechanism may directly oppose some of the protein tyrosine/ serine kinase paths activated by AT1R. These alternative mechanisms in sum are potentially responsible for the diversion in signal transduction between these two receptors. Regardless of the route of action, our results suggest that in Rat-1 fibroblasts stably transfected with BKB2R, BK slightly decreases connective tissue growth factor (CTGF) mRNA level while in ATIR transfected cells Ang II increases CTGF mRNA markedly. To determine whether mutant hybrids can be formed between these two receptors which encompass some of the function of the donor receptor but bind the ligand of the recipient receptor, a series of hybrids were formed with BKB2R the recipient and AT1R the donor receptor. Some of these hybrids show resistance to exchanges with the AT1R and form receptors which either do not bind (IC1 exchanges) or demonstrate poor function but normal internalization (proximal C-terminus exchanges). However, other hybrids have proven very functional. For example, the IC2, IC3 and distal C-terminus of the BKB2R IC face can be replaced simultaneously with the AT1R resulting in an hybrid which binds BK, continues to signal, is internalized and resensitized. Formation of this and other less extensive hybrids is discussed. Some of these hybrids possess the capacity to function as the AT1R as exemplified by their ability to upregulate CTGF expression as wild-type (WT) AT1R.

• Feng YH, Sun Y, Douglas JG
• Gbeta gamma -independent constitutive association of Galpha s with SHP-1 and angiotensin II receptor AT2 is essential in AT2-mediated ITIM-independent activation of SHP-1.
• Proc Natl Acad Sci U S A. 2002; 99: 12049-54
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• Conventional mode of activation of SH2 domain-containing phosphatase 1 (SHP-1) by a single transmembrane (TM) inhibitory receptor such as killer cell inhibitory receptor, Fcgamma receptor type IIb1, and paired Ig-like receptors of inhibitory types requires tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory (ITIM) motifs in the cytoplasmic domains of the inhibitory receptors. Contrary to this paradigm, AT(2), a G protein-coupled 7TM receptor that does not undergo tyrosine phosphorylation in response to angiotensin II (Ang II) stimulation, also activates SHP-1. Here we show that SHP-1 constitutively and physically associates with AT(2) receptor in transfected COS-7 cells. On stimulation by Ang II, SHP-1 becomes activated and dissociated from AT(2) receptor, independent of pertussis toxin. Cotransfection of transducin G(betagamma) inhibits SHP-1/AT(2) association and the SHP-1 activation, whereas cotransfection of C-terminal of beta-adrenergic receptor kinase, which abrogates G(betagamma) signaling, facilitates SHP-1 activation. Surprisingly, SHP-1/AT(2) association and the SHP-1 activation requires the presence of G(alphas) as shown by differential coimmunoprecipitation, dominant negative G(alphas), constitutively active G(alphas), and G(alpha) peptides. A mutant AT(2) receptor D141A-R142L that is inactive in G(alpha) protein activation constitutively associates with SHP-1 and activates it. Together, these results indicate that G(alphas) alone, rather than exclusively in the form of G(alphabetagamma) heterotrimer may facilitate signal transduction for G protein-coupled receptors, suggesting a novel mechanism distinct from the classic paradigm of heterotrimeric G proteins. The AT(2)-mediated ITIM-independent activation of SHP-1 that is distinct from the conventional mode of activation, may represent a general paradigm for activation of SHP-1/2-class tyrosine phosphatases by G protein-coupled receptors.

• Scott RO, Thelin WR, Milgram SL
• A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor.
• J Biol Chem. 2002; 277: 22934-41
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• Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin.

• Pulakat L, Gray A, Johnson J, Knowle D, Burns V, Gavini N
• Role of C-terminal cytoplasmic domain of the AT2 receptor in ligand binding and signaling.
• FEBS Lett. 2002; 524: 73-8
• Display abstract

• A stop codon at position 322 was introduced to generate a truncated, C-terminal-deleted AT2 receptor. Expression studies in Xenopus oocytes showed that C-terminal-deleted AT2 had reduced affinity to [(125)I]angiotensin II (K(d)=1.7 nM) and enhanced binding of the AT2-specific peptidic ligand [(125)I]CGP42112A (K(d)=0.097 nM). AT2 activation by angiotensin II resulted in reduction of cGMP levels in oocytes and this reduction was further enhanced by C-terminal deletion, implying that the C-terminus may have a negative effect on the AT2-mediated cGMP reduction. Moreover, interaction of the AT2 with the ATP-binding domain of the human ErbB3 receptor in yeast two-hybrid assay was abolished by C-terminal deletion. In summary, the C-terminal cytoplasmic tail of AT2 modulates its ligand binding and signaling properties.

