Abstract
G protein-coupled receptors (GPCRs) bind a broad array of extracellular molecules and transmit intracellular signals that initiate physiological responses. The signal transduction functions of GPCRs are inherently related to their structural plasticity, which can be experimentally observed by spectroscopic techniques. Nuclear magnetic resonance (NMR) spectroscopy in particular is an especially advantageous method to study the dynamic behavior of GPCRs. The success of NMR studies critically relies on the production of functional GPCRs containing stable-isotope labeled probes, which remains a challenging endeavor for most human GPCRs. We report a protocol for the production of the human histamine H1 receptor (H1R) in the methylotrophic yeast Pichia pastoris for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed H1R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H1R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H1R show well-dispersed and resolved signals consistent with a properly folded protein, and 19F-NMR data register a response of the protein to differences in efficacies of bound ligands.
Highlights
G protein-coupled receptors (GPCRs) initiate physiological processes by recognizing and binding a wide range of extracellular stimuli, including hormones, neurotransmitters and numerous other small molecules
We present a protocol for the expression and purification of the human H1 histamine receptor in Pichia pastoris, which can be applied to economically produce stable-isotope labeled GPCR samples for Nuclear magnetic resonance (NMR) experiments
Purified H1 receptor (H1 R) responded to the addition of known ligands, as monitored by fluorescence thermal shift assays, with the addition of the ligand histamine resulting in the largest increase in the protein melting temperature
Summary
G protein-coupled receptors (GPCRs) initiate physiological processes by recognizing and binding a wide range of extracellular stimuli, including hormones, neurotransmitters and numerous other small molecules. Over one third of FDA-approved drugs target human. Histamine receptors are class A GPCRs and one of the earliest GPCR targets of rational drug development to treat illness. Four histamine receptor subtypes regulate vital physiological processes including gastric acid secretion, smooth muscle relaxation, the release of neurotransmitters and neutrophil distribution [2]. The human H1 receptor (H1 R) subtype is expressed in several different tissue types including in the brain, vascular smooth muscle, airway, liver and in lymphocytes [3]. H1 R activation by endogenous histamine is blocked by widely recognized drugs known as antihistamines to prevent or reduce allergic responses. The strong interest in identifying novel H1 R drugs has led to the development of over 40 H1 R antagonists approved for use as therapeutics [7,8]
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