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.     

The cannabinoid CB1 receptor is expressed in pancreatic delta-cells

Antagonists of cannabinoid CB1 receptor (CB1, CNR1) promote weight loss and decrease hyperglycemia in patients with type 2 diabetes. While the endocannabinoid system may modulate islet hormone secretion, the cell-type expressing CB1 receptor in islets has not been fully resolved. In this study, we verified receptor gene expression in rodent islets and cell lines and examined the distribution of CB1 receptor in mouse, rat, and human islets by confocal immunofluorescence (IF) microscopy. IF demonstrated CB1 receptor was present in β-cell lines, but co-localized solely with somatostatin in the islet δ-cells of Zucker rats, C57BL/6 mice, and humans; no CB1 receptor expression was observed in α-, β-, or pp-cells. Similarly, a rat somatostatinoma cell line, MSL-G2-Tu6, was found to express CB1 receptor. We also found monoacylglycerol lipase (MAGL) to be expressed in δ-cells and fatty acid amide hydrolase (FAAH) to be expressed in α-cells. The specific expression of CB1 in δ-cells suggests that the ECS may play a role in modulating islet hormone secretion. As there are some differences between our findings and previous reports, further studies, including detailed physiological studies of the effects of the ECS on islet function, are warranted.     

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.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

Purification and characterization of the recombinant human aldose reductase expressed in baculovirus system

Large quantities of recombinant human aldose reductase were produced using Spodoptera frugiperda cells and properties of the enzyme were characterized. Direct purification of the recombinant aldose reductase by affinity column chromatography using Matrex gel orange A yielded a single 36 kDa band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. The isoelectric point of the recombinant enzyme was 5.85 which is identical to the human muscle aldose reductase. Following the treatment with an acylamino-acid releasing enzyme, the blocked NH2-terminal amino acid was identified to be acetylalanine. The successive NH2-terminal sequence and that of the COOH-terminal peptide concurred with the expected translated sequence. Kinetic analyses of the recombinant enzyme activity for various substrates and the cofactor, NADPH, demonstrated a good agreement with the previously reported kinetic data on the purified human aldose reductase. A high concentration of (NH4)2SO4 elicited a significant increase in both Km and Kcat for DL-glyceraldehyde as well as D-glucose. Although IC50 values for most of the aldose reductase inhibitors with recombinant enzyme were found to fall within the comparable range of those obtained with nonhuman mammalian enzymes, the IC50 value for epalrestat was more than 10-fold higher in the recombinant enzyme. These results indicate that the recombinant human aldose reductase expressed in the baculovirus system possesses structurally and enzymatically similar properties as those reported for the native human enzyme and should serve as a superior enzyme preparation to nonhuman mammalian enzymes for the screening of the efficacy and potency of newly developed aldose reductase inhibitors.     

Biochemical and mass spectrometric characterization of the human CB2 cannabinoid receptor expressed in Pichia pastoris--importance of correct processing of the N-terminus

This study was conducted to optimize the expression of human CB2 cannabinoid receptors in methylotrophic yeast Pichia pastoris (P. pastoris). Two major species of expressed CB2 proteins were seen on Western blot, i.e., a 42 kDa band which matches the calculated molecular weight for tagged CB2, and a 52/55 kDa doublet. Treatment of membranes with N-glycosidase F or inclusion of tunicamycin in the culture medium during induction resulted in the disappearance of the 55 kDa, but not the 52 kDa band, suggesting that the 3 kDa extra in the 55 kDa band is due to N-glycosylation, but the 10 kDa extra in the 52 kDa band is not due to N-glycosylation. Anti-FLAG M1 antibody had a much higher preference for the 42 kDa band over the 52/55 kDa doublet, and a 10 kDa fragment recognized by anti-FLAG M2 antibody was generated by CNBr digestion of the 52/55 doublet. These data strongly support the hypothesis that the 10 kDa increase in molecular weight was due to unprocessed alpha-factor sequence. This conclusion was further validated by finding several peptide sequences for alpha-factor fragments at the N-terminal of the CB2 receptor using pepsin/chymotrypsin digestion and LC/MS/MS approaches. Importantly, unprocessed alpha-factor was found to be associated with poor ligand binding. In addition, controlling the level of CB2 protein expression was found to be critical for minimizing the presence of unprocessed alpha-factor sequence. The information gained from this study should aid the proper expression of not only CB2 receptor but also other members of the GPCR family in P. pastoris.     

Ligand based structural studies of the CB1 cannabinoid receptor.

The structural characterization of G-protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three-dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane-associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X-ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug-receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site-directed mutagenesis of GPCRs allows for three-dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.     

