(Received for publication, May 11, 1995; and in revised form, June 20, 1995)
From the
We have further investigated the molecular basis of increased
differentiation-regulated expression of SGTL1, a
Na/glucose cotransporter, in the renal epithelial cell
line LLC-PK
. Treatment of confluent monolayers either with
the differentiation inducer hexamethylene bisacetamide (HMBA) or with
cyclic AMP-elevating agents promoted increased levels of the SGLT1
mRNA, the immunodetectable 75-kDa cotransporter subunit, and the
transport activity. Two molecular species of SGLT1 mRNA (2.2 and 3.9
kilobases (kb)) are transcribed from the same gene in LLC-PK
cells and differ only in the length of the 3`-untranslated
region. The larger transcript is less stable (t = 2 h)
than the smaller one (t = 10 h) in control, confluent
monolayers. The 3.9-kb species was stabilized from degradation after
either cyclic AMP elevation (t = 14 h) or HMBA addition (t = 8 h), with negligible effects on the stability of
the 2.2-kb species (t = 11 h). Inhibition of
translation by cycloheximide resulted in a 10-fold increase in the t of the 3.9-kb transcript and a 2-fold increase in that of
the 2.2-kb species in control monolayers. Our results demonstrate that
post-transcriptional regulation of message stability plays a major role
in differentiation-dependent SGTL1 expression promoted by
either HMBA or cyclic AMP.
The SGTL1 gene family consists of a number of
structurally related transporters (Hediger et al., 1987;
Wright, 1993) which catalyze Na-coupled secondary
active transport. SGTL1, a Na
/glucose cotransporter,
is restricted in expression to intestinal and renal proximal tubule
apical membranes (Lever, 1992) where it plays an essential role in
concentrative transepithelial transport of glucose. The inherited human
disorder glucose/galactose malabsorption results from a single mutation
in SGTL1 (Turk et al., 1991), which maps on human
chromosome 22 (Hediger et al., 1989). Expression of this
transporter is developmentally regulated in the LLC-PK
polarized epithelial cell line of renal proximal tubule origin
(Mullin et al., 1980; Amsler and Cook, 1982). Whereas SGLT1
mRNA is undetectable in sparse, actively dividing cultures of
LLC-PK
cells (Yet et al., 1994) the development of
a confluent cell density is accompanied by tight junction formation,
membrane polarization, and expression of SGTL1 together with
other differentiated markers characteristic of renal proximal tubule
(Mullin et al., 1980; Yoneyama and Lever, 1984). Levels of
SGTL1 transport activity (Amsler and Cook, 1982, 1985; Peng and Lever,
1993), the 75-kDa transporter protein subunit (Wu and Lever, 1989; Peng
and Lever, 1993), and SGLT1 mRNA (Yet et al., 1994) are
significantly increased by independent and synergistic mechanisms after
elevation of cAMP levels or treatment with the differentiation inducer
hexamethylene bisacetamide (HMBA). (
)An LLC-PK
cell mutant deficient in protein kinase A activity was also
impaired in Na
/glucose cotransport activity (Amsler et al., 1991). Shioda et al.(1994) have recently
demonstrated that activation of protein kinase C results in the loss of
SGLT1 mRNA.
Alternative cleavage and polyadenylation results in the
accumulation of two SGTL1 transcripts in LLC-PK cells (Ohta et al., 1990), a 3.9-kilobase (kb) form and
a 2.2-kb form. In the present study, we demonstrate that the 3.9-kb SGTL1 transcript is relatively unstable in comparison with the
2.2-kb form. Induction of increased SGTL1 expression following
exposure to either cyclic AMP-elevating agents or HMBA is accompanied
by a pronounced stabilization of the 3.9-kb SGTL1 transcript
with negligible effects on the 2.2-kb form. These results indicate that
post-transcriptional mechanisms play an important role in regulating
levels of SGTL1 following cell differentiation.
Filters were prehybridized
for 3 h and then hybridized overnight at 55 °C in hybridization
solution containing 50% formamide, 5 SSPE (20
SSPE
= 3 M NaCl, 0.2 M NaH
PO
, 0.02 M EDTA, pH 7.4), 1
PE (1
PE = 50 mM Tris-HCl, pH 7.5, 0.1%
sodium pyrophosphate, 1% SDS, 0.2% polyvinylpyrrolidone, 0.2% Ficoll,
and 5 mM EDTA) and 150 µg/ml denatured salmon sperm DNA.
