Thromboxane A2 G Protein-Coupled Receptor Production and Crystallization for Structure Studies.

G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands.

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/106 cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

A novel nuclear signaling pathway for thromboxane A2 receptors in oligodendrocytes: evidence for signaling compartmentalization during differentiation.

The present study investigated G protein expression, localization, and functional coupling to thromboxane A(2) receptors (TPRs) during oligodendrocyte (OLG) development. It was found that as OLGs mature, the expression levels of G(q) increase while those of G(13) decrease. In contrast, the expression levels of G(s), G(o), and G(i) do not change significantly. Localization studies revealed that G(q), G(13), and G(i) are present only in the extranuclear compartment, whereas G(s) and G(o) are found in both the extranuclear and the nuclear compartments. Purification of TPR-G protein complexes demonstrated that TPRs couple to both G(q) and G(13) in the extranuclear compartment but only to G(s) in the nuclear compartment. Furthermore, functional analysis revealed that stimulation of nuclear TPR in OLGs stimulates CREB phosphorylation and myelin basic protein transcription and increases survival. Collectively, these results demonstrate that (i) OLGs selectively modulate the expression of certain G proteins during development, (ii) G proteins are differentially localized in OLGs leading to subcellular compartmentalization, (iii) TPRs couple to G(q) and G(13) in the extranuclear compartment and to G(s) only in the nucleus, (iv) mature OLGs have a functional nuclear TPR-G(s) signaling pathway, and (v) nuclear TPR signaling can stimulate CREB phosphorylation and myelin gene transcription and increase cell survival. These findings represent a novel paradigm for selective modulation of G protein-coupled receptor-G protein signaling during cell development.

A simple, quick, and high-yield preparation of the human thromboxane A2 receptor in full size for structural studies.

Human thromboxane A2 receptor (TP), a G protein-coupled receptor (GPCR), is one of the most promising targets for developing the next generation of anti-thrombosis and hypertension drugs. However, obtaining a sufficient amount of the full-sized and active membrane protein has been the major obstacle for structural elucidation that reveals the molecular mechanisms of the receptor activation and drug designs. Here we report an approach for the simple, quick, and high-yield preparation of the purified and active full-sized TP in an amount suitable for structural studies. Glycosylated human TP was highly expressed in Sf-9 cells using an optimized baculovirus (BV) expression system. The active receptor was extracted and solubilized by different detergents for comparison and was finally purified to a nearly single band with a ratio of 1:0.9 +/- 0.05 (ligand:receptor molecule) in ligand binding using a Ni column with a relatively low yield. However, a high-yield purification (milligram quantity) of the TP protein, from a modulate scale of transfected Sf-9 cell culture, has been achieved by quick and simple purification steps, which include DNA digestion, dodecyl-maltoside detergent extraction, centrifugation, and FPLC purification. The purity and quantity of the purified TP, using the high-yield approach, were suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution one-dimensional and two-dimensional (1)H NMR spectroscopic analyses. These studies provide a basis for the high-yield expression and purification of the GPCR for the structural and functional characterization using biophysics approaches.