(Received for publication, August 26, 1994; and in revised form, January 17, 1995)
From the
A novel heterotrimeric G-protein -subunit has been cloned,
and its function has been confirmed by expression and purification.
This
-subunit is only detected in the olfactory epithelium, the
vomeronasal epithelium and, to a lesser extent, the olfactory bulb. It
is absent from all other tissues studied including the nasal
respiratory epithelium. During development, expression of G
8 in
the olfactory epithelium parallels neurogenesis, peaking shortly after
birth and declining in the adult. In situ hybridization
studies localize expression of this novel
-subunit to the sensory
neurons; hybridization is strongest in the region of the epithelium
that contains immature neurons. Unlike proteins that are expressed only
in mature olfactory neurons (e.g. olfactory marker protein or
Golf
), expression of G
8 in the olfactory epithelium is
relatively unaffected by olfactory bulbectomy. In the vomeronasal
epithelium expression of G
8 is also highest in the developing
neurons. Taken together, these findings are consistent with a very
specific role for G
8 in the development and turnover of olfactory
and vomeronasal neurons.
Heterotrimeric G-proteins ()are central to a wide
variety of receptor-effector coupling pathways(1, 2) .
It was believed that the
-subunit of these proteins was the only
critical determinant of G-protein receptor and G-protein effector
interaction. However, it is now becoming clear that the diverse
-subunits (2) also have distinct roles. One of the
first examples of this was the mating response pathway in yeast where
molecular genetic experiments demonstrated that the
-subunits
are responsible for signaling and that the
-subunit has an
inhibitory role(3) . Interactions between different
G
s and specific G-protein-linked receptors have been shown to
vary in vitro(4) . Recently, in vivo coupling
of specific receptors to effectors has also been shown to be determined
by the nature of the
-subunit (5) and the
-subunit (6) . A number of different effector enzymes are influenced by
-subunits, for example different subtypes of PLC
(7, 8) and adenylate cyclase (9) differ in
sensitivity to
-subunits. Another role that
-subunits appear to play is in desensitization of
G-protein-linked receptor pathways by recruitment of the G-protein
receptor kinase,
-adrenergic receptor kinase-1, to the
membrane(10, 11) , and possibly of other more diverse
proteins involved in signal transduction(12) . The diversity of
- and
-subunits also seems able to influence the interaction
between effectors and
-subunit (13, 14) .
The role of heterotrimeric G-proteins in control of cell fate and
development has been documented in several organisms. For example,
G-proteins mediate cell-cycle arrest in haploid Saccharomyces
cerevisiae(3) and the growth and development of Dictyostelium discoidium(15) . In multicellular
organisms, G-proteins have also been implicated as mediators of
development; for example the developmental mutant of Drosophila, concertina, results from a defect in the
gene for a G-protein
-subunit(16) .
The understanding
of olfactory signal transduction has advanced rapidly over the last few
years. Proteins that appear to have a role in G-protein-mediated
coupling of olfactory receptors to cAMP-controlled ion flux have been
cloned from olfactory epithelium and have been shown to be highly
enriched in the sensory cilia(17, 18, 19) .
More recently, much attention has focused on what appears to be a very
large family of G-protein-linked olfactory
receptors(20, 21, 22) . However, no coupling
between these receptors and the cAMP second messenger pathway has been
shown yet. Other signaling pathways have also been suggested to have a
role in olfactory signal transduction. These pathways include pertussis
toxin-sensitive G-protein-dependent stimulation of PLC(23) , a
pathway which may involve stimulation of PLC-2 by G-protein
-subunits in many cells(7, 8) . Therefore,
we were interested in the diversity of G-protein
-subunits in the
olfactory epithelium.
Among vertebrate neurons, the olfactory and
vomeronasal neurons are unique in that they turnover throughout life.
