Internalization and Recycling of the CB1 Cannabinoid Receptor

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Abstract

Tolerance develops rapidly to cannabis, cannabinoids, and related drugs acting at the CB1 cannabinoid receptor. However, little is known about what happens to the receptor as tolerance is developing. In this study, we have found that CB1 receptors are rapidly internalized following agonist binding and receptor activation. Efficacious cannabinoid agonists (WIN 55,212-2, CP 55,940, and HU 210) caused rapid internalization. Methanandamide (an analogue of an endogenous cannabinoid, anandamide) was less effective, causing internalization only at high concentration, whereas delta9-tetrahydrocannabinol caused little internalization, even at 3 microM. CB1 internalized via clathrin-coated pits as sequestration was inhibited by hypertonic sucrose. Internalization did not require activated G protein alpha(i), alpha(o), or alpha(s) subunits. A region of the extreme carboxy terminus of the receptor was necessary for internalization, as a mutant CB1 receptor lacking the last 14 residues did not internalize, whereas a mutant lacking the last 10 residues did. Steps involved in the recycling of sequestered receptor were also investigated. Recovery of CB1 to the cell surface after short (20 min) but not long (90 min) agonist treatment was independent of new protein synthesis. Recycling also required endosomal acidification and dephosphorylation. These results show that CB1 receptor trafficking is dynamically regulated by cannabimimetic drugs.

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Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice.

C Ledent, G Vassart, J-F Aubert … (1999)

The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced. These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.

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Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation

RG Anderson, JE Heuser (1989)

Two seemingly unrelated experimental treatments inhibit receptor mediated endocytosis: (a) depletion of intracellular K+ (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson. 1983. Cell. 33:273- 285); and (b) treatment with hypertonic media (Daukas, G., and S. H. Zigmond. 1985. J. Cell Biol. 101:1673-1679). Since the former inhibits the formation of clathrin-coated pits (Larkin, J. M., W. D. Donzell, and R. G. W. Anderson, 1986. J. Cell Biol. 103:2619-2627), we were interested in determining whether hypertonic treatment has the same effect, and if so, why. Fibroblasts (human or chicken) were incubated in normal saline made hypertonic with 0.45 M sucrose, then broken open by sonication and freeze-etched to generate replicas of their inner membrane surfaces. Whereas untreated cells display typical geodesic lattices of clathrin under each coated pit, hypertonic cells display in addition a number of empty clathrin "microcages". At first, these appear around the edges of normal coated pit lattices. With further time in hypertonic medium, however, normal lattices largely disappear and are replaced by accumulations of microcages. Concomitantly, low density lipoprotein (LDL) receptors lose their normal clustered distribution and become dispersed all over the cell surface, as seen by fluorescence microscopy and freeze-etch electron microscopy of LDL attached to the cell surface. Upon return to normal medium at 37 degrees C, these changes promptly reverse. Within 2 min, small clusters of LDL reappear on the surfaces of cells and normal clathrin lattices begin to reappear inside; the size and number of these receptor/clathrin complexes returns to normal over the next 10 min. Thus, in spite of their seeming unrelatedness, both K+ depletion and hypertonic treatment cause coated pits to disappear, and both induce abnormal clathrin polymerization into empty microcages. This suggests that in both cases, an abnormal formation of microcages inhibits endocytosis by rendering clathrin unavailable for assembly into normal coated pits.