Other GPCRs

The GPR55 receptor is a GPCR with little homology to the classical cannabinoid receptors. It was cloned almost a decade ago and has been detected in both the brain and peripheral tissues although a comprehensive investigation of its distribution is lacking (Sawzdargo et al., 1999).

Anandamide binds to GPR55 and activates coupling to GTPgS with an EC50 of 18 nM (Drmota et al., 2004; Ryberg et al., 2007). Coupling of this GPCR to G13 as well as G12 and Gq subtypes of G proteins has been indicated (Baker et al., 2006; Brown and Wise, 2001; Lauckner et al., 2008; Ryberg et al., 2007). At micromolar concentrations, anandamide stimulation of GPR55 increases intracellular Ca2+ in transiently transfected HEK 293 cells (Lauckner et al., 2008). The same response, when elicited by alternative cannabinoids, was dependant on Gq, G12, PLC and RhoA activation and subsequent release of Ca2+ from internal stores by IP3 (Lauckner et al., 2008) and similarly, in GPR55 stably transfected HEK 293 cells, anandamide stimulation resulted in activation of cdc42, rac1, and RhoA (Ryberg et al., 2007).

Recently, further insights into the signaling pathways involved in GPR55 stimulation by anandamide have been elucidated. Waldeck-Weiermair et al. (2008) describe a role for integrin clustering in CB1 and GPR55 receptor signaling; when integrins are unclustered, signaling via the CB1 receptor activates spleen tyrosine kinase (Syk), an inhibitor of PI3K and as GPR55 signaling is dependent on PI3K activity, when the CB1 receptor is activated GPR55 signaling is inhibited. The inhibition of GPR55 signaling is lifted when integrins are induced to form clusters and dissociate from the CB1 receptor (by addition of Mn2+ or removal of Ca2+ from the extracellular space). The authors suggest that this involves the physical movement of the CB1 receptor complex, including Syk, away from integrin-associated GPR55, allowing activation of PI3K which proceeds to activate PLC via a tyrosine kinase Bmx/Etk. PLC is responsible for the generation of IP3 which stimulates release of Ca2+ from intracellular stores, subsequently activating the transcription factor NFAT. Upregulation ofp42/44 MAPK occurs under both integrin clustering conditions but it is not clear if this is due to continued CB1 receptor signaling independent of integrin association, through GPR55 signaling pathways, or a combination of both (Waldeck-Weiermair et al., 2008). This work highlights that careful characterization of cell lines and models as well as experimental protocols are important in determining cell signaling pathways. In this case, the expression profile of other membrane proteins strongly influences the ability of GPR55 to respond to stimuli and is further modulated by the presence or absence of various ions during stimulation (Waldeck-Weiermair et al., 2008).

Anandamide has been suggested to mediate a non-CB1/CB2 or TRPV1 receptor-mediated vasorelaxation of blood vessels (White et al., 2001).

GPR55 has been suggested as a candidate for the ''endothelial anandamide receptor'' involved in vasodilatory effects (Hiley and Kaup, 2007; Offertaler et al., 2003). Although few publications focusing on GPR55-mediated vasodilation are available, the results are controversial. Many of the vasodilatory effects mediated by the unidentified endothelial receptor are dependent on coupling to Gi/o which has not been observed with GPR55. In addition, GPR55 knockout mice fails to exhibit any changes in basal blood pressure or heart rate, and isolated knockout blood vessels continue to exhibit vasodilatory responses to agonists of the receptor (Johns et al., 2007). On the other hand Waldeck-Weiermair et al. (2008) saw an increase in the anandamide-induced intracellular Ca2+ response when GPR55 was over-expressed in endothelial cells and a corresponding decrease in response when GPR55 expression was downregulated with siRNA. This indicates that in this model, at least, GPR55 is the likely endothelial anandamide receptor. Although GPR55 may be involved in the vasorelaxation effects of anandamide there appears to still be an unidentified receptor or receptors that also mediate this effect.

Although only briefly noted by Overton and colleagues, anandamide, but not methanandamide, induced a mild response in a yeast reporter assay with GPR119, another orphan GPCR (Overton et al., 2006). Additional evidence and characterization ofthis interaction may provide an alternative candidate for the endothelial receptor.

It is apparent that anandamide modulates the activity of many targets besides the better characterized interactions with CB1, CB2, and TRPV1 receptors. Many of these appear to be specific to the nervous system and functioning of neurons. As research progresses in this area, it would be expected that some of the currently suggested direct targets for anandamide action will be confirmed and others placed further down the signal trans-duction pathways stimulated by this molecule. The role of anandamide in altering lipid bilayer character should not be overlooked as a mechanism to achieve structural constraints and changes in the activity of integral membrane proteins, particularly given that the majority of putative anandamide receptors are ion channels.

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