Olfactory neurons are replaced through differentiation of the basal
cells of the olfactory epithelium(24, 25) . The
vomeronasal organ possesses a neuroepithelium like that of the
olfactory epithelium. However, the role of this organ appears to be in
perception of stimuli related to social and/or reproductive behavior in
many species(26, 27) . As in the olfactory epithelium,
the neural receptor cells of the vomeronasal organ appear to turnover
throughout life with the principal regions of neurogenesis being at the
junctions between sensory and nonsensory
epithelia(28, 29) . In studying the diversity of
G-protein subunits that may have roles in olfaction, we have
characterized a novel -subunit which was expressed specifically in
neural cells in the olfactory and vomeronasal epithelia. The expression
of this G-protein
-subunit was not limited to mature neurons as is
the case for proteins believed to be involved in olfactory signal
transduction, but was highest in developing neurons, suggesting a
signaling role for a novel heterotrimeric G-protein in neurogenesis in
these tissues.
Figure 2:
Comparison of the predicted protein
sequence of G8 to previously reported G
s. The sequence of the
novel G
was aligned with the sequences of several mammalian and
one insect (D
-1) subtypes of G
subunit(53, 54, 55, 56, 57, 58, 59) .
Residues in other G
s identical to those in G
8 are indicated
by a solid line above the sequence, residues that are similar
by a colon above the sequence. Gaps introduced into the
sequences to optimize the alignment are represented by periods. The sequences of G
4 and G
-S1 are
incomplete. The regions of sequence of G
2 used for design of the
partially degenerate primers, oligo 1 and 2, are underlined.
An
oligonucleotide specific for the new -subunit (oligo 3) was
designed, 5`-end labeled and was used to screen approximately 150,000
plaques of an olfactory cDNA library in
gt10 (hybridization 45
°C: 5
NET (0.15 M NaCl, 15 mM Tris-HCl,
pH 8.3, 1 mM EDTA), 5
Denhardt's, 100 µg/ml
yeast tRNA, 0.25% SDS; stringency washes, 40 °C, 0.5
SSC
(0.15 M NaCl, 15 mM sodium citrate pH 7.0). Several
hybridizing plaques were purified, and the cDNA inserts were excised
and subcloned into pBluescript for analysis. None of these initial
isolates represented a full-length transcript of the
-subunit,
therefore 5`-RACE was carried out. Oligo 3 was used to prime first
strand cDNA synthesis using total olfactory RNA as a template (10
µg of RNA, 10 pmol of oligo 3). Residual nucleotides and primer
were removed by two rounds of dilution and concentration using
ultrafiltration (Centricon 100). The cDNA was tailed using terminal
transferase and dATP. PCR amplification was carried out using oligo 4
and oligo 5 (95 °C, 180 s; three cycles 95 °C, 20 s; a linear
ramp lasting 90 s from 45 to 72 °C; 72 °C, 600 s; then 35
cycles 95 °C, 15 s, 50 °C, 30 s, 72 °C, 60 s, followed by
72 °C, 600 s). The single
150-bp product was gel purified and
cloned into pBluescript for sequence analysis.
Oligo 6 was
synthesized on the basis of the sequence of the 5`-RACE product and was
used to screen 100,000 clones of the olfactory
-Zap II
library (hybridization 65 °C: 5
NET, 5
Denhardt's, 100 µg/ml yeast tRNA, 0.25% SDS; stringency
washes: 60 °C, 0.5
SSC). A single hybridizing plaque was
isolated. Using the in vivo excision protocol, the insert was
obtained in pBluescript. The sequence of this clone was determined for
both strands using Sequenase II. To check whether the 5`-non-coding
region of this clone, not contained within the 5`-RACE product, was an
alternative (longer) form of G
, PCR was carried out using oligo 7
and oligo 3. Total olfactory epithelium RNA (10 µg) was used as a
template for cDNA synthesis using an oligo(dT) primer. PCR
amplification was carried out with 150 pmol of each primer and 10 ng of
cDNA (95 °C, 270 s; 30 cycles 95 °C, 30 s; 55 °C, 30 s; 72
°C, 30 s; followed by 72 °C, 600 s).