• Seong HA, Gil M, Kim KT, Kim SJ, Ha H
• Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38).
• Biochem J. 2002; 361: 597-604
• Display abstract

• We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family [Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295-301]. Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38. In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38. In addition, ZPR9 physically interacts with itself in mammalian cells. The ZPR9 cDNA hybridized with a mRNA species of approx. 1.7 kb in Northern-blot analysis. The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen,liver and brain. Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus. These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo.

• Guo DF, Sun YL, Hamet P, Inagami T
• The angiotensin II type 1 receptor and receptor-associated proteins.
• Cell Res. 2001; 11: 165-80
• Display abstract

• The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.

• Knowle D, Ahmed S, Pulakat L
• Identification of an interaction between the angiotensin II receptor sub-type AT2 and the ErbB3 receptor, a member of the epidermal growth factor receptor family.
• Regul Pept. 2000; 87: 73-82
• Display abstract

• To identify the proteins that interact and mediate angiotensin II receptor AT2-specific signaling, a random peptide library was screened by yeast-based Two-Hybrid protein-protein interaction assay technique. A peptide that shared significant homology with the amino acids located between the residues Gly-Xaa-Gly-Xaa-Xaa-Gly721 and Lys742, the residues predicted to be important for ATP binding of the ErbB3 and ErbB2 receptors, was identified to be interacting with the AT2 receptor. The interaction between the human ErbB3 receptor and the AT2 receptor was further confirmed using the cytoplasmic domain (amino acids 671-782) of the human ErbB3 receptor. Moreover, an AT2 receptor peptide that spans the amino acids 226-363, (spans the third ICL and carboxy terminal domain) could also interact with the AT2 receptor in a yeast Two-Hybrid protein-protein interaction assay. Studies using mutated and chimeric AT2 receptors showed that replacing the third intracellular loop (ICL) of the AT2 receptor with that of the AT1 abolishes the interaction between the ErbB3 and the AT2 in yeast Two-Hybrid protein-protein interaction assay. Thus the interaction between the AT2 receptor and the ErbB3 receptor seems to require the region spanning the third ICL and carboxy terminus of the AT2 receptor. Since the third ICL of the AT2 receptor is essential for exerting its inhibitory effects on cell growth, possible involvement of this region in the interaction with the cytoplasmic domain of the ErbB3 receptor suggests a novel signaling mechanism for the AT2 receptor mediated inhibition of cell growth. Furthermore, since both the AT2 and the ErbB3 receptors are expressed during fetal development, we propose that the existence of direct interaction between these two receptors may play a role in the regulation of growth during the initial stages of development.

• Gosselin MJ, Leclerc PC, Auger-Messier M, Guillemette G, Escher E, Leduc R
• Molecular cloning of a ferret angiotensin II AT(1) receptor reveals the importance of position 163 for Losartan binding.
• Biochim Biophys Acta. 2000; 1497: 94-102
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• A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I- inverted question markSar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist inverted question markSar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.

• Hosoda Y, Fujino I, Akagawa K, Kuwahara A
• Molecular cloning of guinea pig angiotensin type 1 receptor.
• Biol Signals Recept. 2000; 9: 231-9
• Display abstract

• The primary structure of cDNA encoding of the angiotensin type 1 receptor (AT(1)R) was cloned from guinea pig liver. Guinea pig AT(1)R (GP-AT(1)R) cDNA clone contains a 1,077-bp open reading frame which encodes a protein consisting of 359 amino acid residues. GP-AT(1)R amino acid sequence showed a 92% level of identity among mammalian species. GP-AT(1)R is expressed in liver, kidney, adrenal gland, heart and colon.

