ABSTRACT
Internalization and intracellular trafficking of G protein-coupled receptors (GPCR) plays an important role in the signal transduction. These processes are often highly dynamic and take place rapidly. In the past 10 years, it became obvious that internalized GPCRs are also capable of signaling via arrestin or heterotrimeric G proteins within the endosomal compartment. Real-time imaging of receptors in living cells can help to evaluate the temporal and spatial localization. We achieved a two-color pulse-chase labeling approach, which allowed the tracking of the human neuropeptide Y2 receptor (hY2R) in the same cell at different times. The ability to visualize the internalization pathway of two separately labeled and separately stimulated subsets of hY2R in a time-resolved manner revealed a rapid trafficking. Fusion of the two hY2R subsets was already observed 10 min after stimulation in the early endosomal compartment without subsequent separation of the fused receptor populations. The results demonstrate that the cells do not discriminate between receptors that were stimulated and internalized at different time points.
Subject(s)
Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide Y/metabolism , Single Molecule Imaging/methods , Color , Endosomes/metabolism , Humans , Methods , Protein Transport , Receptors, Neuropeptide Y/analysis , Time FactorsABSTRACT
Peptide-tag based labelling can be achieved by (i) enzymes (ii) recognition of metal ions or small molecules and (iii) peptide-peptide interactions and enables site-specific protein visualization to investigate protein localization and trafficking.
Subject(s)
Peptides/chemistry , Proteins/chemistry , Staining and Labeling , Animals , Catalysis , Enzymes/chemistry , Enzymes/metabolism , Humans , Ions/chemistry , Ions/metabolism , Metals/chemistry , Metals/metabolism , Peptides/metabolism , Protein Binding , Proteins/metabolismABSTRACT
Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.
Subject(s)
Fluorescent Dyes/chemistry , Membrane Proteins/chemistry , Cysteine/chemistry , Fluoresceins/chemistry , HEK293 Cells , Humans , Membrane Proteins/metabolism , Peptides/chemical synthesis , Peptides/chemistry , Protein Conformation , Pyrazines/chemistry , Receptors, Neuropeptide/chemistry , Receptors, Neuropeptide/metabolism , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/metabolism , Rhodamines/chemistry , Solid-Phase Synthesis TechniquesABSTRACT
The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N-terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N-terminally tagged with a 22 aa long Cys-E3 peptide (acceptor), which is capable of forming a coiled-coil with a reporter-armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y2 receptor on living cells.
Subject(s)
Membrane Proteins/analysis , Membrane Proteins/chemistry , Peptides/chemistry , Staining and Labeling , Cell Survival , HEK293 Cells , HumansABSTRACT
Hydroxy-mediated methoxy formation or stabilization is probably an important process in many methanol adsorption systems. Hydrogen atoms originating from the scission of the methanol O-H bond react with the substrate and form water. This process may result 1) in the production of additional surface defects as reactive centers for methoxy formation and 2) in the stabilization of methoxy groups by suppression of methanol formation.