Labeled probes were added to the hybridization solution to give a final
concentration of 10
cpm/ml. Blots were given two 15-min
washes at room temperature in 0.1
SSC, 0.1% SDS, then washed
for 15 min at 55 °C and were given a final 30 min-wash at 65
°C. Filters were exposed at -80 °C to X-Omat AR films
with double intensifying screens. The hybridization signals on the
autoradiograms were quantitated by computer-assisted image processing
analysis (Bio Image® System, Millipore). For quantitative
experiments two different exposures were analyzed.
For determination
of mRNA half-life, poly(A) RNA was prepared from
monolayers at various times following addition of 5 µg/ml
actinomycin D, and SGLT1 mRNA levels were determined by quantitation of
Northern blots. Half-life was estimated by the equation: t = 0.693/k, where k = 2.303
slope of the plot of log
mRNA versus time).
Both
SGTL1 and SGLT2 cotransporter isoforms are expressed in LLC-PK cells (Kong et al., 1993; Mackenzie et al.,
1994). The Na
-dependent glucose transport properties
of this cell line, notably sensitivity to competitive inhibition by
galactose (Moran et al., 1988) and a 2:1
Na
:glucose stoichiometry (Moran et al., 1982)
are characteristic of properties expressed by the cloned SGTL1 isoform
(Ikeda et al., 1989), but not the SGLT2 isoform (Mackenzie et al., 1994), and suggest that SGTL1 is functionally the
dominant isoform expressed in these cultures.
Addition of HMBA to
newly confluent cultures at 3 days after seeding at a density of 1
10
cells/cm
induced an increase in
Na
-coupled glucose transport activity, measured by
Na
-coupled
-MGP uptake, as a function of exposure
time and concentration (Fig. 1, A and B). HMBA
effects on transport exhibited a 4-day lag period, followed by an
increase in transport activity for another 8 days (Fig. 1A). Addition of 1 mM IBMX to either
subconfluent cultures (0.6-0.8
10
cells/cm
) (Fig. 1C) or early
confluent cultures (10
cells/cm
) (Fig. 1D) for 12 days caused a 4-5-fold increase
in symport activity after a 4-day lag period. If IBMX was added to
post-confluent cultures (2.0-2.2
10
cells/cm
), the lag period was shortened to 2 days,
reaching a peak of 5-fold induction beginning at day 4 (Fig. 1E). This result revealed that cell density does
not appreciably change the magnitude of induction (4
6-fold), but
the lag period was shortened at higher cell density. By contrast, in
the case of HMBA, optimal induction was observed when early confluent
cells were used, and a reduced induction of symport activity was noted
the higher the cell density at the time of inducer addition (not
shown). Since the cell density at the time of inducer addition
significantly influences the magnitude of induction and exhibits a
different optimum for each inducer, in all subsequent experiments
unless otherwise stated, we added 5 mM HMBA to newly confluent
cultures at a density of 10
cells/cm
(3 days
after initial seeding), and 1 mM IBMX to post-confluent
cultures at a density of 2
10
cells/cm
(4-5 days after initial seeding).
Figure 1:
Induction of
Na/glucose symport activity by HMBA and IBMX. A, time course of induction by HMBA; B, HMBA
concentration dependence. (
), untreated control; (
), HMBA (2
mM); (
), HMBA (5 mM). C-E,
effect of cell density on symporter induction by IBMX. IBMX was added
to medium at (C) 2 days (subconfluent), (D) 3 days
(early confluent), and (E) 4 days (late confluent) after
plating LLC-PK
cells at a density of 10
cells/cm
. (
), control; (
), IBMX-induced. F, synergistic interaction between HMBA and IBMX. Early
confluent cells were treated for 8 days with 1 mM IBMX (I) or HMBA (H) (2 mM or 5 mM as
indicated), separately or in combination, for comparison with untreated
control cultures. All values are means ± S.E. of triplicate
determinations averaged from two
experiments.
HMBA and IBMX have synergistic effects on induction of symporter expression. Addition of IBMX in the presence of either a suboptimal level of HMBA (2 mM), which by itself does not influence symport activity, or optimal levels of HMBA (5 mM), resulted in a significantly greater induction of transport activity than produced by either agent alone (Fig. 1F).
Western blot analysis using a
polyclonal antibody specific for the 75-kDa pig kidney
Na/glucose cotransporter subunit confirmed the
separate and synergistic effects of these inducers (Fig. 2).