• Thomas WG, Pipolo L, Qian H
• Identification of a Ca2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT1A) receptor.
• FEBS Lett. 1999; 455: 367-71
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• To identify regulators of the type 1A angiotensin II receptor (AT1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT1A receptor carboxyl-terminus. An approximately 20 kDa cytoplasmic protein interacted with the fusion protein in a Ca2+-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca2+/calmodulin and a peptide corresponding to a membrane proximal portion of the AT1A receptor carboxyl-terminus with analogy to known calmodulin-binding sequences were synthesised and tested for calmodulin-binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca2+-dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT1A receptor by Ca2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT1A receptor function.

• Galiegue S et al.
• Cloning and characterization of PRAX-1. A new protein that specifically interacts with the peripheral benzodiazepine receptor.
• J Biol Chem. 1999; 274: 2938-52
• Display abstract

• Using a cytoplasmic domain of the peripheral benzodiazepine receptor (PBR) as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a new protein that specifically interacts with PBR. We named it PRAX-1, for peripheral benzodiazepine receptor-associated protein 1. PRAX-1 is a 1857-amino acid protein, the sequence of which was structurally unrelated to any known proteins. The gene encoding PRAX-1 is located in the q22-q23 region of the long arm of the human chromosome 17. The PRAX-1 mRNA is 7.5 kilobase pairs, predominantly expressed in the central nervous system, pituitary gland, and thymus. At the protein level, we found the PRAX-1 as a single 220-250-kDa protein in the brain and in many different human cell lines tested using specific antibody raised against PRAX-1. Parallel analysis of the PRAX-1 mRNA and protein expression performed in mouse and rat gave similar results. Immunocytochemistry analysis carried out to define the distribution of the PRAX-1 protein in the rat brain showed that PRAX-1 was prevalent in the mesolimbic system, specially abundant in the CA1 subfield of the hippocampus. Exhibiting several domains involved in protein-protein interaction (three proline-rich domains, three leucine-zipper motifs, and an Src homology region 3-like domain), the PRAX-1 may be looked upon as a new adaptator protein. We show that both the Src homology region 3-like domain and a proline-rich domain in PRAX-1 are required for the interaction with PBR. PRAX-1 is a cytoplasmic protein that also partially colocalizes with PBR in the mitochondria, as determined by confocal microscopy and Western blotting. Altogether our observations support a model of interaction implicating PBR and this newly described protein, PRAX-1. As being the first cytoplasmic protein associated with PBR, PRAX-1 is a new tool that opens new fields for exploring PBR biological roles.

• Millican DS, Bird IM
• Isolation of an ovine genomic sequence containing the full-length angiotensin II type-1 receptor.
• Endocr Res. 1998; 24: 387-90
• Display abstract

• We have isolated from a genomic library using PCR amplification an 1171 base sequence containing a putative ovine AT1-R protein coding sequence of 1080 bases. As expected the protein coding sequence is of greater than 99% homology to the partial protein coding sequence reported by Robillard et al, with only one base difference. Relative to other species, highest homology at the level of the cDNA protein coding sequence is to bovine (97.6%) and lowest homology to rat Type 1a (83.3%). The predicted protein amino acid sequence in turn encodes a protein with the properties of a seven alpha-helix transmembrane receptor (by TMPred) sharing closest homology (98.6%) to the bovine receptor and lowest to the rat Type 1a (90.2%). As expected from such a high degree of interspecies homology, amino acids identified by site-directed mutagenesis of the human or rat AT1A-R as involved in binding and action of AII are very highly conserved in the ovine sequence. In addition, both bovine and ovine AT1-R are known to exhibit lower affinity for DuP753 than human AT1-R, and in bovine AT1-R this has been suggested to coincide with the amino acid substitutions Ala->Thr (163) and Leu->Met (262) relative to the human sequence. Our ovine AT1-R cDNA sequence shares these same bovine substitutions.

• Ruiz-Opazo N
• Identification of a novel dual angiotensin II/vasopressin receptor.
• Nephrologie. 1998; 19: 417-20
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• The isolation and molecular characterization of the Ang II/AVP receptor elucidates the structure of a novel dual receptor coupled to adenylate cyclase and responding with equal sensitivity to Ang II and AVP. The cloning strategy in conjunction with site directed mutagenesis have permitted the delineation of the Ang II and AVP binding domains within the receptor polypeptide. Pharmacological characterization of the receptor defines the AngII/AVP receptor as a novel AT1/V2 type of receptor. The renal immunocytochemical distribution of the Ang II/AVP receptor to the outer medullary thick ascending limb tubules and inner medullary collecting ducts suggests a prominent role in renal tubular sodium and fluid reabsorption.