RNA from a
variety of tissues (10 µg) was denatured (10 min, 85 °C) in 30
µl of hybridization buffer (80% formamide, 0.4 M NaCl, 40
mM PIPES, pH 6.7, 1 mM EDTA) containing antisense RNA
for the -subunit, Golf
, and actin. Sense cRNA for the
-subunit was mixed with 10 µg of yeast tRNA and was denatured
with the same mixed antisense probe. Denatured RNA was cooled rapidly
to 45 °C and was incubated for 16 h to allow hybridization.
Hybridized samples were cooled to room temperature and treated with
RNase-T1 (300 µl, 2 µg/ml; 60 min), followed by proteinase K
(0.6 mg/ml; 30 min, 37 °C). RNA was denatured, fractionated on 6%
acrylamide sequencing gels containing 8 M urea, and protected
RNA was visualized using autoradiography.
Figure 1:
The cDNA sequence of the novel
G-protein -subunit, G
8. The nucleotide sequence of the
full-length clone of the novel G-protein
-subunit was determined
from both strands. The amino acid sequence predicted for the protein is
shown above the nucleotide sequence. The start site for the longest
clone obtained from screening of the
gt10 library is indicated by double underlining. The positions of the primers used in the
cloning and generation of the full coding sequence for expression and
generation of probes are indicated by underlining. The product
of 5`-RACE was identical to the sequence shown except that the
5`-residue, indicated by an arrow, was not A but G. The
isoprenylation motif at the C terminus of the protein and the consensus
Olf1-binding site are also highlighted.
An
oligonucleotide was synthesized on the basis of the sequence of 5`-RACE
(5` to the coding region) and was used to rescreen the olfactory cDNA
library. A single full-length clone of the novel -subunit
(containing the sequence determined by 5`-RACE and that of all partial
clones) was isolated from
100,000 plaques. The sequence of this
clone (determined for both strands) is shown in Fig. 1. The
predicted protein sequence of G
8 shares features present in other
G
s, most notably, the 3`-isoprenylation site (-CaaX) and the small
size (
7 kDa). At the amino acid level, the predicted sequence
identity to known mammalian G
s is in the range
25-70%.
The most similar known G
-subunit is G
2. An alignment of the
novel G
and other subtypes from several species is shown in Fig. 2. The full-length clone isolated from the library
contained sequence that extended beyond the 5` end of the RACE product.
An appropriate sized product was obtained when olfactory cDNA was used
as a template for PCR amplification with a primer within this region of
5`-extended sequence and a second in the coding sequence of the G
(data not shown).
Figure 3:
Heterologous expression of G8.
G
8 and G
2 were expressed as heterodimers with G
1 in
insect larval cells (Sf9). A, purified heterodimers
were analyzed by SDS-PAGE, 16% Tricine gel (60) stained with
Coomassie Blue. Lane 1, G
1
8; lane 2,
G
1
2; M molecular weight standards. B, the
efficacy of
-heterodimers at stimulating the
rhodopsin-dependent activation of transducin: the concentration
dependence of the initial rate of
-dependent stimulation of
rhodopsin-mediated activation of transducin was determined using a
10-min standard reaction. This contained 30 nM regenerated
rhodopsin, 0.2 µM transducin, 2 µM GTP-
-S, 0.1% cholate, and either G
1
8 or
G
1
2 (indicated in the inset) at concentrations
shown. Activation of transducin was determined by GTP-
-S binding.
Full activation of transducin was achieved by 1 h of incubation with
500 nM brain G
and 2 µM rhodopsin and
corresponded to the calculated saturation. Data points are the
mean ± standard deviation of triplicates; curves are
single-site fits with half-maximal values of 120 nM for
G
1
8 and 40 nM for G
1
2. C, time
course of G
-dependent stimulation of rhodopsin-mediated
activation of transducin: standard reactions containing 90 nM G
1
8 (three independent expressions and purifications
indicated by filled or open triangles) or 30 nM G
1
2 (two independent expressions and purifications
indicated by filled or open circles) were incubated
for 90 min. Aliquots were removed at indicated times and transducin
activation measured by GTP-
-S binding. Curves are simple
exponential fits of the means obtained for the three preparations of
G
1
8 and the two preparations of G
1
2. For comparison
the activation of 0.2 µM transducin by 30 nM rhodopsin in the absence of
-heterodimers is
shown.
