Functional investigation of cannabinoid receptors

Abstract

The endocannabinoid system performs various regulatory functions and has been implicated in a growing number of physiological roles. In an animal model for cutaneous contact hypersensitivity (CHS), we show that mice lacking both known cannabinoid receptors display exacerbated allergic inflammation. Cannabinoid receptor antagonists increased allergic inflammation, whereas receptor agonists attenuated inflammation. <br /> The results with CHS demonstrate a protective role of the endocannabinoid system in contact allergy in the skin and suggest a target for therapeutic intervention.<br /> Unfortunately, it is difficult to assess CB2 receptor expression by immunostaining, because we still often observe immunostaining in tissues from CB2 knock-out mice. To overcome this problem the aim of this work was to generate mice with fluorescent tagged CB2 receptors. <br /> During this work various cell lines expressing murine CB2, CB2-eCFP and CB2-eGFP receptors were generated. The cellular distribution and localization of the fusion proteins was analyzed by fluorescence microscopy. Furthermore, agonist dependent Erk-MAPK phosphorylation using CB2 and CB2-eCFP cell lines was also studied.<br /> The fusion protein was found to be localized in the cell membrane as well as in intracellular compartments. This pattern was strikingly analogous to the one observed with a similar CB1-eGFP fusion protein, indicating that the fusion does not change cellular localization.<br /> To determine whether downstream signalling pathways were affected by the C-terminal fusion, CB2 and CB2-eCFP expressing CHO cells were treated with increasing concentrations of the CB2 specific agonist HU-308. A similar pattern of phosphorylation was observed, indicating that the downstream signalling was not altered. <br /> Having established that fluorescence tagging does not affect functionality of CB2 receptor; CB2-eCFP and CB2-eGFP knock-in mice were generated. Unfortunately, only a weak mRNA expression for CB2-eCFP in spleen, lymph nodes and thymus, and either no or a very low CB2-eCFP protein expression by immunoprecipitation was observed. No CB2-eCFP expression was found in peritoneal and bone marrow derived macrophages, dendritic cells, splenocytes, and lymph node cells by FACS analysis, and also no specific fluorescence in tissue sections of spleen, lymph node, spinal cord, and thymus. <br /> The in-vitro experiments indicate that the functionality of the CB2 receptor was not altered by eCFP fusion. Thus, the tagging strategy should to be useful for the detection of CB2 receptors in living cells or tissues. However, the very weak expression of CB2-eCFP receptors in knock-in mice indicates that the fusion affects expression at translation level and/or stability of the modified protein. During this work, CB2-eGFP knock-in mice were also generated, which are under current analysis.