Biomedical Engineering Reference
In-Depth Information
Fig. 5.1
G
α15
-mediated calcium imaging. When an odorant binds and activates the OR, G
α15
dis-
sociates from the OR and activates phospholipase C (
PLC
), which hydrolyses the membrane phos-
pholipid PIP2 to form inositol triphosphate (
IP3
) and diacylglycerol (
DAG
). IP3 binds to gated
calcium channels on the endoplasmic reticulum, resulting in an increase in calcium in the cytosol
5.4
Heterologous Cell Types
HEK293 and Other Mammalian Cell Lines
A high-throughput method for
screening ORs would involve the expression of ORs in a heterologous expression
system so as to eliminate the need to generate transgenic mouse lines with the recep-
tor of interest labeled with a marker for gene expression. However, as previously
stated, this method of expression was complicated by the difficulty of expressing
ORs at the surface of the plasma membrane, as they would be broken down in the
endoplasmic reticulum, preventing functional expression at the cell membrane.
The first successful attempt at expressing ORs within a heterologous system uti-
lized HEK-293 cells [
5
,
30
]. A method was developed in which N-terminus rhodopsin
tags were attached to OR coding regions, thus facilitating the translocation of proteins
to the plasma membrane in HEK cells. HEK cells were transfected with a construct
containing an N-terminus rhodopsin tag attached to an altered coding region of the
rat M4 OR in which transmembrane domains
(TM)
II-VII (regions implicated in OR
ligand-binding) were first replaced with that of the beta-2 adrenergic receptor, and then
the rat I7 OR (the cognate ligands of which had been elucidated through adenovirus-
mediated in vivo expression; see Sect. 5.2). When cells transfected with the beta-
2 adrenergic receptor chimera along with G
α15
were stimulated with isoproterenol,
an adrenergic agonist, a transient increase in intracellular Ca2+ levels was observed
through the inositol triphosphate (IP
3
) receptor-mediated pathway (Fig.
5.1
), an ef-
fect that was repeated when the cells transfected with the I7 chimera were stimulated
with octanal. This confirmed that the rhodopsin tag facilitated cell surface expression
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