Probing the binding pocket of the rat Nk1 tachykinin receptor with a spin labeled Substance P analogue
Biophysics
Shafer A M; Bennett V J; Voss J C
Biophysical Journal
2002
2002-01
Journal Article
n/a
Probing the binding pocket and endocytosis of a G protein-coupled receptor in live cells reported by a spin-labeled substance P agonist
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
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.
Shafer A M; Bennett V J; Kim P; Voss J C
Journal of Biological Chemistry
2003
2003-09
Journal Article
<a href="http://doi.org/10.1074/jbc.M212712200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M212712200</a>
Characterization of a conformationally sensitive TOAC spin-labeled substance P
activation; adrenergic-receptor; agonist; amino-acid; binding-site; Biochemistry & Molecular Biology; Endocrinology & Metabolism; EPR; ESR; GPCR; neurokinin-1; nk-1; peptide-synthesis; Pharmacology & Pharmacy; protein-coupled-receptors; receptor; Substance P; tachykinin nk1 receptor; TOAC spin label
To probe the binding of a peptide agonist to a G-protein coupled receptor in native membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethyl-piperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. The affinity of the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC derivative is similar to 250-fold lower. Both peptides activate receptor signaling, though the potency of the 9-TOAC peptide is substantially lower. The utility of these modified ligands for reporting conformational dynamics during the neurokinin-1 receptor activation was explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that maintain the receptor in its high-affinity binding state. In contrast, 4-TOAC substance P binding is observed by solution EPR under both low- and high-affinity receptor states, with evidence of a more strongly immobilized peptide in the presence of GDP. In addition, to better understand the conformational consequences of TOAC substitution into substance P as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC versions of the peptide were constructed, and the molecular dynamics calculated via simulated annealing to explore the influence of the TOAC substitutions on backbone structure. (C) 2008 Elsevier Inc. All rights reserved.
Shafer A M; Nakaie C R; Deupi X; Bennett V J; Voss J C
Peptides
2008
2008-11
Journal Article
<a href="http://doi.org/10.1016/j.peptides.2008.08.002" target="_blank" rel="noreferrer noopener">10.1016/j.peptides.2008.08.002</a>