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GLIM, Spring 1996, volume 2
Swarthmore College student publications (1874 - 2013)
1996-04-01
reformatted digital
subunits as probes, David T. Jones and
Randall R. Reed were able to clone a
unique G protein which they designat-
ed Gig Vones & Reed, 1989). G.,
which is the G protein that activates
adenylate cyclase in most systems, has
88 percent of its sequence in common
with Goj¢ and therefore the two G pro-
teins are very similar in structure and
mechanism of action (Jones & Reed,
1989).
By assuming that the receptors
belonged to the G protein family, Linda
Buck and Richard Axel, in what was
one of the most important advances in
the field of olfactory research, cloned
and described the first receptors (Buck
and Axel, 1991). Buck and Axel used
PCR to amplify the conserved regions
of G-protein coupled receptors and
then used those fragments to screen a
cDNA library made from rat olfactory
epithelium cells. The cloned receptors
shared many features in common with
other members of the G protein linked
receptor family including, the position
of several cysteine residues, which form
sulfide bonds in the first and second
extracellular loops, a potential palmi-
toylation site in the C terminal as well
as other sequence similarities (Buck and
Axel, 1991). Within the group of olfac-
tory receptors, the largest differences in
sequence occurred between the third,
fourth, and fifth transmembrane span-
ning regions. Thus, these parts of the
receptors are probably involved in lig-
and binding (see figure one, page 11)
(Buck and Axel, 1991). Northern blot
analysis in which RNA from all major
organ systems of the body was examined
revealed that the receptors were only
found in olfactory epithelium (Buck
and Axel, 1991). Nevertheless, just
because these receptors were expressed
exclusively in the olfactory system, it
did not mean that the receptors Axel
and Buck found were olfactory recep-
tors. One could argue that what Axel
and Buck found were some type of
receptor that just happened to be in the
olfactory epithelium, but which did not
actually bind odor.
In order to counter these possibili-
ties, a team led by H. Breer and his col-
leagues using the same methods as Buck
and Axel cloned more putative olfacto-
ry receptors and expressed these recep-
tors in Spodoptera frugiperda (Sf9) cells
BY ERIC ELENKO @ PROF. SIWICKI @ NEUROBIOLOGY, Bio. 29 @ SPRING “95
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using baculovirus as a vector (Raming
et al., 1993). After exposing the trans-
fected cells to oderants, the researchers
found a large increase in the amount of
inositol triphosphate. Since there was a
change in a particular second messenger
in response to being exposed to a lig-
and, the cloned putative receptors had
to be true odorant receptors (Raming et
al., 1993).
In addition, Breer found that the
transfected receptor (odorant receptor
five) was able to produce an increase in
the amount of intracellular inositol
triphosphate in response to several dif-
ferent odors. Thus, one receptor
appeared able to bind many different
ligands (Ramming et al., 1993).
Breer’s findings were significant,
since a long simmering controversy in
olfactory receptor research is how olfac-
tory neurons are able to bind and
process the millions of diverse ligands
involved in olfaction. one side has
argued that each olfactory neuron
expresses many different types of recep-
tors and each receptor has a narrow
range of ligands that it can bind; the
other side has argued that each neuron
only expresses one type of receptor
which is capable of binding many differ-
ent ligands (Lance, 1994). Thus, Breer’s
findings support the idea of one recep-
tor type on one neuron. In addition, in
situ hybridization of rat cells in the
olfactory epithelium with probes specif-
ic to odorant receptors found that there
was only a minuscule number of cells
which bound probe specific for two
olfactory receptors (Lancet, 1994). A
third line of evidence came from a series
of elegant experiments performed by a
team of scientists led by Richard Axel.
The researchers showed that for recep-
tor 17, only the maternal in a given
paternal allele is expressed but not both
(Chess et al., 1994). If there is inactiva-
tion of one of the alleles, which is usu-
ally accomplished by cis acting tran-
scriptional factors, then the two alleles
replicate at different times during the S
phase of the cell cycle. In fact, Axel
found that there was asynchronous
reproduction of several receptors, indi-
cating receptor 17 (Chess et al., 1994).
If only one allele was expressed on one
chromosome, then this suggests that
only one receptor type would be
expressed per neuron.
GLIM, Spring 1996, volume 2
Swarthmore College student publications (1874 - 2013)
1996-04-01
reformatted digital