Probing the Binding Pocket and Endocytosis of a G Protein-coupled Receptor in Live Cells Reported by a Spin-labeled Substance P Agonist

Aaron M. Shafer, Vicki J. Bennett, Phillip Kim, John C Voss

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)34203-34210
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number36
DOIs
StatePublished - Sep 5 2003

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Substance P
G-Protein-Coupled Receptors
Endocytosis
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Peptides
Tachykinin Receptors
Neurokinin-1 Receptors
Molecular Probes
Endosomes
Guanosine Triphosphate
Lysosomes
Spectrum Analysis
Down-Regulation
Chemical activation
Spectroscopy
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Probing the Binding Pocket and Endocytosis of a G Protein-coupled Receptor in Live Cells Reported by a Spin-labeled Substance P Agonist. / Shafer, Aaron M.; Bennett, Vicki J.; Kim, Phillip; Voss, John C.

In: Journal of Biological Chemistry, Vol. 278, No. 36, 05.09.2003, p. 34203-34210.

Research output: Contribution to journalArticle

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AB - 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.

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