Results shown in Fig. 2A indicated that addition of
either IBMX or HMBA to newly confluent cultures for 8 days induced a 7-
and 8-fold increase in levels of the 75-kDa symporter subunit,
respectively (lanes 2 and 3). The protein kinase A
inhibitor H-89 (50 µM) (Chijiwa et al., 1990)
inhibited spontaneous expression of the 75-kDa subunit in control cells (lane 4) and abolished IBMX-mediated induction (lane
6) but had only a moderate effect in the presence of HMBA
(
20%) (lane 5). Addition of cAMP-elevating agents, either
forskolin or IBMX, to post-confluent cultures for 4 days resulted in an
increased expression of the 75-kDa symporter subunit, by 2.5- and
7.2-fold, respectively (Fig. 2B, lanes 3 and 4). Combination of IBMX and forskolin did not produce an
additional effect compared to that of IBMX alone (Fig. 2B, lane 5). Treatment of cells with
IBMX (1 mM) in the presence of 2 mM HMBA (a
suboptimal dose) induced a higher expression of the transporter subunit (Fig. 2B, lane 6) than observed with either
inducer alone, showing a synergistic effect (compare with lanes 2 and 4).
Figure 2:
Levels of the immunodetectable 75-kDa
Na/glucose symporter subunit. A, effect of
inducers and inhibitors. Cells were plated at 10
cell/cm
, and treated for 8 days with the indicated
additions at 3 days after seeding (early confluent). Lane 1,
control; lane 2, 1 mM IBMX; lane 3, 5 mM HMBA; lane 4, H-89 (50 µM); lane 5,
HMBA plus H-89; lane 6, IBMX plus H89. B, synergy
between HMBA and IBMX on expression of the transporter protein. Cells
were plated at 10
cells/cm
and treated for 4
days with the indicated additions at 4 days after seeding
(late-confluent). Lane 1, control; lane 2, 2 mM HMBA; lane 3, forskolin (100 µM); lane
4, 1 mM IBMX; lane 5, IBMX plus forskolin; lane 6, IBMX plus 2 mM HMBA. Samples (50 µg/lane)
of cell extracts were resolved on 12% SDS-polyacrylamide gel
electrophoresis and subjected to Western blot analysis using a
polyclonal antibody to the pig renal Na
/glucose
cotransporter. Densitometric values are shown
below.
Our earlier report demonstrating increased steady-state SGLT1 mRNA levels following inducer treatment utilized roller bottle-grown cells for mRNA isolation (Yet et al., 1994). Since the rate of differentiation is strongly influenced by growth conditions, we have reinvestigated several parameters of SGLT1 mRNA induction, using cells grown on dishes, for optimal comparison with results from transport assay and Western blot analysis also obtained using cell monolayers on dishes. A 324-bp antisense RNA probe specific for the SGTL1 isoform (Yet et al., 1994), which did not cross-hybridize with SGLT2, was used. Blots were stripped and rehybridized with a probe for GAPDH mRNA, as a control for recovery and integrity of the samples.
The steady-state levels of SGLT1 mRNAs increased as a
function of exposure time to inducers (Fig. 3). HMBA stimulated
a maximal induction (3-fold) peaking at day 8, with a 4-day lag
period before increased message levels could be detected. Since GAPDH
mRNA levels increased within the first 4 days (Fig. 3A, lower panel), it is possible that an increase in SGLT1 mRNA
within this time period was masked by the normalization. In the case of
HMBA, the peak in mRNA levels preceded the peak in transport activity
by 4 days (compare Fig. 1A and 3A). In the
case of IBMX, 1 mM, an induction of SGLT1 mRNA was observed
after a 2-day lag and reached a peak (8-fold increase) at 4 days of
exposure (Fig. 3B), coincident with the peak in
transport activity of postconfluent cells (compare with Fig. 1E). Maximal induction was observed at 5 mM HMBA (Fig. 4A) and 1 mM IBMX (Fig. 4B), when measured at the peak induction periods
of 8 and 4 days, respectively. The dose-response pattern observed for
the increase in transport activity resembled that observed for the
increase in message in the case of HMBA (Fig. 1A) and
IBMX (not shown).
Figure 3:
Time
course of induction of SGLT1 mRNA. Cells were exposed to either (A) 5 mM HMBA or (B) 1 mM IBMX.
Poly(A) RNA (1.5 µg) samples were subjected to
Northern blot analysis using a
P-labeled SGTL1-specific antisense RNA probe (upper panels) and
reprobed with an antisense GAPDH RNA probe (lower panels).
mRNA levels were quantitated by optical densitometry. Values for SGLT1
mRNA are normalized using GAPDH as internal standard and expressed as
means ± S.E. of two independent experiments. Empty
symbols, untreated controls; solid symbols,
inducer-treated. (
,
), 2.2-kb SGTL1 message;
(
,
), 3.9-kb SGTL1 message; (
,
),
GAPDH mRNA.
