(Received for publication, September 15, 1995; and in revised form, November 8, 1995)
From the
We have used a [H]forskolin binding assay
to assess G
-adenylyl cyclase interactions in intact
wild-type (WT) and kin
S49 cells under conditions
that desensitize the
-adrenergic receptor
(
-AR) system. This assay provides a measurement of
G
-adenylyl cyclase interaction that does not rely on
the determination of second messenger accumulation or enzyme activity
in broken cells. Kin
S49 cells lack protein kinase A
(PKA) activity and provide a unique system in which to study the
relative importance of this enzyme in
-AR
desensitization. Although both WT and kin
S49 cells
display similar kinetics of cAMP accumulation and agonist-induced
cell-surface
-AR loss, we found that these cell types
exhibited very different extents of desensitization of forskolin
binding following agonist treatment. Specifically, 10 µM isoproterenol (37 °C, 30 min) induced the loss of 70% of
[
H]forskolin binding sites in WT cells but only
30% in kin
cells. This loss of sites in WT cells
displayed a t
of
7 min, was agonist
concentration-dependent (EC
60 nM), was not
mimicked by 8-Br-cAMP, and could be blocked by the PKA inhibitor, H89.
The difference between WT and kin
cells in
agonist-induced desensitization of the
-AR pathway was
also noted in studies of cAMP accumulation in cells. In addition,
preincubation of intact cells with isoproterenol did not inhibit
guanine nucleotide-dependent [
H]forskolin binding
in permeabilized cells. Overall, data obtained from
[
H]forskolin binding assays demonstrate the
involvement of PKA in the agonist-dependent uncoupling of
-AR and G
; thus we conclude that PKA plays
an important role in the homologous desensitization of the
-AR-G
-adenylyl cyclase pathway in intact
cells.
Desensitization of receptor-mediated cellular responses is a
complex process which can involve multiple pathways including: (a) receptor uncoupling from G protein, (b)
sequestration of receptors away from the plasma membrane, and (c) down-regulation of signaling
proteins(1, 2, 3, 4) . It has been
demonstrated that uncoupling of receptor and G protein occurs rapidly
(within 10 min) in response to receptor activation and appears to
involve receptor phosphorylation(5) . In the case of the
-adrenergic receptor (
-AR) (
)system, agonist-occupied receptors activate the
stimulatory G protein, G
, which in turn promotes the
production of cAMP by adenylyl cyclase. Increased cAMP levels activate
the cAMP-dependent protein kinase (PKA) resulting in the
phosphorylation of several proteins, including the
-AR
and other receptors. In addition, agonist-occupied
-AR
are phosphorylated by one or more isoforms of the
-adrenergic
receptor kinase (
-ARK, also termed G protein receptor kinase; GRK)
resulting in the uncoupling of receptor and
G
(6, 7, 8, 9, 10) .
Therefore, both PKA and
-ARK can contribute to desensitization of
the
-AR system.
In contrast to the effect of
-ARK, PKA-mediated
-AR phosphorylation is most
often associated with heterologous desensitization (that which is
agonist nonspecific and therefore not dependent on
-AR
occupancy), but is generally thought to be of little importance in
homologous desensitization (agonist-specific and dependent on
-AR
occupancy)(11, 12, 13, 14) .
Therefore, while the
-AR can be phosphorylated by both
PKA and
-ARK, most recent work has focused on the importance of
-ARK and related proteins (reviewed in (6) and (15) -17) as mediators of homologous desensitization.
The kin S49 murine lymphoma cell lacks PKA
activity and thus provides a unique system in which to study the
relative importance of PKA in mediating various cellular responses.
Importantly, both WT and kin
S49 cells express only
the
-AR isoform and display similar profiles of
-AR agonist-induced desensitization of cAMP production and
cell-surface
-AR loss(14, 18) . The
ability of the cell-permeable cAMP analog, 8-Br-cAMP, to mimic the
effect of epinephrine in decreasing
-AR agonist-induced adenylyl
cyclase activity in membranes prepared from treated cells suggest that
the effect of PKA activation is not dependent on the presence of
agonist(11) . These results suggest that PKA is not involved in
homologous desensitization of the
-AR.
