Biologically active derivatives of angiotensin for labeling cellular receptors

Re, Galardy; Ss, Stafford; Ml, Schaefer; H, Ho; Ka, La Vorgna; Jd, Jamieson

doi:10.1021/jm00210a020

Cited by 23 publications

(17 citation statements)

References 14 publications

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“…The molecular weight of the responsible macromolecule was estimated as approximately 65,000-70,000 after radioiodinated angiotensin II had been crosslinked to it in a crude solubilized extract with disuccinimidyl suberate (11). Capponi and Catt (25) had estimated a similar size for the specific angiotensin II-binding molecule that they labeled in dog adrenal and uterine membranes by using a radioiodinated photoaffinity analog (26). In this communication we report two independent techniques, specifically based on recognition of angiotensin II, by which we have isolated a protein of this molecular weight in almost homogeneous form.…”

Section: Discussionmentioning

confidence: 84%

Isolation of an angiotensin II-binding protein from liver.

Sen¹,

Bull²,

Soffer³

1984

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

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A protein that specifically binds angiotensin II has been isolated in nearly homogeneous form by two independent approaches after solubilization from rabbit liver particles by treatment with digitonin. The protein purified by either of these methods resembles in size the single radioactive macromolecular component made by using disuccinimidyl suberate to crosslink radioiodinated angiotensin II with its receptor in the solubilized extract. In the first technique, angiotensin II as an affinity ligand specifically extracted the protein from a preparation that had been freed of angiotensin-degrading activity. In the second approach, the angiotensin II-protein complex was specifically precipitated by anti-angiotensin II antibodies and staphylococcal protein A-Sepharose. The protein could be eluted from the affinity column with angiotensin II or 4 M MgCl2. The angiotensin TI-protein complex dissociated in the presence of sulfhydryl-containing reagents, and these could therefore be used to elute it from either the chemical or the immunoaffinity-based matrix. This effect of sulfhydryl-containing reagents and the paradoxical observation that the isolated protein after denaturation exhibited a slower electrophoretic mobility in its reduced form than in its unreduced form suggest that the binding configuration of this protein may be sensitive to reduction.Angiotensin II is the biologically active component of the renin-angiotensin system (1, 2). Several mechanisms may contribute to its role as an important vasopressor agent. Among these are a contractive effect on blood vessels (1), a stimulation of cells of the adrenal zona glomerulosa to elaborate aldosterone (3-6), and a direct action on the central nervous system (7). These and other effects of angiotensin II are thought to be mediated by specific receptors on the surface of its target cells (8). Such receptors have been detected and their binding properties have been characterized in particulate fractions from various cell types (reviewed in ref. 9). In addition, they have recently been solubilized in active form from bovine adrenal (10) and rabbit hepatic (11) membranes. However, despite the fact that it is thought to mediate directly and thus determine the specificity of action of angiotensin II, the responsible receptor is the only component of the renin-angiotensin system that has not been purified and characterized in the isolated state. It has therefore not yet been possible to address directly certain important questions such as whether or not a chemically identical receptor molecule is responsible for the action of angiotensin II on different cell types.As a result of the development of inhibitors of angiotensinconverting enzyme (12, 13), it has already become apparent that suppression of the renin-angiotensin system represents an important approach to antihypertensive therapy (reviewed in ref. 14). However, this enzyme is a dipeptidyl carboxypeptidase capable of acting on many peptide substrates in addition to angiotensin I (reviewed in ref. 15). Ther...

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Section: Discussionmentioning

confidence: 84%

Isolation of an angiotensin II-binding protein from liver.

Sen¹,

Bull²,

Soffer³

1984

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

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“…Three of these probes, representing full agonist, partial agonist, and antagonist, had a photolabile Bpa at the amino terminus of the pharmacophoric domain of the peptide in the identical position of residue 24 of CCK-33 (standard numbering convention for CCK peptides). The fourth probe was also a fluorescent full agonist, but with the Bpa residue positioned in the middle of the peptide pharmacophoric domain, in the site of Gly 29 . The fifth probe was a radioiodinatable-Bpa 24 agonist, fully analogous to one of the key fluorescent compounds in the series.…”

Section: Resultsmentioning

confidence: 99%

“…The only difference from the fully characterized analogues was the addition of a fluorescent bodipy moiety with a glycine spacer at the amino terminus of the peptides, a position known to be fully tolerant of such a modification (19). These probes represent the following: BodipyBpa 24 agonist, Bodipy-Gly-Bpa-Gly-Asp-Tyr(SO 3 )-Nle-Gly-Trp-NleAsp-Phe-NH 2 ; Bodipy-Bpa 24 -partial agonist, Bodipy-Gly-Bpa-Gly-AspTyr(SO 3 )-Nle-Gly-Trp-Nle-Asp-phenylethyl ester; Bodipy-Bpa 24 -antagonist, Bodipy-Gly-Bpa-Gly-Asp-Tyr(SO 3 )-Nle-Gly-(D-Trp)-NleAsp-phenylethyl ester; and Bodipy-Bpa 29 agonist, Bodipy-Gly-AspTyr(SO 3 )-Nle-Bpa-Trp-Nle-Asp-Phe-NH 2 . Each peptide was synthesized to the level of the photolabile Bpa residue, deprotected, and purified to homogeneity by HPLC.…”