The efficacy of purified G1
8 at stimulating
rhodopsin-mediated activation of transducin was compared with that of
G
1
2 expressed and purified under identical conditions. Both
-heterodimers had a profound effect on the initial rate of
GTP-
-S binding to transducin (Fig. 3B). For the
conditions shown, 30 nM rhodopsin and 0.2 µM transducin, the half-maximal rate of binding was achieved at a
G
1
8 concentration of
120 nM, whereas this rate
was reached when
40 nM G
1
2 was added. In time
course experiments, the effectiveness of three separate preparations of
G
1
8 was compared with that of two preparations of
G
1
2 (Fig. 3C). In all cases 90 nM G
1
8 resulted in very similar stimulation of GTP-
-S
binding by transducin to that produced by 30 nM G
1
2.
Figure 4:
Tissue distribution of G8: Northern
analysis. Northern analysis of the tissue distribution of G
8
indicated that its expression was specific to the olfactory epithelium
and was not found in other tissues. A, 20 µg of total RNA
from brain (1), heart (2), intestine (3),
kidney (4), liver (5), lung (6), olfactory
epithelium (7), and testis (8) was probed for
transcripts that hybridized at high stringency with cDNA probes to the
full coding sequence of G
8. B, 2 µg of
A
-RNA from brain (1), olfactory epithelium (2), olfactory epithelium of bulbectomized rats (3),
and testis (4) was probed under identical conditions to A; the blot was stripped and reprobed for
-actin (center panel) and was stripped and hybridized with a mixed
Golf
and OMP probe (lower panel; both cDNAs used to
generate probes were of similar length but the relative specific
activity of the Golf
probe was 10
higher than that of
OMP).
RNase protection was used to make a more detailed
quantitation of the expression of G8 in the olfactory epithelium,
in other nasal epithelia, and in a wide variety of other adult rat
tissues (Fig. 5). G
8 mRNA was detected at comparable levels
in the olfactory and vomeronasal epithelia; a much lower level was
present in the olfactory bulb, and no G
8 mRNA was detected in any
other tissue. This pattern of expression was entirely consistent with
that detected using Northern analysis (Fig. 4A). Based
on protection of antisense G
8 cRNA by known amounts of sense cRNA,
G
8 was estimated to be expressed at a level of about 10
copies/µg total RNA (
2 molecules in 10
of
mRNA). Preliminary experiments to investigate the relative amounts of
G
8, Golf
, and
-actin expression in the olfactory
epithelium indicated that in the adult rat a ratio of about 1:100:1000,
G
8/Golf
/
-actin was present. Therefore, in order to carry
out RNase protection assays shown in Fig. 5, the specific
activities of the Golf
and
-actin probes were reduced 100 and
1000-fold, respectively, by increasing the concentration of GTP in the
labeling reaction. The relative specific activities of
actin/Golf
/G
8 probes used in the RNase protection assays
shown in Fig. 6were 1:20:1000. The relatively even protection
of cRNA probes for actin and G
8 indicated that in the olfactory
epithelium of adult rats the expression of actin is about three orders
of magnitude higher than that of G
8; quantitation of Golf
and
G
8 is less precise but it appears that about 10-fold higher levels
of Golf
are expressed than of G
8 ( Fig. 5and Fig. 6). The adult vomeronasal epithelium contained a similar
ratio of G
/actin RNA to that observed in the olfactory epithelium
but, unlike in the olfactory epithelium, no expression of Golf
was
detected. The olfactory bulb RNA contained
10-fold less G
RNA
relative to actin RNA. G
8 RNA was not detected in several regions
of the brain, nor was it present in a number of other tissues including
the nasal respiratory epithelium. However, in contrast to G
8,
which was only expressed in the olfactory tract, considerable
expression of Golf
could be detected in the brain (Fig. 6A).