In the
current studies, we have used a [H]forskolin
binding assay to define more precisely the role of PKA in desensitizing
the
-AR system in intact S49 cells. Since
high-affinity forskolin interaction with adenylyl cyclase requires the
activation of G
, the amount of
[
H]forskolin associated with cells can be used as
an index of G
-adenylyl cyclase
interaction(19, 20, 21) . This assay,
therefore, both provides a direct means of assessing hormone-stimulated
G
-adenylyl cyclase interaction in intact cells that is
independent of second messenger accumulation and maintains components
of the intracellular milieu important for regulating adenylyl cyclase
activity and that may be altered in membrane preparations. Using
forskolin binding assays, we compared G
-adenylyl
cyclase interactions in WT and kin
S49 cells under
conditions that desensitize the
-AR system. We
reasoned that a decrease in the
-AR-stimulated
[
H]forskolin binding would reflect the functional
desensitization of the
-AR pathway and that a
difference in the extent of desensitization between WT and
kin
cells would demonstrate the importance of PKA in
this process. Using this [
H]forskolin binding
assay, we found that, in contrast to previous
studies(11, 12, 13, 14) , PKA plays
an important role in homologous desensitization of the
-AR.
In response to isoproterenol, WT and kin
S49 cells display a similar amount of
[
H]forskolin binding (19, and data not shown).
This increase is dependent upon
-AR-mediated activation of the
stimulatory G protein, G
(19, 25) .
However, following the preincubation of cells with isoproterenol (10
µM) for various times (5-60 min), WT and
kin
cells differ in the extent to which the agonist
promotes an increase in forskolin binding sites (Fig. 1).
Specifically, whereas only 28 ± 3% of control
isoproterenol-stimulated [
H]forskolin binding
remained following a 60-min preincubation of WT S49 cells with agonist,
kin
S49 cells retained 64 ± 7% of these sites (p < 0.05 versus WT). In each case, the extent of
desensitization of forskolin binding reached maximum by about 30 min of
incubation with agonist and displayed a t
7
min. When WT cells were treated with the PKA inhibitor H89 (3
µM) the extent of isoproterenol-induced desensitization of
[
H]forskolin binding was similar to that observed
for kin
cells (Table 1). Importantly, this
concentration of H89 had no effect either on the activity of
recombinant GRK
(
-ARK
, data not shown) or
on the extent of desensitization observed in kin
cells (data not shown).
Figure 1:
Desensitization of
isoproterenol-stimulated forskolin binding in WT and kin S49 cells. WT and kin
S49 cells were
preincubated with 10 µM isoproterenol for the indicated
times. Following pretreatment, 40 nM
[
H]forskolin was added, the incubation was
continued for an additional 10 min, and radioactivity associated with
cells was determined as described under ``Experimental
Procedures.'' Radioactivity associated with cells in the absence
of isoproterenol (i.e. non-stimulated binding; WT =
1679 ± 116, kin
= 1627 ± 197
sites per cell) was subtracted and the data normalized to the amount of
isoproterenol-stimulated [
H]forskolin bound to
cells preincubated in buffer alone (WT = 1241 ±
143; kin
= 1599 ± 229 sites
per cell). The average ± S.E. of three separate experiments
performed in triplicate is shown. The curve represents a
mathematical fit of the data to a one-phase exponential decay
equation.
Since PKA activation is dependent on
cAMP production, we compared the concentration dependence for
isoproterenol-induced desensitization of forskolin binding with that
previously described for isoproterenol-stimulated cAMP
production(25, 26) . Importantly, the ability of
isoproterenol to induce desensitization of
[H]forskolin binding (EC
=
56.5 ± 2.1 nM) parallels its ability to stimulate cAMP
production (cf. Fig. 2and (25) and (26) ). Together with data presented in Fig. 1, these
results indicate that the different extents of isoproterenol-induced
desensitization of [
H]forskolin binding observed
in WT and kin
cells results from cAMP-dependent
activation of PKA. Furthermore, PKA activity is responsible for about
50% ((% desensitization in WT - % desensitization in
kin
)/% desensitization in WT
100%; Fig. 1) of the total desensitization of
[
H]forskolin binding observed in intact WT S49
cells; the desensitization occurring in kin
cells is
presumably due to the activity of
-ARK and related proteins (5) .
Figure 2:
Desensitization of forskolin binding in WT
S49 cells depends on agonist concentration. WT S49 cells were
preincubated for 60 min at 25 °C with the indicated concentration
of isoproterenol. Following pretreatment, 40 nM
[H]forskolin was added together with 10
µM isoproterenol and the incubation was continued for 10
min and processed as described under ``Experimental
Procedures.'' Points were determined in triplicate and
radioactivity associated with cells in the presence of 10
µM forskolin (nonspecific; 232 ± 102 cpm, 928
± 394 sites per cell) subtracted. The curve represents
a mathematical fit of the data to a sigmoidal curve. Similar results
were obtained in two separate experiments.
