Molecular Biology of Cannabinoid Receptors 97levels of constitutive activity compared to WT CB 1. This result supports the hy-
pothesis of aχ1 rotamer “toggle” switch (W6.48χ1 g+, F3.36χ 1 trans)→(W6.48
χ 1 trans, F3.36χ1 g+) for activation of CB 1.
7.2
Residues Involved in Activation of CB 1
Studiestodatehaveindicatedthatnotonlyaresetsofdifferentaminoacidsinvolved
in the binding of several cannabinoid ligands, but that these ligands promote
interactions with different G proteins (Bonhaus et al. 1998; Glass and Northup 1999;
Griffin et al. 1998; Kearn et al. 1999; Mukhopadhyay et al. 2000; Selley et al. 1996; Tao
etal.1999).Thedifferentsitesofligand–receptorinteractionmaypromotedifferent
receptor conformations, which in turn result in selective interaction with different
G proteins. Evidence that different receptor conformations can promote distinct
G protein interactions is provided by a study in which a mutation produced a
constitutivelyactiveCB 1 receptor that coupled to GsinpreferencetoGi(Abadji et al.
1999).ThepredominantcouplingoftheWTCB 1 receptor is to Gi;couplingtoGscan
usually only be demonstrated in the presence of pertussis toxin, which uncouples
receptors from Gi/oproteins (Glass and Felder 1997). A swap of two adjacent
residues in the carboxyl terminus of the third intracellular loop/bottom of helix 6,
L6.34(341)A/A6.35(342)L, resulted in a receptor that produced minimal inhibition
of adenylyl cyclase in the presence of agonist, but instead showed increased basal
levels of cAMP in the absence of agonist (Abadji et al. 1999).
UsingsyntheticpeptidesderivedfromtheCB 1 receptor,Howlett’slaboratoryhas
demonstrated that the amino terminal side of the intracellular (i3) loop can interact
with Gi, leading to the inhibition of adenylyl cyclase and that the juxtamembrane
portion of the C-terminus is critical for G protein activation (Howlett et al. 1998).
As in many other GPCRs, the CB 1 receptor C terminal region may assume a
helical structure. In fact, this helical segment is quite clear in the Rho crystal
structure (Palczewski et al. 2000). Synthetic peptides derived from this region
can autonomously inhibit adenylyl cyclase by regulation of Giand Goproteins
(Mukhopadhyay et al. 1999, 2000). Residues R400, K402, and C415 have been
implicated as potential sites for G protein activation (Mukhopadhyay et al. 1999).
Interestingly, the analogous region of CB 2 does not activate Gi(Mukhopadhyay et
al. 1999, 2000).
Residues in the C-terminus have also been shown to be important in G protein
couplingandsequestration(NieandLewis2001a,b).TruncationoftheCB 1 receptor
atresidue417attenuatesGproteincoupling,andtruncationatresidue400abolishes
theinhibitionofcalciumchannelsproducedbyCB 1 receptorsexpressedinsuperior
cervical ganglia neurons (Nie and Lewis 2001a). Truncation at residue 417 also
enhances constitutive activity and G protein sequestration of receptors (Nie and
Lewis 2001b). These mutations did not affect trafficking of the receptor to the cell
surface.
In contrast, mutation of D2.50(164) to N abolished G protein sequestration and
constitutive activity without disrupting agonist activity of CB 1 receptors expressed