AI Article Synopsis
- The study identifies a 55-kDa GH-releasing factor (GRF) receptor in sheep pituitary membranes and human cell lines, using photoaffinity cross-linking methods.
- Findings show that cross-linking is specific to GRF and reduces when tested with guanosine 5'-O-(3-thiotriphosphate), indicating that the receptor is a G-protein-coupled receptor.
- The receptor was confirmed as an N-linked glycoprotein, with size shifts observed after deglycosylation treatment, which helps differentiate it from previous studies that used chemical cross-linking techniques.
Article Abstract
Photoaffinity cross-linking methods presented here demonstrate a 55-kilodalton (kDa) GH-releasing factor (GRF) receptor in ovine pituitary membranes and in cell lines expressing the cloned human pituitary receptor complementary DNA. Covalent cross-linking of photoprobe to this high affinity site is strongly competed by 1 nM GRF. Competition shows strong specificity for GRF over related peptides. Reduced cross-linking in the presence of guanosine 5'-O-(3-thiotriphosphate) suggests that this is a G-protein-coupled receptor. Detection of cross-linking to this receptor required detergent extraction to reduce high nonspecific binding of GRF photoprobe. Partial deglycosylation of the cross-linked receptor with neuraminidase caused a shift in apparent size to 52 kDa. Complete deglycosylation with N-glycosidase caused a shift to 45 kDa, demonstrating that this receptor is an N-linked glycoprotein and agreeing with the protein size and single glycosylation site predicted from the cloned complementary DNA sequence. These sizes differ from those found in previous reports which used chemical cross-linking to identify GRF receptor. This photoaffinity cross-linking method will facilitate studies of receptor function and tissue distribution. Photoaffinity cross-linking can also be used to map regions of the receptor molecule and bound GRF that are in close proximity.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1210/endo.135.3.8070391 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Enzymatic Cleavage of Double-Stranded DNA-Encoded Libraries (DELs) to Single-Stranded DELs with Compounds at the 3' End: Its Application in Photo-Crosslinking Selection.
Chemistry
December 2024
Chemical Research Laboratories, Nissan Chemical Corporation, 10-1 Tsuboi-Nishi 2-chome, Funabashi, Chiba, Japan.
DNA-encoded library (DEL) technology is a crucial tool in pharmaceutical research, rapidly identifying compounds that bind to a target of interest from an extensive pool of compounds. In this study, we propose a new method for generating single-stranded DELs (ssDELs) with compounds at the 3' end. The introduction of uniquely designed hairpin-shaped headpieces containing deoxyuridine (NC-HP) and the use of a cleavage enzyme facilitate the conversion from double-stranded DELs (dsDELs) to such ssDELs.
View Article and Find Full Text PDFPhotoactivatable mRNA 5' Cap Analogs for RNA-Protein Crosslinking.
Adv Sci (Weinh)
September 2024
Centre of New Technologies, University of Warsaw, Banacha 2c, Warsaw, 02-097, Poland.
Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA-based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5'-end, called 5'-cap.
View Article and Find Full Text PDFRecent advances in photoaffinity labeling strategies to capture Glycan-Protein interactions.
Curr Opin Chem Biol
June 2024
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, K7L 3N6, Canada; Department of Chemistry, Queen's University, Kingston, K7L 2S8, Canada; Department of Surgery, Queen's University, Kingston, K7L 2V7, Canada. Electronic address:
Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan-protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands.
View Article and Find Full Text PDFA Cell-Permeable Photosensitizer for Selective Proximity Labeling and Crosslinking of Aggregated Proteome.
Adv Sci (Weinh)
May 2024
State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, China.
Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, this work reports on a cell-permeable photosensitizer (P8, Rose Bengal derivative) for selective photo induced proximity labeling and crosslinking of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect.
View Article and Find Full Text PDFUnexpected Cyclization Product Discovery from the Photoinduced Bioconjugation Chemistry between Tetrazole and Amine.
J Am Chem Soc
January 2024
Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, Innovative Drug Research Center, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, P. R. China.
Bioconjugation chemistry has emerged as a powerful tool for the modification of diverse biomolecules under mild conditions. Tetrazole, initially proposed as a bioorthogonal photoclick handle for 1,3-dipolar cyclization with alkenes, was later demonstrated to possess broader photoreactivity with carboxylic acids, serving as a versatile bioconjugation and photoaffinity labeling probe. In this study, we unexpectedly discovered and validated the photoreactivity between tetrazole and primary amine to afford a new 1,2,4-triazole cyclization product.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!