Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist

Title

Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist

Creator

Shafer A M; Bennett V J; Kim P; Voss J C

Publisher

Journal of Biological Chemistry

Date

2003
2003-09

Description

To probe the molecular nature of the binding pocket of a G protein-coupled receptor and the events immediately following the binding and activation, we have modified the substance P peptide, a potent agonist for the neurokinin-1 receptor, with a nitroxide spin probe specifically attached at Lys-3. The agonist properties and binding affinity of the spin-labeled substance P are similar to the native peptide. Using electron paramagnetic resonance (EPR) spectroscopy, the substance P analogue is capable of reporting the microenvironment found in the binding pocket of the receptor. The EPR spectrum of bound peptide indicates that the Lys-3 portion of the agonist is highly flexible. In addition, we detect a slight increase in the mobility of the bound peptide in the presence of a non-hydrolyzable analogue of GTP, indicative of the alternate conformational states described for this class of receptor. The down-regulation of neurokinin-tachykinin receptors is accomplished by a rapid internalization of the activated protein. Thus, it was also of interest to establish whether spin-labeled substance P could serve as a real time reporter for endocytosis. Our findings show the receptor agonist is efficiently endocytosed and the loss of EPR signal upon internalization provides a real time monitor of endocytosis. The rapid loss of signal suggests that endosomal trafficking vesicles maintain a reductive environment. Whereas the reductive capacity of the lysosome has been established, our findings indicate this capacity in early endosomes as well.

Subject

antagonist-binding; Biochemistry & Molecular Biology; disulfide bonds; human neurokinin-1 receptor; induced; internalization; loop-gap; molecular characterization; nk-1 receptor; pathway; resonator; tachykinin nk1 receptor; xenopus oocytes

Format

Journal Article

Search for Full-text

Users with a NEOMED Library login can search for full-text journal articles at the following url: https://libraryguides.neomed.edu/home

Rights

Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).

Pages

34203-34210

Issue

36

Volume

278

Citation

Shafer A M; Bennett V J; Kim P; Voss J C, “Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist,” NEOMED Bibliography Database, accessed June 12, 2021, https://neomed.omeka.net/items/show/7141.

Social Bookmarking