• Moriuchi R, Shibata S, Himeno A, Johren O, Hoe KL, Saavedra JM
• Molecular cloning and pharmacological characterization of an atypical gerbil angiotensin II type-1 receptor and its mRNA expression in brain and peripheral tissues.
• Brain Res Mol Brain Res. 1998; 60: 234-46
• Display abstract

• In the gerbil brain, most of the [125I]Sarcosine1-Angiotensin II binding sites are atypical, not sensitive to displacement with selective Angiotensin II AT1 and AT2 receptor ligands. A similar atypical binding profile exists in the gerbil kidney, where binding is highly expressed. We isolated a 2197 base pair clone from a gerbil kidney cDNA library which encodes a 359 amino acid protein with higher than 90% homology to other mammalian angiotensin II AT1 receptors. When expressed in COS-7 cells, stimulation by Angiotensin II of both the cloned gerbil receptor or the human AT1 receptor enhanced IP3 production to a similar degree. In COS-7 cells, the gerbil receptor also had a ligand affinity profile similar to that of the human AT1 receptor, but showed greatly reduced affinity for losartan (IC50=3480+/-174 nM). In the gerbil brain, in situ hybridization revealed receptor mRNA in circumventricular organs, selective hypothalamic, midbrain and brain stem areas, and in the hippocampus, where high mRNA expression was detected in the stratum pyramidale of the CA1 and CA2 subfields, and in the stratum granulosum of the dentate gyrus. The expression pattern of receptor mRNA corresponded well with that of atypical [125I]Sar1-Ang II binding. In situ hybridization and Southern blot experiments using riboprobes against the open reading frame and the 3'-untranslated region of the cloned gerbil Ang II receptor cDNA suggest that gerbils have, like other rodents, two AT1 receptor subtypes. The receptor mRNA distribution of the cloned gerbil Ang II receptor corresponds to the distribution of AT1A receptors described in other rodent species.

• Su B, Martin MM, Elton TS
• Human AT1 receptor gene regulation.
• Adv Exp Med Biol. 1996; 396: 11-21

• Ichiki T, Kambayashi Y, Inagami T
• Molecular cloning and expression of angiotensin II type 2 receptor gene.
• Adv Exp Med Biol. 1996; 396: 145-52

• Razdan K, Kroll MH
• Molecular cloning of a novel platelet protein showing homology to the angiotensin II receptor C-terminal domain.
• J Biol Chem. 1996; 271: 2221-4
• Display abstract

• Oligoscreening of a cDNA library obtained from 4 beta-phorbol 12-myristate 13-acetate-stimulated human erythroleukemia (HEL) cells resulted in the isolation of a novel clone coding for a protein with a calculated molecular mass of 8110 Da. This protein of 71 amino acids shows significant homology to the carboxyl-terminal regulatory domain of angiotensin II type 1 receptors. The homology encompasses four regions of amino acid residues thought to serve as consensus sequences for phosphorylation by serine/threonine kinases such as protein kinase C, which are key mediators of intracellular signaling. Reverse transcription-polymerase chain reaction identified the transcript in human platelets, human megakaryocytic DAMI cells, and HEL cells. High stringency Northern blotting revealed a tissue-specific distribution of three transcript species, with predominant expression in skeletal muscle and pancreas. Rabbit anti-peptide antiserum was used to immunoblot protein lysates from washed resting platelets and from 4 beta-phorbol 12-myristate 13-acetate-stimulated DAMI and HEL cells. These immunoblots revealed the presence of an intense approximately 8-kDa protein band in platelets and HEL cells and a faint band of identical size in DAMI cells.

