Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus

Olson, John M.; Kennedy, Suzanne J.; Cabral, Guy A.

doi:10.1016/s0006-2952(03)00200-4

Cited by 10 publications

(3 citation statements)

References 37 publications

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“…These experiment unequivocally demonstrate that CB2 is glycosylated in our system. The detection of bands representing glycosylated and nonglycosylated versions of CB2 have previously been reported (13,14,29). It should be noted that in heterologous expression systems, the presence of both glycosylated and nonglycosylated expressed integral membrane proteins is relatively common.…”

Section: Expression Of Cb2mentioning

confidence: 88%

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

Filppula

Yaddanapudi

Mercier

et al. 2004

The Journal of Peptide Research

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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.

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Section: Expression Of Cb2mentioning

confidence: 88%

“…Four independent expression constructs were generated in an attempt to optimize the amount of purified, functional (13,14,29). It should be noted that in heterologous expression systems, the presence of both glycosylated and nonglycosylated expressed integral membrane proteins is relatively common.…”

Section: Expression Of Cb2mentioning

confidence: 99%

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

Filppula

Yaddanapudi

Mercier

et al. 2004

The Journal of Peptide Research

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“…The presumed open reading frame for the CB2R protein is within exon 3, which splices with each of the above-noted exons. Interestingly, two major proteins at 40 and 26 kDa were demonstrated using a Semliki Forest Virus system in hamster kidney cells [11]. The predicted molecular weight of the murine CB2R protein is 38.2 kDa [3].…”

Section: Cannabinoid 2 Receptor Structurementioning

confidence: 99%

The Cannabinoid 2 Receptor as a Potential Therapeutic Target for Sepsis

Kasten

Tschöp²,

Tschöp³

et al. 2010

EMIDDT

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The sepsis syndrome represents an improper immune response to pathogens and is associated with an unacceptably high rate of mortality. Although supportive care is of benefit to the septic patient, there are no viable therapeutics available that target the immune system suitable for the whole septic population. Recently, using a physiologically relevant murine mouse model, the cannabiniod 2 receptor has been shown to play a critical role in the host response to sepsis. Here, the structure, expression, signaling, and function of the CB2 receptor on leukocytes will be reviewed. Further, the effects mediated by the CB2 receptor during sepsis will be reviewed. Altogether, alterations in inflammation and the host response during sepsis by the CB2 receptor support its use as a possible therapeutic agent.

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Regulation of cannabinoid CB2 receptor constitutive activity in vivo: repeated treatments with inverse agonists reverse the acute activation of JNK and associated apoptotic signaling in mouse brain

2017

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Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus

Cited by 10 publications

References 37 publications

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

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

The Cannabinoid 2 Receptor as a Potential Therapeutic Target for Sepsis

Regulation of cannabinoid CB2 receptor constitutive activity in vivo: repeated treatments with inverse agonists reverse the acute activation of JNK and associated apoptotic signaling in mouse brain

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