Figure 4:
Induction of SGLT1 mRNA as a function of
inducer concentration. A, samples (3 µg) of
poly(A) RNA from LLC-PK
cells treated for
8 days with the indicated concentrations of HMBA, or B,
samples (1.5 µg) of poly(A
) RNA from LLC-PK
cells treated for 4 days with indicated concentrations of IBMX,
were analyzed by Northern blot using
P-labeled SGTL1-specific and GAPDH antisense RNA
probes.
To further examine whether the inducer-stimulated increase in steady-state SGLT1 mRNA levels is associated with elevation of cyclic AMP levels and activation of protein kinase A, cells were treated for 4 days with either the adenylyl cyclase activator forskolin, or the cyclic AMP analogs dibutyryl cyclic AMP or 8-bromo-cyclic AMP. All of these cyclic AMP-elevating agents resulted in increased steady-state SGLT1 mRNA levels (Fig. 5A). The protein kinase A inhibitor H-89 down-regulated SGLT1 mRNA in control cultures, and blocked the induction of SGLT1 mRNA by IBMX (Fig. 5B). These results strongly indicated that cyclic AMP elevation and resultant protein kinase A activation is required to induce SGLT1 mRNA levels.
Figure 5:
Regulation of SGLT1 mRNA levels by cAMP
and protein kinase A. Samples (1.5 µg) of poly(A)
RNA from untreated control cells or cells treated for 4 days with
various compounds as indicated were analyzed by Northern blot using a
P-labeled SGTL1 antisense RNA probe. A, lane 1, untreated control; lane 2, 1 mM IBMX; lane 3, 100 µM forskolin; lane
4, 100 µM dibutyryl cAMP (dbcAMP); lane
5, 250 µM 8-bromo-cAMP (8Br-cAMP). B, the protein kinase A inhibitor H-89 down-regulates SGLT1
mRNA levels in LLC-PK
cells. Lane 1, untreated
control; lane 2, 1 mM IBMX; lane 3, 1 mM IBMX plus 50 µM H-89; lane 4, 50 µM H-89.
Figure 6:
Stabilization of the 3.9-kb SGLT1 transcript after IBMX treatment. Cells were treated with 1 mM IBMX for 4 days before addition of 5 µg/ml actinomycin D (Act D). At the indicated time intervals after addition of
ActD, poly(A) RNA was isolated. SGLT1 mRNA levels were
analyzed by Northern blot analysis and quantitated by densitometry. A, autoradiogram; B, half-life was determined from
densitometric data. Values are means ± S.E. of two to three
experiments. Empty symbols, control; solid symbols,
IBMX-treated cells. (
,
), 3.9-kb SGLT1 mRNA; (
,
), 2.2-kb SGLT1 mRNA.
Figure 7:
HMBA differentially stabilizes the SGLT1 3.9-kb message. Cells were treated with 5 mM HMBA for 8 days before addition of 5 µg/ml actinomycin D (Act D) and SGLT1 mRNA half-life determination as described in
the legend to Fig. 6. A, autoradiogram from a typical
experiment; B, scanning densitometry. Values are means
± S.E. of two or three experiments. Empty symbols,
control; solid symbols, HMBA-treated. (,
), 3.9-kb
SGLT1 mRNA; (
,
), 2.2-kb mRNA.
Due to the low abundance of the SGTL1 message and the limited sensitivity of the nuclear run-on assay, a measurable transcriptional activity under both control and inducer-treated conditions was not detectable, although positive controls demonstrated an unchanged rate of GAPDH transcription after inducer treatment (data not shown). Thus we were unable to evaluate the possible contribution of transcriptional activation to changes in steady-state SGTL1 message levels.
Figure 8:
Effect of
cycloheximide on the stability of SGLT1 mRNA. Cultures were treated
with either A, HMBA, 5 mM, for 8 days or B,
IBMX, 1 mM for 4 days. Cycloheximide (CHX), 50 µg/ml, was
added to medium 2 h before addition of 5 µg/ml actinomycin D (Act D) and additions were present thereafter. Half-lives of
the SGTL1 transcripts were determined as described in the
legend to Fig. 6. Open symbols, control; solid
symbols, inducer-treated. Dashed lines, with CHX; solid lines, without CHX. (,
), 3.9-kb SGLT1 mRNA;
(
,
), 2.2-kb SGLT1 mRNA.
