Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.     

Expression of CB2 cannabinoid receptor in Pichia pastoris

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification.     

Expression of CB2 cannabinoid receptor in Pichia pastoris

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification.     

Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor

The human peripheral cannabinoid receptor (CB2) was expressed as a fusion with the maltose-binding protein (at the N-terminus), thioredoxin A (at the C-terminus) and two small affinity tags (a Strep-tag and a polyhistidine tag). Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were dependent on location and type of tags in the expression construct, and were as high as 1-2mg per liter of bacterial culture. The recombinant receptor was ligand binding-competent, and activated cognate G-proteins in an in vitro coupled assay. The fusion CB2-125 protein was purified by immobilized metal affinity chromatography on a Ni-NTA resin. Maltose-binding protein, thioredoxin and a decahistidine tag were removed from the fusion by treatment with Tobacco etch virus (Tev) protease. Purification to over 90% homogeneity of the resulting CB2, containing an N-terminal Strep-tag was achieved by affinity chromatography on a StrepTactin resin. Circular dichroism spectroscopy indicated an alpha-helical content of the purified recombinant protein of approximately 54%. The expression and purification protocol allows for production of large (milligram) quantities of functional peripheral cannabinoid receptor, suitable for subsequent structural characterization. Preliminary results of reconstitution experiments indicate that the CB2 has retained its ligand-binding properties.     

Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233)

To develop an approach to obtain milligram quantities of purified isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor for NMR structural analysis, we chose a truncated CB receptor fragment, CB2(180-233), spanning from the fifth transmembrane domain (TM5) to the associated loop regions of cannabinoid CB2 receptor. This highly hydrophobic membrane protein fragment was pursued for developmental studies of membrane proteins through expression and purification in Escherichia coli. The target peptide was cloned and over-expressed in a preparative scale as a fusion protein with a modified TrpDeltaLE1413 (TrpLE) leader sequence and a nine-histidine tag at its N-terminal. An experimental protocol for enzyme cleavage was developed by using Factor Xa to remove the TrpLE tag from the fusion protein. A purification process was also established using a nickel affinity column and reverse-phase HPLC, and then monitored by SDS-PAGE and MS. This expression level is one of the highest reported for a G-protein coupled receptor and fragments in E. Coli, and provided a sufficient amount of purified protein for further biophysical studies.     

Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system.

The cannabinergic system is present in a variety of organs and tissues that perform a wide range of essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane-bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)-terminal His-tagged CB2 construct displayed a B(max) value of 9.3 pmol/mg with a K(D) of 7.30 nM using [3(H)]CP-55(940), a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high-level expression systems when developing membrane-bound protein purification strategies. This work will aid in the identification of receptor-ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.     

Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2

A biotin-protein ligase recognition site (BRS) was inserted into a polypeptide comprised of the maltose-binding protein, the peripheral cannabinoid receptor (CB2), thioredoxin A, and a polyhistidine tag at the carboxy terminus. Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were approximately 1mg per liter of bacterial culture. The biotinylated CB2-fusion fully retained its ligand-binding capacity. Introduction of the BRS at the C-terminus of the CB2 fusion protein (construct CB2-109) resulted in its complete in vivo biotinylation; the biotinylated protein was streptavidin-binding competent. Positioning of the BRS near the N-terminus of CB2 (CB2-112) resulted in a very low level of biotinylation in vivo. However, the detergent solubilized and purified CB2-112 fusion protein were successfully biotinylated in vitro by action of a BirA biotin-protein ligase. The biotinylated CB2-112 fusion protein was cleaved by the tobacco etch virus protease at specifically inserted sites, and deposited onto monomeric avidin agarose beads. Biotinylation of the recombinant CB2 receptor enabled not only purification but also immobilization of the GPCR on a solid support in homogeneous orientation which is beneficial for subsequent structural characterization.     

Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor

The CB1 and CB2 cannabinoid receptors belong to the GPCR superfamily and are associated with a variety of physiological and pathophysiological processes. Both receptors, with several lead compounds at different phases of development, are potentially useful targets for drug discovery. For this reason, fully elucidating the structural features of these membrane-associated proteins would be extremely valuable in designing more selective, novel therapeutic drug molecules. As a first step toward obtaining information on the structural features of the drug-receptor complex, we describe the full mass spectrometric (MS) analysis of the recombinant human cannabinoid CB2 receptor. This first complete proteomic characterization of a GPCR protein beyond rhodopsin was accomplished by a combination of several LC/MS approaches involving nanocapillary liquid chromatography, coupled with either a quadrupole-linear ion trap or linear ion trap-FTICR mass spectrometer. The CB2 receptor, with incorporated N-terminal FLAG and C-terminal HIS6 epitope tags, was functionally expressed in baculovirus cells and purified using a single step of anti-FLAG M2 affinity chromatography. To overcome the difficulties involved with in-gel digestion, due to the highly hydrophobic nature of this membrane-associated protein, we conducted in-solution trypsin and chymotrypsin digestions of purified and desalted samples in the presence of a low concentration of CYMAL5. This was followed by nanoLC peptide separation and analysis using a nanospray ESI source operated in the positive mode. The results can be reported confidently, based on the overlapping sequence data obtained using the highly mass accurate LTQ-FT and the 4000 Q-Trap mass spectrometers. Both instruments gave very similar patterns of identified peptides, with full coverage of all transmembrane helices, resulting in the complete characterization of the cannabinoid CB2 receptor. Mass spectrometric identification of all amino acid residues in the cannabinoid CB2 receptor is a key step toward the "Ligand Based Structural Biology" approach developed in our laboratory for characterizing ligand binding sites in GPCRs using a variety of covalent cannabinergic ligands.     

Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli

The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.     

Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.     

Expression of human peripheral cannabinoid receptor for structural studies

Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%-90% homogeneity by affinity chromatography followed by ion-exchange chromatography. By high-resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques.     

Behaviroal, pharmacological, and molecular characterization of an amphibian cannabinoid receptor

Investigation of cannabinoid pharmacology in a vertebrate with a phylogenetic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granulosa, the roughskin newt, was used here to characterize an amphibian cannabinoid receptor. Behavioral experiments demonstrated that the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor activity and courtship clasping behavior. Inhibition of clasping was dose-dependent and potent (IC(50) = 1.2 microgram per animal). Radioligand binding studies using [(3)H]CP-55940 allowed identification of a specific binding site (K(D) = 6.5 nM, B(max) = 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (K(D), nM) : CP-55940 (3.8) > levonantradol (13.0) > WIN55212-2 (25.7) > anandamide (1,665) approximately anandamide 100 microM phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose-dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function.     

Distinct expression profiles of the peripheral cannabinoid receptor in lymphoid tissues depending on receptor activation status

Using two distinct anti-CB2 receptor Abs, we investigated the expression patterns of the peripheral cannabinoid receptor CB2 in human secondary lymphoid organs. Immunohistochemical analysis using an N-terminal specific anti-CB2 Ab revealed high protein expression in the germinal centers (GCs) of secondary follicles. A C-terminal specific anti-CB2 Ab, which only recognizes a nonphosphorylated inactive receptor, showed positivity in the mantle zones (MZs) and marginal zones (MGZs) of the secondary follicles where resting cells reside, and in the primary follicles. In contrast, no positivity was observed in GCs using the C-terminal Ab, suggesting that active CB2 receptors are mainly present on cells in the GCs. Dual immunohistochemical analysis revealed that B lymphocytes express the CB2 protein abundantly. In contrast to B cells in the MZ or MGZ, CB2-expressing cells in the GCs coexpress the costimulatory membrane protein CD40, which is mainly expressed in the GCs and at very low levels in the MZs and MGZs and the proliferation marker Ki-67. Using the human Raji B cell line as a model, we demonstrate in a transwell assay that moderate migration occurs upon stimulation of the CB2 receptor with the endocannabinoid 2-arachidonoylglycerol, which is enhanced by CD40 costimulation. Our findings, that GC-related cells express active CB2 and that CB2-dependent migration requires CD40 costimulation, suggest that CB2 is involved in B cell activation.     

CB1 receptor-G protein association. Subtype selectivity is determined by distinct intracellular domains.

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C-terminal domain (CB(1)401-417). Guanosine 5'-O-(3-thio)triphosphate binding to G proteins can be stimulated by both peptide CB(1)401-417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol. 53, 504-510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry 38, 3447-3455]. In Chaps-solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB(1) receptor-G alpha(i) association and attenuated the CB(1)401-417-induced inhibition of adenylate cyclase. CB(1)401-417 significantly reduced the CB(1) receptor association with G alpha(i3), but not with G alpha(i1) or G alpha(i2). In contrast, third intracellular loop peptides significantly reduced the CB(1) receptor association with G alpha(i1) and G alpha(i2), but not G alpha(i3). These interactions are specific for the CB(1) receptor because a peptide corresponding to the juxtamembrane C-terminal domain of the CB(2) receptor failed to compete for the association of the CB1 receptor with any of the Gi alpha subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB(1) receptor direct the interaction with specific G protein subtypes.     

