Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Siemens, Ivo R.; Swanson, Geoffrey N.; Fluharty, Steven J.; Harding, Joseph W.

doi:10.1111/j.1471-4159.1991.tb03801.x

Cited by 21 publications

(7 citation statements)

References 47 publications

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“…For instance, in previous studies AngIl receptors from rat brain (15) and from NlE-115 cells (14) have been successfully solubilized by using the zwitterionic de- (14). These studies revealed that the AT2 binding site has an apparent molecular mass of 66 kDa as estimated by covalent crosslinking and SDS/PAGE analysis.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Development of polyclonal antibodies against angiotensin type 2 receptors.

Reagan

Théveniau²,

Yang³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Murine neuroblastoma N1E-115 cells are a useful system in which to study neuronal angiotensin II (AngII) receptors. NlE-115 cells possess both type 1 (AT1) and type 2 (AT2) AnglI receptor subtpes, as does mammalian brain. AT2 receptors in brain or NlE-115 cells can be solubilized in 3- [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. In the present study, heparin-Sepharose chromatography was used to partiafly purify solubilized NlE-115 membranes to produce an enriched population of AT2 receptors. Subsequendy, an eluted peak, containng the majority ofAT2 binding activity, was used as an immunogen in the development of protein-directed polyclonal antibodies. The antibodies specifically detected immunoreactive proteins of approximately 110 and 66 kDa in both solubilized NlE-115 cells, as well as the orignal protein material that eluted from the heparin-Sepharose column, whereas no such immunoreactivity was detected in a kidney epithelial cell line that lacks any specific 12SI-labeled AngI (12Ang binding activity. Moreover, the antibodies immuno d with affinit-purified AT2 receptors. These antibodies were also able to immunoprecipitate AT2 receptors from solubilized NlE-115 cells, as revealed by the pharmacologic proffle of 12I-A binding to the precipitated protein. Simllariy, the antibodies were able to immunoprecipitate a 66-kDa protein that had been covalently crosslinked with 12'I-AnH by use ofthe homobinctional crosslinker dithiobis(succinimidyl propionate). Collectively, these results demonstrate the development of a specific AT2 receptor antibody that may be used to further characterize this receptor subtype at both the cellular and molecular levels.The renin-angiotensin system is one of the most important regulators of body fluid and cardiovascular homeostasis (1). Angiotensin II (AngII) is known to exert its diverse effects by acting at membrane-bound receptors present on several peripheral target tissues, such as kidney, adrenal cortex, heart, and vascular smooth muscle (1). In addition to its many peripheral effects, all of the components of the reninangiotensin system have also been localized in the central nervous system (2). Moreover, it is now recognized that many of the physiological, endocrinological, and behavioral actions of AngII are mediated by specific receptors present in the brain (3).The diversity of actions mediated by AnglI was among the first pieces of evidence leading to the suggestion of multiple receptor subtypes for Angll. The unequivocal demonstration of such multiple receptor subtypes, however, came with the development of subtype-specific antagonists (4). The existence of at least two receptor subtypes for AngII has initiated the search for a greater understanding of these receptors at the cellular and molecular levels. Such analyses have revealed that both receptor subtypes, referred to as AT1 andThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S....

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Section: Discussionmentioning

confidence: 99%

“…Previously, we have demonstrated the successful solubilization of functional AT2 receptors from NlE-115 cells by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate (CHAPS) (14,15). These solubilized AT2 receptors retained their high affinity for 1251-labeled AngII (125I-AngII) and specificity for AT2-selective ligands.…”

mentioning

confidence: 99%

Development of polyclonal antibodies against angiotensin type 2 receptors.

Reagan

Théveniau²,

Yang³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Previously, AT 1 receptor dimerization has been suggested by using various approaches. 1) Using Angiotensin affinity labeling, or chemical cross-linking experiments, AT 1 receptor complexes with a molecular weight corresponding to a dimeric complex have been identified in transfected cell lines and in several organs such as liver adrenal pituitary and brain (41)(42)(43)(44)(45). 2) High affinity ligand binding has been reconstituted by co-expression of two receptors (AT 1 -K102A and -K199A) that did not bind ligand when expressed individually (17).…”

Section: Discussionmentioning

confidence: 99%

Oligomerization of Wild Type and Nonfunctional Mutant Angiotensin II Type I Receptors Inhibits Gαq Protein Signaling but Not ERK Activation

