title: Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist.
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.
authors.affiliation: Department of Biological Chemistry, University of California, Davis, California 95616, USA.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Shafer
authors.forename: Vicki J
authors.initials: VJ
authors.lastname: Bennett
authors.forename: Phillip
authors.initials: P
authors.lastname: Kim
authors.forename: John C
authors.initials: JC
authors.lastname: Voss
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004578:Electron Spin Resonance Spectroscopy; D004705:Endocytosis; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D013113:Spin Labels; D013373:Substance P / Q000737:chemistry; D015320:Tachykinins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D008164:Luminescent Proteins; D010455:Peptides; D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1; D013113:Spin Labels; D015320:Tachykinins; D049452:Green Fluorescent Proteins; D013373:Substance P; D006160:Guanosine Triphosphate; D008239:Lysine