Chemical characterization of ligand binding site fragments from turkey β‐adrenergic receptor

Eshdat, Yuval; Chapot, M.-P.; Strosberg, A. Donny

doi:10.1016/0014-5793(89)80276-5

Cited by 3 publications

(3 citation statements)

References 16 publications

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“…The approach involving covalent labelling with specific ligands has provided structural information for adrenergic (Dohlman et al, 1988;Wong et al, 1988;Eshdat et al, 1989;Matsui et al, 1989;Strader et al, 1989) or muscarinic cholinergic (Curtis et al, 1989; Hulme et al, 1989) receptors. Examples of topography of peptide-hormone interaction with their receptors have not yet been reported.…”

Section: Discussionmentioning

confidence: 99%

Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains

Désarnaud

Marie

Lombard

et al. 1993

Biochemical Journal

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We report new structural data about the rat liver angiotensin II receptor, which belongs to the AT1 subclass. This receptor has been purified at analytical or semi-preparative levels by a previously described strategy involving its photolabelling with a biotinylated azido probe and selective adsorption of the covalent probe-receptor complexes to immobilized streptavidin [Marie, Seyer, Lombard, Desarnaud, Aumelas, Jard and Bonnafous (1990) Biochemistry 29, 8943-8950]. Chemical or enzymic deglycosylation of the purified receptor has shown a shift in its molecular mass from 65 kDa to 40 kDa. Fragmentation of the purified receptor was carried out with V8 protease from Staphylococcus aureus, CNBr and trypsin. It was possible to find trypsin-treatment conditions which allowed production of a 6 kDa probe-fragment complex with a satisfactory yield. Attempts to localize this small fragment (5 kDa after subtraction of the probe contribution) in the recently published rat AT1 receptor sequence are reported. As expected, this fragment is not glycosylated; moreover, its further fragmentation by CNBr induces a very slight decrease in its size. These data support the hypothesis that a receptor sequence comprising the third transmembrane domain and adjacent portions of extra- and intracellular loops is involved in photolabelling by the C-terminal azidophenylalanine of the angiotensin-derived probe. These preliminary results are discussed in terms of future prospects for the characterization of hormone-binding domains of angiotensin II receptors.

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Section: Discussionmentioning

confidence: 99%

Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains

Désarnaud

Marie

Lombard

et al. 1993

Biochemical Journal

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“…2. lane b). Due to the nature of the affinity label and data from previous studies in which this reagent was used (Dohlman et al, 1987;Eshdat et al, 1989), this reagent was not expected to bind covalently to a region overlapping with the peptides used for immunization. Indeed, the ligand-binding domain of the receptor is formed by a pocket of hydrophobic transmembrane helices, whereas potential immunogenic peptides were selected from hydrophilic parts of the protein.…”

Section: Immunoprecipitation Assaymentioning

confidence: 99%

Antibodies for the Immunochemistry of the Human β3‐Adrenergic Receptor

Guillaume

Petitjean

Haasemann

et al. 1994

European Journal of Biochemistry

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Based on the amino acid sequence deduced from the recently cloned human P3-adrenergic receptor (hup3AR) gene, polyclonal antibodies were prepared against synthetic peptides, corresponding to regions of huP3AR presumed to be exposed at the outer or the inner side of the membrane on the basis of the putative three-dimensional structure of the previously characterized /31 and P2 adrenergic receptors. Affinity-purified antibodies directed against N-terminal, extracellular or intracellular loops and C-terminal peptides reacted specifically with the hup3AR and not with either the human Pl or p2 adrenergic receptor. Using these antibodies, it was demonstrated that the receptor is present at the surface of Chinese Hamster Ovary (CHO) cells transfected with the huP3AR gene; in addition, the presence of the receptor protein was established in a human tissue (gall bladder). Immuno-affinity chromatography of solubilized CHO hup3AR-containing cell membranes allowed the isolation of hup3AR protein with an overall yield of 30%. The degree of purity of the receptor was more than 80%, as assessed by N-terminal sequencing of the protein eluted from the column. Sequence analysis demonstrated the absence of a methionine residue at the N-terminal position, and suggested that the side chain of the asparagine residue at position 7 is glycosylated.

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“…In those studies the disulfide bond was easily reduced in the presence of DTT. Similarly, hydroxylamine readily cleaves ester groups, and its ability to remove an affinity label from a fragment of the /3-adrenergic receptor fragment was interpreted to indicate an ester linkage (22).…”

Section: Discussionmentioning

confidence: 99%

"Cleavable Trifunctional" Approach to Receptor Affinity Labeling: Regeneration of Binding to A1-Adenosine Receptors

Jacobson

Fischer²,

Ji³

1995

Bioconjugate Chem.

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A general approach for reversible affinity labeling of receptors has been developed. The objective is to carry out a series of chemical modifications resulting in a covalently-modified, yet functionally-regenerated, receptor protein that also may contain a reporter group. The ligand recognition site of A1-adenosine receptors in bovine brain membranes was probed to demonstrated the feasibility of this approach. Use of disulfide or ester linkages, intended for cleavage by exposure of the labeled receptor to either reducing reagents or hydroxylamine, respectively, was considered. Binding of the antagonist radioligand [3H]CPX was preserved following incubation of the native receptor with 3 M hydroxylamine, while binding was inhibited by the reducing reagent dithiothreitol (DTT) with an IC50 of 0.29 M. Hydroxylamine displaced specific agonist ([3H]PIA) binding in a noncovalent manner. Specific affinity labels containing reactive isothiocyanate groups were synthesized from XCC (8-[4-](carboxymethyl)-oxy]phenyl]-1,3-dipropylxanthine) and shown to bind irreversibly to A1-receptors. The ligands were structurally similar to previously reported xanthine inhibitors (e.g., DITC-XAC: (1989) J. Med. Chem. 32, 1043) except that either a disulfide linkage or an ester linkage was incorporated in the chain between the pharmacophore and the isothiocyanate-substituted ring. These groups were intended for chemical cleavage by thiols or hydroxylamine, respectively. Radioligand binding to A1-receptors was inhibited by these reactive xanthines in a manner that was not reversed by repeated washing. Hydroxylamine or DTT restored a significant fraction of the binding of [3H]CPX in A1-receptors inhibited by the appropriate cleavable xanthine isothiocyanate derivative.

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Chemical characterization of ligand binding site fragments from turkey β‐adrenergic receptor

Cited by 3 publications

References 16 publications

Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains

Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains

Antibodies for the Immunochemistry of the Human β3‐Adrenergic Receptor

"Cleavable Trifunctional" Approach to Receptor Affinity Labeling: Regeneration of Binding to A1-Adenosine Receptors

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