Purification and characterization of the recombinant extracellular domain of human nerve growth factor receptor expressed in a baculovirus system

To obtain the large quantities of the extracellular domain of the nerve growth factor receptor (NGF-R) necessary for structural analyses, we produced this protein in the baculovirus expression system. A cDNA coding for the extracellular domain of the human NGF-R was first introduced into transfer vector pVL941. Recombinant baculovirus was produced by cotransfecting Spodoptera frugiperda cells with the transfer vector and DNA of Autographa californica nuclear polyhedrosis virus. Recombinant viral plaques were selected by morphology and dot hybridization. The expression of recombinant extracellular domain (RED) was analyzed by Western blot analysis using anti-NGF-R monoclonal antibody. Insect cells infected with recombinant virus synthesized RED and secreted it into the culture supernatant. RED was isolated by ammonium sulfate precipitation, immunoaffinity chromatography, and anion exchange chromatography yielding 4 mg of RED/liter of suspension culture. Since there was no apparent effect of endoglycosidase H or F on RED electrophoretic mobility, and since RED did not bind concanavalin A or soybean lectin, there appears to be little or no glycosylation of RED. Purified RED bound 125I-NGF as well as anti-NGF-R monoclonal antibodies. Sedimentation analysis and gel exclusion chromatography revealed that RED is an asymmetric molecule and may be a dimer.     

Constitutive endocytic cycle of the CB1 cannabinoid receptor

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.     

Cannabis reinforcement and dependence: role of the cannabinoid CB1 receptor.

Awareness of cannabis dependence as a clinically relevant issue has grown in recent years. Clinical and laboratory studies demonstrate that chronic marijuana smokers can experience withdrawal symptoms upon cessation of marijuana smoking and have difficulty abstaining from marijuana use. This paper will review data implicating the cannabinoid CB1 receptor in regulating the behavioral effects of Delta(9)-tetrahydrocannobinol (THC), the primary psychoactive component of cannabis, across a range of species. The behavioral effects that will be discussed include those that directly contribute to the maintenance of chronic marijuana smoking, such as reward, subjective effects, and the positive and negative reinforcing effects of marijuana, THC and synthetic cannabinoids. The role of the CB1 receptor in the development of marijuana dependence and expression of withdrawal will also be discussed. Lastly, treatment options that may alleviate withdrawal symptoms and promote marijuana abstinence will be considered.     

Loss of cannabinoid receptor CB1 induces preterm birth

Background

Preterm birth accounting approximate 10% of pregnancies in women is a tremendous social, clinical and economic burden. However, its underlying causes remain largely unknown. Emerging evidence suggests that endocannabinoid signaling via cannabinoid receptor CB1 play critical roles in multiple early pregnancy events in both animals and humans. Since our previous studies demonstrated that loss of CB1 defers the normal implantation window in mice, we surmised that CB1 deficiency would influence parturition events.

Methods and Findings

Exploiting mouse models with targeted deletion of Cnr1, Cnr2 and Ptgs1 encoding CB1, CB2 and cyclooxygenase-1, respectively, we examined consequences of CB1 or CB2 silencing on the onset of parturition. We observed that genetic or pharmacological inactivation of CB1, but not CB2, induced preterm labor in mice. Radioimmunoassay analysis of circulating levels of ovarian steroid hormones revealed that premature birth resulting from CB1 inactivation is correlated with altered progesterone/estrogen ratios prior to parturition. More strikingly, the phenotypic defects of prolonged pregnancy length and parturition failure in mice missing Ptgs1 were corrected by introducing CB1 deficiency into Ptgs1 null mice. In addition, loss of CB1 resulted in aberrant secretions of corticotrophin-releasing hormone and corticosterone during late gestation. The pathophysiological significance of this altered corticotrophin-releasing hormone-driven endocrine activity in the absence of CB1 was evident from our subsequent findings that a selective corticotrophin-releasing hormone antagonist was able to restore the normal parturition timing in Cnr1 deficient mice. In contrast, wild-type females receiving excessive levels of corticosterone induced preterm birth.

Conclusions

CB1 deficiency altering normal progesterone and estrogen levels induces preterm birth in mice. This defect is independent of prostaglandins produced by cyclooxygenase-1. Moreover, CB1 inactivation resulted in aberrant corticotrophin-releasing hormone and corticosterone activities prior to parturition, suggesting that CB1 regulates labor by interacting with the corticotrophin-releasing hormone-driven endocrine axis.     

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.     

Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli

Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100. Affinity chromatography using an amylose column and Ni2+ affinity column was used to purify the sigma-1 receptor. The sigma-1 receptor purified by this method is a 26 kDa polypeptide as assessed by SDS-PAGE, binds sigma ligands with high affinity and can be specifically photoaffinity labeled with the sigma-1 receptor photoprobe, [125I]-iodoazidococaine. Ligand binding using [3H]-(+)-pentazocine indicated that approximately half of the purified protein in Triton X-100 bound to radioligand. The MBP-sigma-1 receptor and the sigma-1 receptor in 0.5% triton were maximally stable for approximately two weeks at -20 degrees C in buffer containing 30% glycerol.     

Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. The enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. To perform more extensive biochemical characterization and to develop new approaches for enzyme therapy, a method of producing and purifying recombinant alpha-galactosidase A suitable for scale-up manufacture for use in humans is needed. Previously, a catalytically active recombinant human alpha-galactosidase A was expressed using a baculovirus vector and purified using conventional chromatography. However, the level of expression was too low to permit economical production and the chromatographic techniques used for enzyme purification were not suitable for enzyme to be used in humans. Therefore, the cDNA of the enzyme was cloned to an improved baculovirus vector and the enzyme was expressed in a 15-liter bioreactor using optimized growth conditions. Infection of insect cells by the baculovirus resulted in a significant fivefold increase in the level of secreted recombinant alpha-galactosidase A activity that is compatible with economic manufacturing. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (Poros 20-CM, Poros 20-HQ) and hydrophobic chromatography (Toso-ether, Toso-butyl) using the BioCAD HPLC workstation. These chromatographic steps are readily scalable to larger volumes and are appropriate for the purification of the recombinant human alpha-galactosidase A to be used in clinical trials of enzyme replacement therapy for Fabry disease patients.     

Purification and characterization of human Syk produced using a baculovirus expression system

The cytoplasmic tyrosine kinase p72syk (Syk) plays an essential role in signaling via a variety of immune and nonimmune cell receptors. Syk is activated in response to the engagement of the appropriate cell surface receptors and can phosphorylate downstream targets and recruit additional SH2-domain-containing proteins. In order to study the characteristics of Syk in vitro, we have overexpressed untagged, full-length human Syk in a recombinant baculovirus expression system. The enzyme was purified to 95% purity using a novel two-step affinity chromatography process using reactive yellow and phosphotyrosine columns. Yields of 3-10 mg purified Syk were obtained from 1 liter of infected insect cells. Western blotting, internal protein sequencing, and the specific tyrosine phosphorylation of a Syk peptide substrate indicated authenticity of the purified protein. The enzymatic properties of Syk were in good agreement with published data for the human enzyme, as the apparent K(m) of Syk for ATP was 10 microM and the peptide substrate was 3 microM. The recombinant protein also showed similar biochemical characteristics to the native protein isolated from B-cells such as autophosphorylation. Proteolytic cleavage of purified recombinant Syk was used to generate the kinase domain by micro-calpain. We therefore describe an efficient expression system and purification methodology to produce biologically active human Syk.     

Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli.

The adenosine A(2a) receptor belongs to the seven transmembrane helix G-protein-coupled receptor family, is abundant in striatum, vasculature and platelets and is involved in several physiological processes such as blood pressure regulation and protection of cells during anoxia. For structural and biophysical studies we have expressed the human adenosine A(2a) receptor (hA2aR) at high levels inserted into the Escherichia coli inner membrane, and established a purification scheme. Expression was in fusion with the periplasmic maltose-binding protein to levels of 10-20 nmol of receptor per L of culture, as detected with the specific antagonist ligand [(3)H]ZM241385. As the receptor C-terminus was proteolyzed upon solubilization, a protease-resistant but still functional receptor was created by truncation to Ala316. Addition of the sterol, cholesteryl hemisuccinate, allowed a stable preparation of functional hA2aR solubilized in dodecylmaltoside to be obtained, and, increased the stability of the receptor solubilized in other alkylmaltosides. Purification to homogeneity was achieved in three steps, including ligand affinity chromatography based on the antagonist xanthine amine congener. The purified hA2aR fusion protein bound [(3)H]ZM241385 with a K(d) of 0.19 nm and an average B(max) of 13.7 nmol x mg(-1) that suggests 100% functionality. Agonist affinities for the purified solubilized receptor were higher than those for the membrane-bound form. Sufficient pure, functional hA2aR can now be prepared regularly for structural studies.     

Diarylimidazolyl oxadiazole and thiadiazole derivatives as cannabinoid CB1 receptor antagonists

Since the CB1 receptor antagonist SR141716 (rimonabant) was reported to modulate food intake, CB1 antagonism has been considered as a new therapeutic target in the treatment of obesity. Several series of derivatives based on diarylimidazolyl oxadiazole and thiadiazole scaffolds were synthesized and tested for CB1 receptor binding affinity. SAR studies directed toward the optimization of imidazole scaffolds resulted in the discovery of 10s which showed highest potency for CB1 receptor binding affinity (IC₅₀ = 1.91 nM) prepared to date.     

Localization of the CB1 cannabinoid receptor in the rat brain. An immunohistochemical study

The presence of central cannabinoid receptor (CB1), involving the N-terminal 14 amino acid peptide, was demonstrated in the rat brain by immunohistochemistry. Intensely stained neurons were observed in the principal neurons of the hippocampus, striatum, substantia nigra, cerebellar cortex, including the Purkinje cells. Moderate CB1-IR cell bodies and fibers were present in the olfactory bulb, cingulate, entorhinal and piriform cortical areas, amygdala and nucleus accumbens. The perivascular glial fibers have shown moderate to high density CB1-IR in olfactory and limbic structures. Low density was detected in the thalamus and hypothalamus and area postrema. The CB1 receptor was widely distributed in the forebrain and sparsely in the hindbrain. These new data support the view that the endogenous cannabinoids play an important role in different neuronal functions as neuromodulators or neurotransmitters.     

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the beta-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant alpha-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular 31P-1H and 1H-1H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.