Effects of an F238L point mutation on intracellular trafficking and signaling of the cannabinoid type 1 receptor
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Abstract
The CB1 receptor is involved in many physiological functions, such as learning, memory and
regulation of emotion. Furthermore, it plays a role in various pathological conditions, e.g. psychiatric
and neurodegenerative. The CB1 receptors availability and its polarization towards the axon and
the presynapse is amongst others regulated by endocytosis. It has been further suggested that
endocytosis and polarization of the CB1 receptor positively correlate with the receptors activity. In
this work, the effect of a F238L mutation in the fourth transmembrane domain of the CB1 receptor
on its trafficking and signaling was examined. The CB1F238L receptor showed a reduced surface
expression in HEK293 cells, which could be rescued by inverse agonist treatment and was found to be
caused by an increased basal endocytosis via lipid rafts/caveolae. In line with this finding, an
increased lipid raft allocation of the mutant CB1 receptor was observed. In accordance with the role
of endocytosis in receptor polarization, the CB1F238L receptor showed increased axonal polarization,
when ectopically expressed in primary hippocampal neurons. However, although the CB1F238L
receptor showed increased endocytosis and polarization, its basal activity was found to be reduced.
It was concluded that there might be other mechanisms for the regulation of CB1 receptor
polarization, additionally to the receptors activity.
The CB1 receptor is known to form heteromers with other GPCRs. Previous work by others showed
the heteromerization of the CB1 receptor and the D2 receptor by co-immunoprecipitation and FRET.
Additionally, activation of the CB1 receptor led to a reduction in the affinity of the D2 receptor for
dopamine. In the present work, the co-immunoprecipitation of the two receptors was reproduced.
Furthermore, it was shown that the mere co-expression of the CB1 receptor affects dopamine
binding to the D2 receptor. In previous experiments, the CB1F238L mutation in rats was shown to
lead to an increased binding potential of a D2 receptor PET radiotracer. In general, the binding
potential of D2 radiotracers has been proposed to depend on D2 receptor expression and the
subcellular localization of the receptor. However, previous experiments did not reveal changes in D2
receptor mRNA levels in the CB1F238L receptor mutant rat. Thus, further experiments were
performed in order to determine the protein level of the D2 receptor in the striatum of the CB1F238L
mutant rat. However, receptor autoradiography and western blot experiments yielded contradictory
data. As the F238L mutation in the CB1 receptor affects the receptors trafficking, and as receptors
can co-traffick in heteromers, it was then hypothesized that differential co-trafficking of the D2
receptor with the CB1F238L receptor could account for the differences observed in the PET
ratiotracer binding potential. To test this hypothesis, HEK293 cells were generated which express the
D2 receptor either alone or together with the CB1wt receptor or the CB1F238L receptor.