To determine if results from isoproterenol-induced
desensitization of [H]forskolin binding assays
correlate with an effect on second messenger production, we compared
cAMP accumulation in WT and kin
cells preincubated
with either isoproterenol or forskolin (Fig. 3). Following 30
min of treatment with isoproterenol (1 µM), WT cells
displayed a much greater decrease in cAMP production following the
addition of forskolin (1 µM) compared to kin
cells (40 versus 70% of control cAMP production
remaining for WT and kin
, respectively; p = 0.03). Treatment of cells with forskolin (1
µM) did not inhibit cAMP production following the addition
of isoproterenol (1 µM), indicating that the
desensitization of cAMP accumulation depends on the presence of
isoproterenol in the preincubation. These results are consistent with
results obtained from [
H]forskolin binding assays
in that WT cells display a greater extent of agonist-dependent
desensitization relative to kin
cells. Thus, we
conclude that
-AR-induced desensitization of both
forskolin binding and cAMP production is dependent on the activity of
PKA.
Figure 3:
Effect of isoproterenol and forskolin
treatment on subsequent cAMP accumulation in WT and
kin S49 cells. WT and kin
S49 cells
were preincubated in the presence or absence of isoproterenol (iso) (1 µM) or forskolin (Fsk) (1
µM) for 30 min at 37 °C. Subsequently,
isobutylmethylxanthine (200 µM final concentration) and
either isoproterenol (1 µM) or forskolin (1
µM) were added to cells preincubated with isoproterenol or
forskolin, respectively. Incubations continued for an additional 7 min
at 37 °C and reactions were terminated by the addition of
trichloroacetic acid (7.5% w/v final concentration). The amount of cAMP
present in samples was quantitated by radioimmunoassay as described
under ``Experimental Procedures.'' Control (100%)
represents cAMP produced following the addition of
isobutylmethylxanthine and both isoproterenol and forskolin to cells
preincubated in buffer (dotted line, absolute amount of cAMP
varied between experiments but typically ranged between 200-400
pmol per 10
WT cells and 400-600 pmol per 10
kin
cells). Shown are the mean ± S.E. of
three individual experiments performed in
triplicate.
While the above data demonstrate the importance of PKA in
-AR-induced desensitization of
G
-adenylyl cyclase interaction, they do not indicate
the site(s) or mechanism (heterologous versus homologous) by
which this effect occurs. The
-AR contains two
consensus PKA phosphorylation sequences and thus could represent one
site for PKA-mediated desensitization(27) . In addition, it has
been suggested that certain isoforms of adenylyl cyclase are subject to
direct regulation by PKA(28) . This is particularly important
in that one such isoform, Type VI, is expressed in S49
cells(29) . To test whether PKA-mediated desensitization of the
-AR pathway in WT S49 cells occurs via homologous or
heterologous pathways, we tested (a) the ability of guanine
nucleotide to stimulate [
H]forskolin binding in
permeabilized cells following isoproterenol pretreatment, and (b) the ability of agents that increase cAMP, and hence
activate PKA, to mimic the effect of isoproterenol preincubation on
[
H]forskolin binding in intact WT cells.
To
determine if the effect of isoproterenol preincubation on subsequent
[H]forskolin binding results from alterations in
the ability of G
to bind guanine nucleotides or
interact with adenylyl cyclase, we examined GTP
S-stimulated
[
H]forskolin binding in saponin-permeabilized WT
S49 cells. As shown in Fig. 4, isoproterenol (10
µM) did not increase [
H]forskolin
binding in permeabilized cells. However,
[
H]forskolin binding in permeabilized cells was
increased by the addition of GTP
S (30 µM) either
alone or with isoproterenol. While isoproterenol did not affect the
total amount of [
H]forskolin bound in the
presence of GTP
S, the agonist decreased the time required to
achieve the full extent of forskolin binding (data not shown). The
amount of guanine nucleotide-stimulated
[
H]forskolin binding in permeabilized cells was
similar to that observed in intact cells incubated in the presence of
isoproterenol. Incubation of cells with isoproterenol (10
µM, 60 min) prior to permeabilization did not affect the
amount of [
H]forskolin bound in the presence of
GTP
S following permeabilization. Since isoproterenol affected the
rate of [
H]forskolin binding to permeabilized
cells, the concentration of isoproterenol (10 µM) used in
the preincubation of intact cells was maintained throughout
permeabilization and subsequent incubation with
[
H]forskolin in order to avoid dissociation of
the
-AR agonist from receptor. These results indicate that the
ability of G
to bind guanine nucleotide and to
interact with adenylyl cyclase is not impaired by isoproterenol
preincubation. Therefore, PKA-mediated desensitization of
[
H]forskolin binding observed in WT cells appears
not to result from PKA-mediated effect on G
or
adenylyl cyclase but rather from a functional uncoupling of the
-AR and G
.
Figure 4:
Isoproterenol treatment of intact cells
does not affect guanine nucleotide-dependent forskolin binding
following cell permeabilization. Intact WT S49 cells were incubated
with or without 10 µM isoproterenol and 40 nM
[H]forskolin for 10 min as described under
``Experimental Procedures'' (left bars).