Section: Methodsmentioning

confidence: 99%

“…This was also observed with partial agonist and antagonist probes (data not shown). This suggests that the Bodipy-BPA 29 agonist site of attachment to the CCK receptor was quite different from that of the other probes or at least has a markedly different charge-to-mass ratio allowing it to migrate quite differently in free solution CE.…”

Section: Identification Of the Cck Receptor Domains Affinity Labeled mentioning

confidence: 99%

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Structurally Related Peptide Agonist, Partial Agonist, and Antagonist Occupy a Similar Binding Pocket within the Cholecystokinin Receptor

Dong

Ding

Pinon

et al. 1999

Journal of Biological Chemistry

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The molecular basis of ligand binding to receptors provides important insights for drug development. Here, we explore domains of the cholecystokinin (CCK) receptor that are critical for ligand binding, using a novel series of fluorescent photolabile probes, receptor proteolysis, and rapid high resolution separation of peptide fragments by capillary electrophoresis. Each probe incorporated the same fluorophore and a photolabile p-benzoylphenylalanine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-33) of CCK analogues representing full agonist, partial agonist, and antagonist of this receptor. Each was used to label the CCK receptor expressed on Chinese hamster ovary-CCKR cells, with the labeled domain then released by cyanogen bromide cleavage. Capillary electrophoresis with laser-induced fluorescence detection achieved an on-capillary mass sensitivity of 1.6 attomoles (10 ؊18 mol), with an excellent signal-to-noise ratio. Each of the biologically divergent, but structurally similar probes saturably and specifically labeled the same receptor domain, consistent with conservation of "docking" determinants. This had an apparent mass of 2.9 kDa, most consistent with the first extracellular loop domain. An additional probe having its site of covalent attachment in a different region of the probe (residue 29 of CCK-33) labeled a distinct receptor fragment with differential migration on capillary electrophoresis (third extracellular loop). Identification of the specific receptor residue(s) covalently linked to the amino-terminal probes must await further fragmentation and sequence analysis.Remarkable diversity exists for structures capable of high affinity binding and activation of G protein-coupled receptors. Understanding the molecular determinants of ligand binding provides important insights that may be useful in structurebased drug design and in understanding the basic mechanisms of receptor activation. Photoaffinity labeling provides the most direct way to identify sites of contact between residues in ligands and a receptor (1); however, traditionally this involves use of large amounts of radioactivity and inefficient and timeconsuming purification procedures. In this work, we developed a simple, rapid, and efficient procedure for analyzing sites of ligand-receptor binding, and applied this to a series of structurally related ligands representing agonists, partial agonist, and antagonist of the CCK receptor.Capillary electrophoresis (CE), 1 initially performed by Jorgenson and Lukacs (2), has emerged in recent years as a versatile and powerful tool for the analysis of biological molecules, because of its unparalleled efficiency for separation, small injection volumes, and low mass detection limits (3, 4). We recently developed a CE method for the separation of peptides cleaved from hydrophobic membrane proteins, such as bacteriorhodopsin, a possible model for the study of G protein-coupled receptors (5). In the present work, we have optimized and extended this to apply to extremely sparse, w...

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“…Although a [Pap"] -derivative of a-MSH had been prepared [7], amphibian pigment cells could not be labelled because of the lack of a suitable melanophore system, resistant to UV illumination but still transparent enough for an efficient photolysis of receptor-bound peptides. In the meantime, several other hormone receptors were successfully labelled using photolysable derivatives of, e.g., glucagon [8], insulin [9], oxytocin [lo], angiotensin [ 111, enkephalin [12], or pentagastrin [13]. With pentagastrin, an irreversible stimulation upon photolysis was reported, whereas in the other cases either an irreversible block of the receptors was observed (oxytocin, angiotensin) or the incorporation was tested by irreversible binding to membrane components.…”

Section: Introduction C-andelanotropinmentioning

confidence: 99%

Irreversible stimulation of Xenopus melanophores by photoaffinity labelling with p‐azidophenylalanine¹³‐α‐melanotropin

Graan

Eberlé

1980

FEBS Letters

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Biologically active derivatives of angiotensin for labeling cellular receptors

Cited by 23 publications

References 14 publications

Isolation of an angiotensin II-binding protein from liver.

Isolation of an angiotensin II-binding protein from liver.

Structurally Related Peptide Agonist, Partial Agonist, and Antagonist Occupy a Similar Binding Pocket within the Cholecystokinin Receptor

Irreversible stimulation of Xenopus melanophores by photoaffinity labelling with p‐azidophenylalanine¹³‐α‐melanotropin

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Biologically active derivatives of angiotensin for labeling cellular receptors

Cited by 23 publications

References 14 publications

Isolation of an angiotensin II-binding protein from liver.

Isolation of an angiotensin II-binding protein from liver.

Structurally Related Peptide Agonist, Partial Agonist, and Antagonist Occupy a Similar Binding Pocket within the Cholecystokinin Receptor

Irreversible stimulation of Xenopus melanophores by photoaffinity labelling with p‐azidophenylalanine13‐α‐melanotropin

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Irreversible stimulation of Xenopus melanophores by photoaffinity labelling with p‐azidophenylalanine¹³‐α‐melanotropin