Figure 5:
Tissue
distribution of G8: RNase protection. 10 µg of total RNA
isolated from a number of different adult rat tissues was analyzed by
RNase protection. The relative specific activities of probes for
G
8, Golf
, and actin were 1000:100:1, respectively. Protected
fragments were the full coding sequence (0.26 kb) for G
8 and were
from the coding sequence of Golf
(0.45 kb) and actin (0.4 kb). A, cerebellum (1), brainstem (2), mid-brain (3), frontal lobe (4), olfactory bulb (5),
eye-cup (6), olfactory epithelium (7), respiratory
epithelium (8), vomeronasal organ (9) heart (10), intestine (11), kidney (12), liver (13), lung (14), muscle (15), spleen (16), testis (17), tongue (18), tRNA (19 and 20). Too little RNA from respiratory epithelium
relative to that from other tissues was used in A, therefore
the assay was repeated with new preparations of RNA in B:
respiratory epithelium (1), vomeronasal epithelium (2), olfactory epithelium (3), olfactory bulb (4), frontal lobe (5), tRNA (6). The
positions of protected bands and the undigested G
8 probe are
marked.
Figure 6:
Developmental changes in G8
expression. 10 µg of total RNA from total brain or from olfactory
epithelium were analyzed by RNase protection at different times before
and after birth. The relative specific activities of probes for
G
8, Golf
, and actin were 1000:20:1, respectively. Protected
fragments were the full coding sequence (0.26 kb) for G
8 and were
from the coding sequence of Golf
(0.45 kb) and actin (0.4 kb). A, comparison of expression of Golf
and G
8 during
development of brain and olfactory epithelium: brain E19.5 (1), brain P6.5 (2), brain adult (3), tRNA (4), olfactory E19.5 (5), olfactory P6.5 (6), olfactory P13.5 (7), olfactory adult (8), tRNA (9), vomernasal adult (10). B, expression of G
8 in the olfactory epithelium was
analyzed in more detail during early development P13.5 (1),
P9.5 (2), P6.5 (3), P4.5 (4), P2.5 (5), P0.5 (6), E21.5 (7), E19.5 (8), whole head E13.5 (10), and tRNA (11).
The positions of protected bands and the undigested G
8 probe are
marked.
Figure 7:
Neural cell-specific localization of
G8 in the olfactory epithelium.
S-Labeled cRNA probes
for the full coding sequence of G
8 were hybridized with adult rat
olfactory epithelium and were washed at high stringency; following
autoradiography, tissue was lightly stained using toluidine blue. A, low magnification bright-field of a region of the olfactory
epithelium hybridized with G
8 sense cRNA shown using dark-field
illumination (B); C, dark-field of an adjacent
section hybridized with antisense cRNA; the boxed region of C is shown at higher magnification in bright-field (D); marked by arrows is the zone of hybridization
clearly seen in dark-field (E); F, bright-field high
magnification of a region of epithelium showing different cell layers
hybridized with sense G
8 cRNA; G, bright-field of an
adjacent section hybridized with antisense cRNA; two representative
clusters of silver grains are arrowed; the positions of the
cell-bodies of the sustentacular cells (s) at the apical
surface of the epithelium, the basal cells at the basolateral surface (b), and the olfactory (o) neurons are indicated. Bar = 36 µm.
A consistent antisense-specific
hybridization of G8 to a subpopulation of olfactory neurons mostly
with nuclei toward the base of the neural cell layer was also detected
in thicker (16 µm) sections with digoxigenin-labeled probes (Fig. 8, A and B). The localization of G
8
was studied after olfactory bulbectomy because significant expression
of G
8 was still detected (Fig. 4). The major histologic
consequence of olfactory bulbectomy was a marked thinning of the
olfactory epithelium resulting from loss of mature neurons. The
expression of G
8 appeared relatively unaffected by bulbectomy (Fig. 8C). Hybridization was not detected in the apical
region of the epithelium that is made up of sustentacular cells but was
strong toward the basolateral surface. In bulbectomized animals, the
region containing G
8 mRNA is made up of immature olfactory neurons
not killed by bulbectomy and also by neurons that started to develop
after bulbectomy.
Figure 8:
Comparison of the cellular localization of
G8 expression in the olfactory and vomeronasal epithelia.