• Martin MM, Su B, Elton TS
• Molecular cloning of the human AT2 receptor.
• Adv Exp Med Biol. 1996; 396: 153-65

• Nahmias C et al.
• Molecular and functional characterization of angiotensin II AT2 receptor in neuroblastoma N1E-115 cells.
• Adv Exp Med Biol. 1996; 396: 167-73

• Zeiner M, Gehring U
• A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.
• Proc Natl Acad Sci U S A. 1995; 92: 11465-9
• Display abstract

• In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

• Konishi H, Kuroda S, Inada Y, Fujisawa Y
• Novel subtype of human angiotensin II type 1 receptor: cDNA cloning and expression.
• Biochem Biophys Res Commun. 1994; 199: 467-74
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• Angiotensin II (AII) plays a major role in regulation of cardiovascular function and fluid homeostasis through the action of an AII type 1 receptor (AT1R). The cDNA encoding a novel subtype of human AT1R (AT1bR) was cloned from a human placental cDNA library. The full-length cDNA clone (1563 bp) encoded a polypeptide that consists of 359 amino acid (aa) residues with 97.2% aa identity to the human AT1aR. All the aa replacements between two human AT1Rs reside within the C-terminal half region of AT1R molecule. The 2.4-knt AT1bR mRNA is expressed in the lung, placenta and liver, and differs from AT1aR mRNA in its tissue distribution. The AT1bR expressed in COS-7 cells is pharmacologically distinct from the human AT1aR.

• Ichiki T, Herold CL, Kambayashi Y, Bardhan S, Inagami T
• Cloning of the cDNA and the genomic DNA of the mouse angiotensin II type 2 receptor.
• Biochim Biophys Acta. 1994; 1189: 247-50
• Display abstract

• Of the two major isoforms of the angiotensin II receptors, type 1 (AT1) and type 2 (AT2), little is known about the structure and features of AT2. We cloned a mouse AT2 cDNA from a mouse fetus cDNA library and an AT2 genomic DNA from a 129SV mouse genomic DNA library. The amino acid sequence of the mouse AT2 (363 residues) deduced from a mouse cDNA clone showed seven membrane-spanning domains. Amino acid identity of the mouse AT2 with mouse AT1 is 37%, and 98% with rat AT2. The genomic DNA (4.4 kb) contained three exons and two introns and the entire coding region was contained in the third exon.

• Ohnaka K, Takayanagi R, Nawata H
• [Molecular biology of human angiotensin II receptor]
• Nippon Rinsho. 1993; 51: 1568-74
• Display abstract

• Pharmacological studies have revealed the presence of two different subtypes (AT1 and AT2) for angiotensin II (Ang II) receptor. Recently, bovine and rat cDNAs of AT1 receptor were cloned by expression cloning. We have isolated a cDNA encoding human AT1 receptor from a human liver cDNA library, using rat AT1 receptor cDNA as a probe. Human AT1 receptor consists of 359 amino acid residues with a relative Mr of 41,060 and seven transmembrane segments, and was highly homologous to those of bovine, rat and mouse AT1 receptors. Southern blot analysis suggested that human AT1 receptor is a single copy gene.

• Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ
• Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors.
• J Biol Chem. 1993; 268: 24539-42
• Display abstract

• Angiotensin II acts on at least two distinct receptor subtypes (AT1 and AT2). Most known effects of angiotensin II in adult tissues are attributable to the AT1 receptor. The function of AT2 receptor is undefined, but its abundant expressions in fetal tissues, immature brain, skin wound, and atretic ovarian follicles suggest a role in growth and development. Previous studies suggested that AT2 receptor may not be G protein-coupled. Here, from a rat fetus expression library, we cloned a cDNA encoding a unique 363-amino acid protein with pharmacological specificity, tissue distribution, and developmental pattern of the AT2 receptor. It is 34% identical in sequence to the AT1 receptor, sharing a seven-transmembrane domain topology. A review of prior data on other receptors suggests that this receptor may belong to a unique class of seven-transmembrane receptors (including somatostatin SSTR1, dopamine D3, and frizzled protein Fz) for which G protein coupling has not been demonstrated. All members of this class exhibit fetal and developmental and/or neuronal-specific expression. A conserved motif in the third intracellular loop, distinguishing this class from "classical" G protein-coupled receptors, may mediate novel intracellular effects.

