102 M.E. Abood
receptor-G protein coupling was subsequently confirmed (Breivogel et al. 1999).
Brain region specificity of receptor downregulation has also been demonstrated
by several laboratories (Breivogel et al. 1999; Oviedo et al. 1993; Rodriguez-de-
Fonseca et al. 1994; Romero et al. 1997). A comprehensive study examining the time
courseofchangesincannabinoid-stimulated[^35 S]GTPγSbindingandcannabinoid
receptor binding in both rat brain sections and membranes, following daily∆^9 -
THC treatments for 3, 7, 14, and 21 days, found time-dependent decreases in both
[^35 S]GTPγSand[^3 H]WIN 55212-2 and [^3 H]SR141716A binding in cerebellum,
hippocampus, caudate-putamen, and globus pallidus, with regional differences in
the rate and magnitude of downregulation and desensitization (Breivogel et al.
1999). In a parallel study, the time course and regional specificity of expression of
the CB 1 receptor was examined (Zhuang et al. 1998). They found that CB 1 mRNA
levels were increased above vehicle control animals at 7 days of treatment (Fan
et al. 1996). However, another laboratory found some regions which showed no
changes in receptor binding, [^35 S]GTPγS activation, or mRNA levels following
chronic cannabinoid administration (Romero et al. 1998a,b).
Several recent studies in transfected cell systems have implicated regions of
the CB 1 receptor involved in receptor regulation following chronic agonist expo-
sure. Rapid internalization of CB 1 receptors was observed after agonist exposure
(Hsieh et al. 1999; Rinaldi-Carmona et al. 1998b). In contrast, chronic treatment
of cells with the inverse agonist SR141716A caused upregulation of cell surface
receptors (Rinaldi-Carmona et al. 1998b). As in other GPCRs, the C-terminal do-
main is critical for receptor internalization; truncation of the terminal 14 amino
acids eliminates receptor internalization (Hsieh et al. 1999). Truncation of the
C-terminus at residue 418 abolished desensitization, as did deletion of residues
418–439 (Jin et al. 1999).
On the other hand, phosphorylation of S426 and S430 (tail region) or S317
(third intracellular loop) resulted in CB 1 receptor desensitization; however, these
sites had no influence on internalization (Garcia et al. 1998; Jin et al. 1999). While
receptor internalization was not affected when G protein signaling was disrupted
by treatment with pertussis toxin, a mutation of the highly conserved aspartate
residue in the second TM domain in which G protein coupling is altered did block
CB 1 receptor internalization (Roche et al. 1999).
Both in vivo and in vitro, different cannabinoid compounds can produce various
degrees of tolerance and desensitization, suggesting their actions at cannabinoid
receptors may not be identical (Dill and Howlett 1988; Fan et al. 1994). In a
comparison of three cannabinoid agonists, the most potent compound (CP 55,940)
Table 3.Amino acids important for desensitization and internalization
Desensitization Internalization
S317 in CB 1 D2.50(164) in CB 1
S426 in CB 1 C-terminus 458–472 in CB 1
S430 in CB 1
S352 in CB 2
C-terminus 418–439