Digoxigenin-labeled cRNA probes for the full coding sequence of G
8
were hybridized (and washed at high stringency) to sections of normal
olfactory epithelium (A and B), epithelium isolated 7
days after bulbectomy (C), and the vomeronasal organ (D-G). A, C, D, and F were
probed with G
8 antisense cRNA; B, E, and G with G
8 sense cRNA. A-C were lightly
stained with eosin and were photographed using normal illumination. In D and E, regions of unstained sections of the
vomeronasal epithelium were photographed using Nomarski optics. F and G show low magnification cross-section through the
whole vomeronasal organ, under normal illumination; arrows indicate the primary regions of neurogenesis. High magnification (A-E), bar = 50 µm; low magnification (F) and (G), bar = 200
µm.
In the vomeronasal epithelium, the expression of
G8 appeared somewhat higher than in the olfactory epithelium (e.g. compare Fig. 8, A and D).
Antisense-specific hybrization of the digoxigenin-labeled probe to the
full coding sequence of G
8 in the perinuclear region of cells
located throughout the sensory vomeronasal epithelium was detected (Fig. 8, D and E). This perinuclear
distribution was consistent with the clustering of silver grains
observed in the olfactory epithelium (Fig. 7G). At low
magnification a clear gradation in the distribution of G
8 mRNA was
detected, with the highest expression being at the boundaries between
the sensory and non-sensory regions of the epithelium (arrowed in Fig. 8F). No specific hybridization was
detected in the convex, non-sensory (respiratory) epithelium (Fig. 8, F and G). Thus, the cellular
distribution of mRNA for G
8 indicates that the G-protein that
contains this
-subunit probably has a very similar role in the
olfactory and vomeronasal epithelia. No hybridization of G
8 was
detected to sections of the olfactory bulb (data not shown) even though
this tissue contained low levels of G
8 mRNA (Fig. 5A).
We investigated whether novel G-protein -subunits might
play a role in olfaction, and as a result identified and cloned a new
-subunit, G
8, (
)from olfactory cDNA. The sequence
of G
8 is typical of known G-protein
-subunits and contains a
conserved site for C-terminal isoprenylation(34) . The -CaaL
motif found at the C terminus of G
8 is predicted to result in
geranyl-geranylation of G
8 in vivo with concomitant
localization of
8-heterodimers to the cell
membrane(35, 36) . The membrane localization and the
similarity of the chromatographic properties of baculovirus expressed
G
1
8 with other
-heterodimers (13, 37) suggest that recombinant G
8 was also
isoprenylated.
Functional assay places G1
8 as intermediate
between G
1
2, brain, or placental
and the retinal
-heterodimer at stimulating transducin activation by
rhodopsin(4, 37) . The 3-fold difference in activity
of G
1
8 and G
1
2 is relatively large considering
their sequence similarity particularly when compared with results from
other in vitro studies of defined
-heterodimers(13, 14, 38) .
However, the specificity of
-heterodimers that has been
reported in vivo(5, 6) has still to be
explained in view of the redundancy that is consistently observed in vitro.
In adult rats there is a very specific expression
of G8 in the olfactory and vomeronasal epithelia, with a trace of
mRNA in the olfactory bulb ( Fig. 4and Fig. 5). The low
levels of G
8 RNA in the olfactory bulb may be associated with
small amounts of mRNA transported along the axon as has been observed
for other neural mRNAs(39) . Of particular significance is the
lack of expression of G
8 in the nasal respiratory mucosa. The
respiratory epithelium is continuous with the olfactory epithelium but
distinct from the olfactory epithelium in that it does not contain
sensory neurons. The absence of G
8 mRNA in brain and other tissues
studied indicates that G
8 is more restricted to the olfactory
system than the G-protein
-subunit Golf
(40) , the
olfactory adenylate cylase(41) , or apparently some of the
olfactory receptors(42) . Therefore, at least in the adult, the
signal transduction pathway, in which G
8 functions, appears to be
olfactory-specific.