In the case of cultures pretreated with inducers for the optimal time period before addition of these inhibitors, a different response was observed. Inhibition of protein synthesis did not prevent the stabilization of the 3.9-kb SGTL1 transcript observed in either IBMX- or HMBA-treated cells, suggesting that preexisting proteins may mediate the stabilization. In fact, an additional 2-fold stabilization was observed after inhibition of protein synthesis, perhaps due to inhibition of the synthesis of destabilizing factors.
The considerable variation in stability of eukaryotic mRNA is an important aspect of the regulation of the expression of genetic information. Linkage of mRNA turnover rates to activation of signal transduction pathways provides a means to rapidly fine-tune rates of protein expression in response to changes in the cell environment.
By use of two alternate polyadenylation sites, transcription of the
Na/glucose cotransporter gene SGTL1 in the
renal kidney cell line LLC-PK
gives rise to two mRNA
transcripts of 3.9 and 2.2 kb that differ in the length of their
3`-untranslated regions (Ohta et al., 1990). In the present
study, we provide evidence that the stability of SGLT1 mRNA is
differentially regulated by cAMP and the differentiation inducer HMBA.
In untreated differentiated cells, the 2.2-kb transcript is quite
stable, with a measured half-life of 10-12 h. By comparison, the
3.9-kb transcript is 5-6-fold less stable with a half-life of
about 2 h. Both HMBA and IBMX significantly increase the half-life of
the larger transcript (3-fold for HMBA and 9-fold for IBMX) but have
only a slight effect on that of the smaller form. Our present results
provide the first demonstration that these agents act, at least in
part, at the post-transcriptional level to stabilize the larger SGTL1 transcript.
It is interesting to note that the magnitude of the cAMP-stimulated increase in the 3.9-kb mRNA half-life is comparable with the observed increase in the steady-state level of the 3.9-kb transcript and also the observed increase in the steady-state level of the 75-kDa transporter subunit (Table 2). There is increasing evidence that the presence of the 3`-untranslated region controls the efficiency of translation (reviewed in Jackson and Standart (1990)). Our observation that the increase in the 3.9-kb transcript alone (not the sum of the 2.2- plus 3.9-kb transcripts) correlates with the increase in the protein (Table 2) suggests the possibility that the 3.9-kb SGTL1 transcript may be preferentially translated. In the case of HMBA, the increase in the transporter subunit is about 2-fold higher than predicted from the levels of the 3.9-kb message, suggesting the possibility that translational or post-translational effects may also be involved in HMBA action in addition to effects on message stability. Although HMBA and cyclic AMP exert synergistic effects on the transport activity and the levels of the transporter subunit ( Fig. 1and Fig. 2), they clearly act on different targets in the cell. HMBA effects on transporter expression appear, at least in part, to involve effects on polyamines and protein kinase C (Peng and Lever, 1993) with only minor involvement of protein kinase A (Fig. 2A); the mechanism of action of HMBA remains poorly understood.
Our
results, taken together with recent findings by Shioda et
al.(1994) that protein kinase C activation destabilizes the SGTL1 message, suggest that two separate and opposing
signaling pathways intersect to regulate post-transcriptional
processing and turnover of the SGTL1 message (Fig. 9).
Thus, activation of protein kinase A in confluent cultures would
up-regulate SGTL1 expression by message stabilization whereas
activation of protein kinase C would down-regulate expression by
message destabilization. Protein kinase A activation may also explain
in part the message stabilization by HMBA since cyclic AMP levels in
LLC-PK cells are also elevated after HMBA treatment (Lever,
1992). As discussed above, HMBA effects cannot be explained solely by
changes in cyclic AMP levels.
Figure 9:
Scheme summarizing the
post-transcriptional regulation of SGLT1 mRNA in LLC-PK cells. PKC and PKA, protein kinases C and
A.
