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J. Biol. Chem. 278 (36): 34203-34210

© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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 {ddagger}, Vicki J. Bennett §, Phillip Kim ¶, and John C. Voss {ddagger} ||

{ddagger}Department of Biological Chemistry, University of California, Davis, California 95616, the §Department of Neurobiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272, and Peptidogenic Research, Livermore, California 94550

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.

Received for publication December 13, 2002. Revision received June 20, 2003.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 530-754-7583; Fax: 530-752-3516; E-mail: jcvoss{at}

The Use of Spin-Labeled Ligands as Biophysical Probes to Report Real-Time Endocytosis of G Protein-Coupled Receptors in Living Cells.
A. Shafer and J. Voss (2004)
Sci. STKE 2004, pl9
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