In situ hybridization was used to
examine the localization of G8 expression in the olfactory and
vomeronasal epithelia. Nonspecific hybridization to other related
proteins might influence interpretation of results. Therefore, to
minimize this potential problem, very high stringency wash conditions
and RNaseA digestion were used following hybridization. The most
similar nucleotide sequence to G
8 is that of G
2; their full
coding sequences are only 62% identical (with no long regions of
continuous identity). Comparison of the results of Northern analysis (Fig. 4) and RNase protection (Fig. 5) indicates that
cross-hybridization of G
8 probes to G
2 (or other sequences)
is unlikely, under stringent conditions. We were unable to detect
hybridization of G
8 probes in the olfactory bulb by in situ hybridization, indicating that the in situ technique does
not, in itself, result in reduced specificity. Moreover, the
localization of G
8 expression that we detected in the olfactory
epithelium (both with
S- and digoxigenin-labeled probes)
was discrete, only a subpopulation of the neural cells showed
significant hybridization (see below). All non-neuronal cells were
negative. This expression pattern was consistent with that found in the
vomeronasal epithelium and also with the developmental and bulbectomy
data (see below). Therefore, it is very likely that the in situ hybridization that we detect reflects the true expression of
G
8.
In the olfactory epithelium, G8 expression is
localized to olfactory neural cells ( Fig. 7and Fig. 8).
However, the expression is not evenly distributed throughout the neural
cell layer of the epithelium, but is concentrated toward the base of
this layer ( Fig. 7and Fig. 8). Olfactory neurons develop
by differentiation of basal cells in the olfactory
epithelium(24) . As neural development proceeds, there is a
migration of the cell body of the neuronal precursors toward the
epithelial surface. Therefore it appears that G
8 is predominantly
expressed in immature neurons. To study this in more detail and to
investigate whether G
8 has a more general role in neurogenesis, we
examined the expression of G
8 during the development of the
olfactory neuroepithelium and the brain. The first olfactory neurons
begin to appear at about E14 in rats(43) . However, the largest
rise in the number of sensory cells occurs in the period shortly after
birth(44) . In the rat brain considerable neurogenesis occurs
over a similar time period. No expression of G
8 above background
was detected in the brain indicating that it is unlikely that G
8
is a molecule essential to neurogenesis in general. However, in the
olfactory epithelium the marked rise in the expression of G
8
shortly post-natum parallels the high rate of neurogenesis shortly
after birth. In adult rats, the number of mature neurons is higher but
the rate of neurogenesis lower than at P13.5; a corresponding decrease
in expression of G
8 was observed. Golf
expression appears to
lag behind that of G
8. Also, in contrast to G
8 expression,
that of Golf
corresponds with the number of mature neurons;
Golf
mRNA was also detectable in brain confirming previous
reports(40) .
Further evidence that G8 is predominantly
expressed in developing neurons comes from olfactory bulbectomy
studies. After olfactory bulbectomy (which results in the degeneration
of mature olfactory neurons (45) and the loss of mRNA species
specifically expressed in these cells of the olfactory epithelium),
there is only a small change in the level of G
8 expression in
comparison to that seen with other olfactory markers (Fig. 4).
This means that the majority of the expression of G
8 is not in
mature olfactory neurons. However, the in situ hybridization
studies are not consistent with expression of G
8 in several other
cell types that make up the epithelium: the perinuclear region and the
majority of the cytoplasm of the sustentacular, basal, and glandular
cells are devoid of G
8 mRNA (Fig. 7). Nevertheless, after
bulbectomy hybridization toward the base of the epithelium is still
observed (Fig. 8). Thus most G
8 mRNA is expressed early in
the maturation of olfactory neurons before the markers of functional
sensory neurons, e.g. OMP or Golf
are found.
