Calcium-dependent ligand binding and G-protein signaling of family B GPCR parathyroid hormone 1 receptor purified in nanodiscs.

GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca(2+) is a weak agonist of PTH1R, and Ca(2+) in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.

BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies.

Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in mammalian cells. Using these vectors, we show that the BacMam system in combination with the 293 GnTI(-) cell line can be used for production of milligram quantities of soluble glycoproteins. Moreover, for crystallization trials, the purified glycoproteins are sensitive to EndoH treatment resulting in a loss of the bulk of the attached N-linked glycosylation. In addition, we also show that a combination of the BacMam system and 293 GnTI(-) cell line can be used for producing milligram quantities of a GPCR-protein ligand complex suitable for crystallization trials.

Molecular recognition of parathyroid hormone by its G protein-coupled receptor.

Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-A structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer alpha-beta-betaalpha fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.

Recombinant expression and purification of the N-terminal extracellular domain of the parathyroid hormone receptor.

Our goal is to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its receptor, the parathyroid hormone receptor type-1 (PTHR1). In order to study this interaction, we are aiming to obtain a three-dimensional structure of the PTH-PTHR1 bimolecular complex. Due to the very low expression levels of endogenous PTHR1, a recombinant form is required for structural analysis. However, the extreme hydrophobicity of the transmembrane regions of PTHR1 makes heterologous expression of PTHR1 difficult. Therefore, we sought to express the N-terminal extracellular domain (N-ECD) of PTHR1, a region that plays a pivotal role in ligand interaction. We expressed the N-ECD in both bacterial (Escherichia coli) and insect (Sf9) cells. The form produced in E. coli, a fusion-protein with thioredoxin, is soluble. However, removal of the fusion partner from a partially purified preparation results in dramatic loss of yield of the N-ECD. Expression in Sf9 cells, however, facilitates purification of a soluble form of the N-ECD. Isothermal calorimetry demonstrates that this N-ECD binds PTH-(1-34), albeit with lower affinity than the full-length receptor. This report describes the expression and purification of milligram quantities of the isolated N-ECD of PTHR1. The receptor fragment retains the ability to bind its cognate peptide ligand, an important pre-requisite for subsequent structural studies.

Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells.

Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1-34) and specifically cross-linked to a radioiodinated bPTH-(1-34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.

Expression of PTHrP and the PTH/PTHrP receptor in growing red deer antler.

Antler growth is highly co-ordinated, so that trabecular bone and antler skin (velvet) develop together, at a rapid rate and in a manner reminiscent of their development in the fetus. Parathyroid hormone-related peptide (PTHrP) is expressed in both bone and skin, and is therefore a candidate to effect co-ordination between these tissues. The aim of this study was to localize the expression of PTHrP and its principal receptor, the parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrPR), in antler ("spiker") of one-year-old red deer. Using immunohistochemistry and in situ hybridization, intense and overlapping expression of PTHrP and its receptor was seen in developing osseocartilaginous structures and in the underlying layers of velvet epidermis. PTHrP was located on both the cell surface and within the nuclei. Our results strongly suggest that PTHrP, acting via the PTH/PTHrPR and possibly other intracrine mechanisms, plays a central role in the co-ordinated regulation of cell division and differentiation of developing antler bone and skin.