Neuron‐type specific cannabinoid‐mediated G protein signalling in mouse hippocampus

Type 1 cannabinoid receptor (CB1) is expressed in different neuronal populations in the mammalian brain. In particular, CB1 on GABAergic or glutamatergic neurons exerts different functions and display different pharmacological properties in vivo. This suggests the existence of neuron‐type specific signalling pathways activated by different subpopulations of CB1. In this study, we analysed CB1 expression, binding and signalling in the hippocampus of conditional mutant mice, bearing CB1 deletion in GABAergic (GABA‐CB1‐KO mice) or cortical glutamatergic neurons (Glu‐CB1‐KO mice). Compared to their wild‐type littermates, Glu‐CB1‐KO displayed a small decrease of CB1 mRNA amount, immunoreactivity and [³H]CP55,940 binding. Conversely, GABA‐CB1‐KO mice showed a drastic reduction of these parameters, confirming that CB1 is present at much higher density on hippocampal GABAergic interneurons than glutamatergic neurons. Surprisingly, however, saturation analysis of HU210‐stimulated [³⁵S]GTPγS binding demonstrated that ‘glutamatergic’ CB1 is more efficiently coupled to G protein signalling than ‘GABAergic’ CB1. Thus, the minority of CB1 on glutamatergic neurons is paradoxically several fold more strongly coupled to G protein signalling than ‘GABAergic’ CB1. This selective signalling mechanism raises the possibility of designing novel cannabinoid ligands that differentially activate only a subset of physiological effects of CB1 stimulation, thereby optimizing therapeutic action.     

Cannabinoid receptors CB1 and CB2 form functional heteromers in brain

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.     

Signal transduction of eicosanoid CB1 receptor ligands.

The eicosanoid ligand, arachidonylethanolamide (anandamide), interacts with the CB1 cannabinoid receptor in the brain to signal its response. Pharmacophoric points of interaction between this agonist and the receptor have been proposed based upon structure-activity relationship studies of ligand binding to the receptor. Three dimensional quantitative structure-activity relationship (3D-QSAR) models have been constructed based upon the corresponding pharmacophoric points predicted for cannabinoid ligands delta9-tetrahydrocannabinol and 9-nor-9beta-hydroxyhexa-hydrocannabinol. A novel data set has been used to test the statistical validity of these models. Once the ligand interacts with the CB1 receptor, signal transduction occurs via G-proteins of the Gi/o family which are shown to be associated with the receptor. Evidence suggests that the juxtamembrane region of the C-terminal of the CB1 receptor is critical for activation of these G-proteins.     

Design, expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ G-protein fusion protein.

We report here the synthesis and characterization of two gene constructs designed to facilitate structure/function studies of the human neuronal cannabinoid receptor, CB1. The first gene, which we call shCB1, is a synthetic gene containing unique restriction sites spaced roughly 50-100 bases apart to facilitate rapid mutagenesis and cloning. A nine amino acid epitope tag (from the rhodopsin C-terminus) is also present in the shCB1 C-terminal tail to enable detection and purification using the monoclonal antibody 1D4. We find that that the shCB1 gene can be transiently expressed in COS cells with yield of approximately 10-15 micro g receptor per 15 cm plate and is wild type like in its ability to bind cannabinoid ligands. Our confocal microscopy studies indicate shCB1 targets to the membrane of HEK293 cells and is internalized in response to agonist. To facilitate functional studies, we also made a chimera in which the C-terminus of shCB1 was fused with the N-terminus of a G-protein alpha subunit, Galphai. The shCB1/Galphai chimera shows agonist stimulated GTPgammaS binding, and thus provides a simplified way to measure agonist induced CB1 activation. Taken together, the shCB1 and shCB1/Galphai gene constructs provide useful tools for biochemical and biophysical examinations of CB1 structure, activation and attenuation.     

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2(271-326))

Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2_271–326, a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidine-selected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2_271–326, was subsequently purified by reverse-phase HPLC and confirmed by SDS–PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2_271–326 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs.     

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (approximately 4mg/L of culture) of functional human adenosine A(2)a receptor fused to a green fluorescent protein (A(2)aR-GFP) from Saccharomyces cerevisiae. In this work, we re-engineered A(2)aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A(2)aR and A(2)aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A(2)aR-GFP-His(10). One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-beta-d-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A(2)aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A(2)a-His(10) receptor was purified in quantities of 6+/-2mg/L of culture, with ligand-binding yields of 1mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A(2)aR yet achieved from any heterologous expression system.