In the
adult vomeronasal epithelium, the level of expression of G8
relative to actin is at least as high as it is in the olfactory
epithelium ( Fig. 5and Fig. 6). The preparation of the
vomeronasal epithelium used for isolation of RNA was relatively crude,
and it is likely that the actual purity of vomeronasal neuroepithelium
used for RNA was substantially lower than that of the olfactory
epithelium. This means that the level of G
8 expression in the
vomeronasal epithelium is as much as 2-5-fold that found in the
adult olfactory epithelium. A similar difference in intensity of signal
was also noted in in situ hybridization studies (Fig. 8). At low magnification it is clear that the highest
level of G
8 expression is localized to the regions at the
boundaries between sensory and non-sensory epithelia. These areas of
maximum G
8 expression are the primary regions of neurogenesis in
rodent vomeronasal epithelia(28, 29) . The rest of the
epithelium is a pseudostratified epithelium similar in organization to,
but much thicker than the olfactory epithelium. Here the distribution
of G
8 mRNA resembled that of the olfactory epithelium (Fig. 8, A and D). Therefore, both in the
olfactory and in the vomeronasal epithelia, G
8 appears to play a
role in signal transduction during neural development.
The level of
expression of G8 in adult rat olfactory epithelium is considerably
lower (at least 10-fold) than that of Golf
( Fig. 5and 6).
Taken together with the localization of Golf
to fully developed
neurons that are lost after olfactory bulbectomy (Fig. 4), and
the absence of Golf
from the vomeronasal epithelium (Fig. 6), it does not appear that the majority of G
8 and
Golf
are associated in a specific heterotrimeric G-protein. We
cannot rule out that some G
8 does occur in such a protein, but it
is much more likely that this G-protein subunit is associated with one
or more of the other G-protein
-subunits that are expressed in the
olfactory epithelium(46) . Thus it is unlikely that G
8 is
a factor that directly mediates coupling of olfactory receptors to cAMP
production in olfactory cells. It will be important to determine
whether G
3 is the principal G-protein
-subunit associated
with Golf
, or whether other
-subunits that were not amplified
by PCR participate in olfactory signal transduction. It is also of
considerable interest that we did not detect expression of Golf
in
the vomeronasal epithelium ( Fig. 5and Fig. 6), whereas
its expression in whole brain was clear. We are currently investigating
whether other olfactory components involved in the cAMP signaling
pathway are present in the vomeronasal epithelium.
Two proteins have
been reported which appear to have a relatively similar distribution in
the olfactory epithelium to G8. The first is a tyrosine
phosphatase (NE-3) that also is found in the adult brain, and to a
lower level, in several other tissues(47) . Like G-proteins,
protein tyrosine phosphatases have been implicated in Drosophila development: both corkscrew a protein tyrosine
phosphatase(48) , and concertina a G-protein subunit (16) are maternal genes required for normal embryonic
development. Similarly, both G-proteins and protein tyrosine
phosphatases are involved in Dictyostelium development(15, 49) . The other protein with a
distribution in the olfactory epithelium similar to G
8 is an
olfactory-specific transcription factor Olf1, which appears to be
expressed both in developing and mature olfactory neurons(50) .
An alternatively spliced form of this protein appears to function as a
transcription factor in pre-B-cells(51) . The expression of
Olf1 is highest in rat olfactory epithelium at times shortly after
birth and declines in the adult (52) in a manner similar to
that seen for G
8. It is interesting to note that the full-length
clone of G
8 isolated from the cDNA library (but not the 5`-RACE
product) contains a strong consensus sequence for Olf1 binding. This
sequence binds Olf1 in vitro, (
)and may play a role
in directing the expression of G
8 to the olfactory tract.
In
summary, we have characterized the expression of a novel G-protein
-subunit that appears to be expressed early in the maturation of
olfactory and vomeronasal neurons. This protein, that is clearly
involved in signal transduction, is most similar to G
2 in
structure, but heterodimers of G
1 and these two different G
s
have quantitatively distinct functional properties. G
8 is
expressed in a fashion that suggests it plays a role in olfactory
neurogenesis. Neurogenesis involves considerable changes in gene
expression, extension of a dendrite to the surface of the epithelium,
and an axon to the olfactory bulb. Control of all these processes,
essential for development and maintenance of a functional olfactory
system, must require controlled response to many signals. Thus the
spatial and temporal pattern of expression of G
8 suggest that this
G-protein subunit is required for olfactory and vomeronasal neurons to
respond to signals that modulate their growth and/or development.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L35921[GenBank].