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.The neurokinin-1 receptor (Nk1r) 1 is a neuropeptide receptor belonging to the G protein-coupled receptor superfamily (1). The receptor, also designated the substance P or tachykinin 1 receptor, has been implicated in numerous physiological functions, which include modulatory roles in the central nervous system and proinflammatory action (for review see Refs. 2 and 3). Although much debate still exists as to which neurokinin represents the physiological ligand for the receptor, the substance P neurokinin has been identified as the primary ligand (4).Because of the significant pathophysiological role of Nk1r, extensive pharmacological research has been carried out to characterize both agonist and antagonist binding properties of the receptor. Previously, extensive mutagenesis of the receptor identified separate binding sites for substance P and the antagonist CP-96,345 (5-9). These studies indicated that substance P interacts almost exclusively with the NH 2 -terminal extracellular residues, with little involvement by residues located in the membrane-spanning domain. The same studies revealed that the antagonist appeared to bind deeply into the protein, within a pocket formed by the transmembrane helices.Although site-directed mutagenesis identified residues important for ligand binding, it cannot differentiate side chains that have a direct coordination of the ligands from residues involved indirectly, such as through allosteric communication. For example, a Y216A substitution located nea...

Site-Specific Insertion of Spin-Labeled l-Amino Acids in Xenopus Oocytes

“…For cytosolic proteins, spin-label reduction must be considered in attempts to observe nitroxyl amino acids incorporated by the TRAMPE method. Within in live cells nitroxide labels are reduced to the EPR-silent diamagnetic species, but reoxidize to the paramagnetic species when exposed to air or mild oxidants (58). This would not be a concern in in vitro systems as long as reductants are not added.…”

Distance Determination in Proteins inside Xenopus laevis Oocytes by Double Electron−Electron Resonance Experiments

“…A nitroxide radical, the most commonly used radical species for SDSL, can be chemically reduced in the cytoplasmic environment. It was reported that the ESR signals of nitroxide radicals attached to the extracellular regions of membrane proteins disappeared after the protein had been internalized from the plasma membrane into the cytosol . Therefore, the persistence of such radical species in cells is a major concern in performing the DEER experiments.…”

“…It was reported that the ESR signals of nitroxide radicals attached to the extracellular regions of membrane proteins disappeared after the protein had been internalized from the plasma membrane into the cytosol. 8 Therefore, the persistence of such radical species in cells is a major concern in performing the DEER experiments. Thus, we first examined the lifetime of nitroxide spin labels in Xenopus oocytes using continuous wave (CW)-ESR.…”

Non-neuronal mammalian tachykinin expression

“…This portion of the tachykinin appears to be less involved in high affinity binding to its receptor. For example, the electron paramagnetic resonance spectrum of a receptor bound substance P analog indicates the N-terminal lys-3 portion is highly flexible (13), an indication of little involvement in binding. The C-terminal portion of the peptide is a highly conserved, amidated, hydrophobic 5-residue sequence required for receptor binding.…”