Type-1 cannabinoid receptors colocalize with caveolin-1 in neuronal cells

Abstract

Type-1 (CB1) and type-2 (CB2) cannabinoid receptors belong to the rhodopsin family of G protein-coupled receptors, and are activated by endogenous lipids termed "endocannabinoids". Recent reports have demonstrated that CB1R, unlike CB2R and other receptors and metabolic enzymes of endocannabinoids, functions in the context of lipid rafts, i.e. plasma membrane microdomains which may be important in modulating signal transduction. Here, we present novel data based on cell subfractionation, immunoprecipitation and confocal microscopy studies, that show that in C6 cells CB1R co-localizes almost entirely with caveolin-1. We also show that trafficking of CB1R in response to the raft disruptor methyl-beta-cyclodextrin (MCD) is superimposable on that of caveolin-1, and that MCD treatment increases the accessibility of CB1R to its specific antibodies. These findings may be relevant for the manifold CB1R-dependent activities of endocannabinoids, like the regulation of apoptosis and of neurodegenerative diseases.

Fig. 1

Localization of CB1R in C6 cells. (A) Western blot analysis of C6 cell membranes (50 μg/lane) from gradient fractions stained for caveolin-1 (anti-CAV1) or CB1 receptor (anti-CB1R). (B) Western blot analysis of C6 cell lysates immunoprecipitated with caveolin-1 specific IgG1 ((mo)CAV1), or irrelevant anti-thyroid hormone receptor antibody (ctrl), and subjected to SDS–PAGE. Immunoblot analysis of whole cell extract (input), immunodepleted supernatants (ID) and immunoprecipitates (IP) was performed using rabbit polyclonal antibodies against CAV1 ((rb)CAV1), CB1R, NAPE-PLD, FAAH and TRPV1. Molecular weights of marker proteins are indicated on the right. C, Colocalization of CAV1 and CB1R by confocal microscopy. The yellow spots indicate that CAV1 and CB1R colocalize, and are present mainly in intracellular vesicles. Membrane fractionation, immunoprecipitation, confocal microscopy and dilution of specific antibodies were as described in Section 2. Reported blots and images are representative of triplicate experiments.

Fig. 2

Trafficking of CAV1 and CB1R upon MCD treatment. C6 cells were treated for the indicated periods of time with 2.5 mM MCD, and immunofluorescence was recorded at 40× magnification. Cells were double stained with antibodies against CB1 receptors (red) and caveolin-1 (green). Reported images are representative of triplicate experiments.

Fig. 3

Effect of MCD on CB1R immunoreactivity. C6 cells were treated for the indicated periods of time with 2.5 mM MCD, and immunofluorescence was recorded at 20× (upper panels) or at 60× (lower panels) magnification. Cell nuclei were stained in blue with DAPI, whereas localization of CB1 receptors is shown as green spots. Reported images are representative of triplicate experiments.