Both stabilizing and destabilizing
effects of cAMP elevation on mRNA turnover have been reported in
various systems (reviewed in Williams et al.(1993)). In
examples of cell differentiation where the precise signaling pathways
are not well understood, changes in mRNA stability accompany
developmental transitions. For example, erythroleukemic differentiation
is associated with stabilization of globin message and destabilization
of non-globin messages (Volloch and Housman, 1981). Activation of
protein kinase C has been associated with regulation of mRNA turnover
in a number of different systems, including the stabilization of
cytokine and lymphokine mRNA's accompanying cell differentiation
(reviewed in Williams et al.(1993)). Phorbol
12-O-tetradecanoate 13-acetate activation of protein kinase C
has been previously implicated in the down-regulation of
Na/glucose cotransport activity in LLC-PK
cells (Amsler and Cook, 1982) and induced a rapid loss of SGLT1
mRNA due to message destabilization (Shioda et al., 1994). In
LLC-PK
cells, protein kinase C, assayed either with histone
(Dawson and Cook, 1987) or a synthetic peptide (Shioda et al.,
1994) as substrate, is mostly in the particulate activated form in
subconfluent, undifferentiated cells and in the inactive, cytosolic
form in confluent, differentiated cultures. Our observations taken
together with those of Shioda et al. (1994) suggest that,
under various conditions which induce message destabilization and
de-differentiation, the SGTL1 message has a half-life of
1.5-2 h, whereas under conditions which induce message
stabilization (differentiation), the SGTL1 message has a
half-life of
12 h. The differentiated cultures used by Shioda et al.(1994), 6
10
cells/cm
,
maintained at confluence for 10 days, are probably at a stage of
differentiation comparable to our HMBA- or IBMX-treated cultures.
The possible effect of protein translation on the stability of SGLT1
mRNA was examined in the present study using the protein synthesis
inhibitor cycloheximide. Cycloheximide treatment stabilized both
transcripts in uninduced cells, resulting in a >10-fold increase in
the half-life of the larger transcript and 1.5-2-fold
increase in the 2.2-kb species. In both HMBA- and IBMX-treated cells,
cycloheximide did not prevent the inducer-augmented stabilization of
the 3.9-kb transcript, indicating that preexisting protein factor(s)
may be involved. However, cycloheximide treatment caused an
approximately 2-fold increase in half-lives of both transcripts in
either HMBA- or IBMX-treated cells compared with inducer-treated cells
in the absence of cycloheximide. Shioda et al.(1994) observed
that inhibition of transcription abrogated the protein kinase C-induced
SGTL1 destabilization, suggesting a requirement for the regulated
expression of phorbol 12-O-tetradecanoate 13-acetate-induced
genes that inhibit SGTL1 gene transcription and/or enhance
SGLT1 mRNA degradation. Since the protein kinase C-mediated SGTL1 message destabilization observed by Shioda et al.(1994)
involved effects on both the 2.2- and 3.9-kb transcripts, whereas our
observed protein kinase A-mediated SGTL1 message stabilization
involved only the 3.9-kb transcript, these kinases clearly influence SGTL1 message stability by different mechanisms and stability
determinants.
Our results suggest that certain labile protein factor(s) may be required for degradation of both the 2.2- and 3.9-kb transcripts in control, differentiated cultures, and the process of decay is translation-associated. The labile factor(s) may be a free cytosolic endonuclase or a destabilizing mRNA binding protein which either directly binds to target mRNA to degrade the mRNA molecule or tags the mRNA molecule for nucleolytic attack. It has also been suggested that initial cleavages of some mRNAs may be triggered by a ribosome-bound nuclease, which would become active when the ribosome reaches a certain site in the coding region (Graves et al., 1987; Yen et al., 1988). Thus the requirement for translation could be explained by either the need for continuing protein synthesis to provide a labile factor, or to unmask a target site for binding of a destabilizing factor or ultimately to activate a ribosome-bound nuclease by unfolding of the mRNA during ribosome movement. Our results suggest that the sensitivity of the SGTL1 message to degradation can be modulated by a cAMP-mediated mechanism, most likely by protein kinase A-mediated protein phosphorylation. In a few instances, specific cis-acting sequences in either the 3`-untranslated region or other portions of the transcript have been shown responsible for hormonal or developmental changes in message stability (reviewed in Jackson(1993) and Williams et al.(1993)). The interaction of regulatory cytoplasmic proteins with these sequences and the modulation of this interaction by various kinase systems would provide a mechanism for linkage to cellular signaling mechanisms. Evidence for such a mechanism has been obtained in the case of GLUT1 mRNA, which encodes a facilitative glucose transporter with no amino acid sequence or functional similarity to SGTL1 (Stephens et al., 1992).
Since the 2.2- and 3.9-kb SGTL1 transcripts differ only in the length of the 3`-untranslated region, it is reasonable to propose that sequences in this region may regulate SGTL1 turnover. Degradation by specific endo- and exonucleases would be modified by altered interaction of cellular RNA binding proteins with these sequences in response to cAMP fluctuation.