In vitro modification of substituted cysteines as tool to study receptor functionality and structure–activity relationships

Rathmann, Daniel; Pedragosa-Badia, Xavier; Beck‐Sickinger, Annette G.

doi:10.1016/j.ab.2013.04.015

Cited by 3 publications

(4 citation statements)

References 48 publications

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“…Two criteria are used to determine whether engineered cysteines are positioned at the surface of the bindingsite pocket: (i) the reaction with the MTSEA reagent alters binding irreversibly and (ii) the reaction is retarded by the presence of the ligand. This approach has been used by us and others to identify residues that line the surface of GPCR binding-site pockets [29][30][31][32][33][34][35][36][37]. Indeed, using SCAM analysis, we have previously identified eleven MTSEA-sensitive residues in TM3, TM4, TM5, TM6 and TM7 of the rat UT receptor (rUT receptor) [34,36] that make up that receptor's binding pocket.…”

Section: Introductionmentioning

confidence: 99%

Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor

Sainsily

Cabana

Holleran

et al. 2014

Biochemical Pharmacology

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The vasoactive urotensin-II (UII), a cyclic undecapeptide widely distributed in cardiovascular, renal and endocrine systems, specifically binds the UII receptor (UT receptor), a G protein-coupled receptor (GPCR). The involvement of this receptor in numerous pathophysiological conditions including atherosclerosis, heart failure, hypertension, renal impairment and diabetes potentially makes it an interesting therapeutic target. To elucidate how UII binds the UT receptor through the identification of specific residues in transmembrane domains (TM) one (TM1) and two (TM2) that are involved in the formation of the receptor's binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue of TM1 (V49((1.30)) to M76((1.57))) and TM2 (V88((2.41)) to H117((2.70))) was mutated, one by one, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant binding inhibition of (125)I-UII to mutant I54C((1.35)) in TM1 and mutants Y100C((2.53)), S103C((2.56)), F106C((2.59)), I107C((2.60)), T110C((2.63)) and Y111C((2.64)) in TM2. These results identify key structural residues in TM1 and TM2 that participate in the formation of the UT receptor binding pocket. Together with previous SCAM analysis of TM3, TM4, TM5, TM6 and TM7, these results have led us to identify residues within all 7 TMs that participate in UT's binding pocket and have enabled us to propose a model of this receptor's orthosteric binding site.

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Section: Introductionmentioning

confidence: 99%

Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor

Sainsily

Cabana

Holleran

et al. 2014

Biochemical Pharmacology

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“…Inositol turnover experiments were conducted in duplicate from transiently transfected COS-7 cells as reported recently [21]. …”

Section: Methodsmentioning

confidence: 99%

C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment

Wanka

Babilon

Burkert

et al. 2017

Cellular Signalling

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The human neuropeptide Y4 receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hY4R desensitization and endocytosis. The role of distinct segments of the hY4R carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hY4R and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif 7.78EESEHLPLSTVHTEVSKGS7.96 was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y4 receptor, which regulates arrestin-3 recruitment and receptor endocytosis.

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“…Fluorescence Microscopy Studies-HEK293 cells were seeded and transfected with cDNA encoding hY 4 R constructs as earlier described (23). The nuclei were stained with Hoechst 33342 (0.5 mg/ml) for 10 min after starving the cells for 20 min in Opti-MEM medium.…”

Section: Methodsmentioning

confidence: 99%

“…Signal Transduction Assays-Signal transduction assays were performed on 24-or 48-well plates as described previously with minor changes (3,23). As transfection reagents, Metafectene and Metafectene Pro (Biontex) were used.…”

Section: Methodsmentioning

confidence: 99%

Pancreatic Polypeptide Is Recognized by Two Hydrophobic Domains of the Human Y4 Receptor Binding Pocket

Pedragosa-Badia

Sliwoski

Nguyen

et al. 2014

Journal of Biological Chemistry

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Background:The Y 4 R is involved in regulation of food intake and gastrointestinal transport. Results: Mutagenesis studies revealed several residues displaying a significant loss of potency for hPP. Conclusion: Tops of TM2, TM6, and TM7 interact with the hY 4 R native agonist hPP. Significance: Characterizing the structure of the Y 4 R binding pocket is crucial for the development of new anti-obesity drugs.

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In vitro modification of substituted cysteines as tool to study receptor functionality and structure–activity relationships

Cited by 3 publications

References 48 publications

Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor

Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor

C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment

Pancreatic Polypeptide Is Recognized by Two Hydrophobic Domains of the Human Y4 Receptor Binding Pocket

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