Hansen

Theilade

Haunsø

et al. 2004

Journal of Biological Chemistry

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The 7-transmembrane or G protein-coupled receptors relay signals from hormones and sensory stimuli to multiple signaling systems at the intracellular face of the plasma membrane including heterotrimeric G proteins, ERK1/2, and arrestins. It is an emerging concept that 7-transmembrane receptors form oligomers; however, it is not well understood which roles oligomerization plays in receptor activation of different signaling systems. To begin to address this question, we used the angiotensin II type 1 (AT 1 ) receptor, a key regulator of blood pressure and fluid homeostasis that in specific context has been described to activate ERKs without activating G proteins. By using bioluminescence resonance energy transfer, we demonstrate that AT 1 receptors exist as oligomers in transfected COS-7 cells. AT 1 oligomerization was both constitutive and receptor-specific as neither agonist, antagonist, nor co-expression with three other receptors affected the bioluminescence resonance energy transfer 2 signal. Furthermore, the oligomerization occurs early in biosynthesis before surface expression, because we could control AT 1 receptor export from the endoplasmic reticulum or Golgi by using regulated secretion/aggregation technology (RPD TM ). Co-expression studies of wild type AT 1 and AT 1 receptor mutants, defective in either ligand binding or G protein and ERK activation, yielded an interesting result. The mutant receptors specifically exerted a dominant negative effect on G␣ q activation, whereas ERK activation was preserved. These data suggest that distinctly active conformations of AT 1 oligomers can couple to each of these signaling systems and imply that oligomerization plays an active role in supporting these distinctly active conformations of AT 1 receptors.The 7-transmembrane (7TM) 1 or G protein-coupled receptors such as the angiotensin II type 1 (AT 1 ) receptor constitute the largest group of cell surface membrane receptors and mediate a vast array of biological effects in response to hormones, neurotransmitters, and sensory stimuli. The 7TM receptors activate or interact with numerous signaling proteins including heterotrimeric G proteins, arrestins, adaptor proteins, and extracellular signal-regulated kinases 1 and 2 (ERK1/2) at the intracellular face of the plasma membrane (1). Although 7TM receptors traditionally have been considered to work as monomeric entities, increasing evidence has shown that these receptors form both homo-and heterodimers or oligomers in vivo and in vitro. Whereas heterodimerization may provide a means to expand pharmacological diversity, the functional role of homodimerization is less well defined (2, 3). 7TM receptor heterodimerization has been shown to modify biological function, receptor trafficking, ligand binding properties, and signal transduction of several 7TM receptors (2, 3). The GABA B "receptor pair" provides an example of two different receptor subunits, the GABA B1 and GABA B2 , that both are necessary for receptor surface expression and function (4). The bradykinin B2 ...

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“…These analyses showed that is was possible to identify AT1R receptor complexes with molecular weights corresponding to both a monomeric and a dimeric form of the receptor. However, these observations were not linked to any functional consequences, and it has been speculated, that the dimers could result from technical artefacts causing protein aggregation (9,23,(49)(50)(51)(52)(53).…”

Section: At1 Receptor Activation and Signallingmentioning

confidence: 99%

Functional interactions between 7TM receptors in the Renin-Angiotensin System—Dimerization or crosstalk?

Lyngsø

Erikstrup

Hansen

2009

Molecular and Cellular Endocrinology

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Abstract:The Renin-Angiotensin System (RAS) is important for the regulation of cardiovascular physiology, where it controls blood pressure, and salt-and water homeostasis. Dysregulation of RAS can lead to severe diseases including hypertension, diabetic nephropathy, and cardiac arrhythmia, and -failure.The importance of the RAS is clearly emphasised by the widespread use of drugs targeting this system in clinical practice. These include, renin inhibitors, angiotensin II receptor type I blockers, and inhibitors of the angiotensin converting enzyme. Some of the important effectors within the system are 7 transmembrane (7TM) receptors (or G-protein-coupled receptors) such as the angiotensin II Receptors type I and II (AT1R and AT2R) and the MAS-oncogene receptor. Several findings indicate that the 7TM receptors can form both homo-and heterodimers, or higher orders of oligomers. Furthermore, dimerization may be important for receptor function, and in the development of cardiovascular diseases. This is very significant, since "dimers" may provide pharmacologists with novel targets for improved drug therapy. However, we know that 7TM receptors can mediate signals as monomeric units, and so far it has been very difficult to establish if our observations reflect actual well defined dimerization or merely reflect close proximity between the receptors and/or various types of functional interaction. In this review, we will present and critically discuss the current data on 7TM receptor dimerization with a clear focus on the RAS, and delineate future challenges within the field.

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Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Cited by 21 publications

References 47 publications

Development of polyclonal antibodies against angiotensin type 2 receptors.

Development of polyclonal antibodies against angiotensin type 2 receptors.

Oligomerization of Wild Type and Nonfunctional Mutant Angiotensin II Type I Receptors Inhibits Gαq Protein Signaling but Not ERK Activation

Functional interactions between 7TM receptors in the Renin-Angiotensin System—Dimerization or crosstalk?

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