• Regoli D, D'Orleans-Juste P, Rouissi N, Rhaleb NE
• Vasoactive peptides and characterization of their receptors.
• Regul Pept. 1993; 45: 323-40

• Griendling KK, Lassegue B, Alexander RW
• The vascular angiotensin (AT1) receptor.
• Thromb Haemost. 1993; 70: 188-92

• Bergsma DJ et al.
• Isolation and expression of a novel angiotensin II receptor from Xenopus laevis heart.
• Mol Pharmacol. 1993; 44: 277-84
• Display abstract

• A Xenopus laevis heart cDNA library was screened using the human angiotensin type 1 (AT1) receptor cDNA coding sequence as a hybridization probe. A cDNA was isolated that encodes a protein of 363 amino acids that shares 63% sequence identity with the human AT1 receptor. Radioligand binding studies with the cloned receptor expressed in COS cells indicated that it is an angiotensin II receptor that possesses pharmacological properties distinct from those of the two known mammalian receptor subtypes, AT1 and AT2. Electrophysiological studies with the recombinant receptor expressed in X. laevis oocytes revealed that the amphibian receptor, like the mammalian AT1 receptor, can functionally couple to a second messenger system, leading to the mobilization of intracellular stores of calcium. However, nonpeptide antagonists selective for the mammalian AT1 and AT2 receptors do not block angiotensin II-stimulated functional responses in injected oocytes, which confirms that the amphibian receptor is a pharmacologically unique angiotensin II receptor. Nevertheless, based on conservation of structural features and motifs and similarity in coupling mechanisms, we speculate that the cloned Xenopus receptor is the amphibian counterpart of the mammalian AT1 receptor, having acquired its unique pharmacology as a consequence of evolutionary divergence.

• Sasamura H, Hein L, Krieger JE, Pratt RE, Kobilka BK, Dzau VJ
• Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome.
• Biochem Biophys Res Commun. 1992; 185: 253-9
• Display abstract

• We report the existence of two structurally distinct forms of the angiotensin receptor AT-1 in the mouse. A Balb/c mouse genomic library was screened by homology screening with a polymerase chain reaction (PCR) amplified probe. Restriction mapping and sequencing of the isolated genes revealed the presence of two receptor isoforms, here named the mouse AT-1a and AT-1b receptors, containing 22 different amino acids. Receptor binding studies performed on COS-7 cells transfected with the two receptors revealed that they had similar binding profiles for angiotensin II, angiotensin III and AT-1 or AT-2 specific antagonists. Because many of the structural differences were in the carboxy terminal putative intracellular domain, we speculate that these isoforms may differ in their regulation, signal transduction, or desensitization mechanisms.

• Mauzy CA, Hwang O, Egloff AM, Wu LH, Chung FZ
• Cloning, expression, and characterization of a gene encoding the human angiotensin II type 1A receptor.
• Biochem Biophys Res Commun. 1992; 186: 277-84
• Display abstract

• The human angiotensin II (AII) type 1a receptor gene and its upstream control sequence has been cloned from a human leukocyte genomic library. The promoter element CAAT and TATA sequences were found at -602 and -538, respectively, upstream from the translational initiation site. The deduced protein sequence is homologous to rat and bovine AT1a receptors (94.7% and 95.3% identity). The expressed gene exhibited high-affinity AII and Dup753 binding and was functionally coupled to inositol phosphate turnover. Northern analysis of human tissues showed AT1 receptor mRNA expression in placenta, lung, heart, liver, and kidney. Using 5' untranslated and coding sequence as probes in a Southern blot analysis, it was established that another AT1 subtype exists in the human genome.

• Bergsma DJ et al.
• Cloning and characterization of a human angiotensin II type 1 receptor.
• Biochem Biophys Res Commun. 1992; 183: 989-95
• Display abstract

• A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.

• Marie J, Desarnaud F, Lombard C, Seyer R, Bonnafous JC, Jard S
• Characterization of the angiotensin II receptor.
• Horm Res. 1990; 34: 95-100
• Display abstract

• We present a brief overview of the present knowledge on the structural and molecular properties of angiotensin II receptors and the various attempts to determine their primary structures, with special reference to our strategy for receptor purification. The strategy involves covalent labeling of the receptor with synthetic biotinylated photoactivatable probes, followed by indirect affinity chromatography on immobilized streptavidin. The various applications of these probes to the study of structural and molecular properties and to the cell biology of angiotensin II receptors are discussed.

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