Saponin-permeabilized cells were incubated with
[
H]forskolin (40 nM) and either 10
µM isoproterenol, 30 µM GTP
S, or both as
described under ``Experimental Procedures'' (middle
bars). To examine the effect of isoproterenol-induced
desensitization on guanine nucleotide-dependent
[
H]forskolin binding in permeabilized cells,
intact WT cells were preincubated in DMEH with buffer or 10 µM isoproterenol for 60 min at 25 °C. The cells were then
permeabilized in reverse buffer containing 10 µM isoproterenol, washed, and incubated in reverse buffer containing
40 nM [
H]forskolin, 10 µM isoproterenol, and 30 µM GTP
S for 10 min (right bars). Radioactivity associated with cells in the
presence of 10 µM forskolin was subtracted from each
point. Data represent the mean ± S.E. of at least three separate
experiments.
Since PKA-mediated
phosphorylation of receptors has been associated with heterologous
desensitization(11, 12, 13, 14) , we
reasoned that increasing intracellular cAMP levels (thereby activating
PKA) with cell-permeable cAMP analogs and phosphodiesterase inhibitors
should mimic the effect of isoproterenol preincubation on
[H]forskolin binding. As shown in Table 1,
preincubation of WT S49 cells with 8-Br-cAMP (1 mM) and
phosphodiesterase inhibitors (isobutylmethylxanthine (100
µM) + RO201724 (100 µM)) for 60 min did
not alter the amount of [
H]forskolin bound
following the addition of isoproterenol. These results agree with those
obtained from cAMP assays showing that preincubation of WT cells with
forskolin does not induce desensitization of the
-AR
response (see Fig. 3). Heterologous desensitization of
-AR-stimulated [
H]forskolin
binding and cAMP production following treatment with agents which
increase intracellular cAMP levels was not observed under these
conditions. Heterologous desensitization in intact cells is often
manifest by an increase in the concentration of hormone required to
elicit a response without changes in the maximal effect of the
agonist(11) . For this reason, the conditions used in these
experiments examining maximal
-AR-stimulated G
-AC
interaction and cAMP production presumably would not detect
heterologous desensitization. Overall, we conclude that increasing cAMP
does not mimic the isoproterenol-induced desensitization of the
-AR system in WT S49 cells, suggesting that PKA is
involved in agonist-dependent (i.e. homologous)
-AR desensitization.
To examine further the role of
PKA in homologous receptor desensitization, we compared the ability of
prostaglandin E, another receptor agonist that increases
cAMP in S49 cells, with the ability of isoproterenol to stimulate
[
H]forskolin binding in cells pretreated with
either prostaglandin E
or isoproterenol. As shown in Fig. 5, isoproterenol-stimulated
[
H]forskolin binding was inhibited by about 70%
following pretreatment of cells with isoproterenol (10 µM,
60 min; *, p < 0.05 versus control). Similarly,
preincubation of cells with PGE
(30 µM, 60
min) inhibited subsequent PGE
-stimulated
[
H]forskolin binding by
75% (*, p < 0.05 versus control). In contrast, isoproterenol
stimulation of [
H]forskolin binding was only
slightly (
25%; p > 0.05) inhibited by pretreatment of
cells with PGE
. Thus, the desensitization of
[
H]forskolin binding following either
isoproterenol or PGE
pretreatment is specific for the
-AR and prostaglandin receptor, respectively. Together
with data presented above, our results demonstrate that in WT S49 cells
PKA serves an important role in homologous receptor desensitization.
Figure 5:
Homologous desensitization of PGE and isoproterenol-stimulated forskolin binding in WT S49 cells.
Intact WT S49 cells were preincubated in DMEH with or without PGE
(30 µM) or isoproterenol (iso) (10
µM) for 30 min at 25 °C (first incubation).
Subsequently, [
H]forskolin (40 nM) and
either buffer, PGE
or isoproterenol, were added and
incubations continued for 10 min (second incubation). Radioactivity
associated with cells in the presence of 10 µM forskolin (i.e. nonspecific binding; buffer preincubated = 2663
± 164, PGE
preincubated = 2800 ± 253,
isoproterenol; preincubated = 2792 ± 269 sites per cell)
was subtracted from each point. Control (100%) represents the amount of
specific [
H]forskolin bound following the
addition of agonist to cells preincubated in buffer (basal = 112
± 24, PGE
-stimulated = 855 ± 180, and
isoproterenol-stimulated = 1583 ± 209 sites per cell).
Shown is the mean ± S.E. of three separate experiments performed
in triplicate.
Desensitization of receptor-mediated cellular responses is a
complex process that can involve multiple pathways. For the
-AR, phosphorylation by both PKA and
-ARK can
uncouple receptor and G
protein(7, 10, 30, 31) . It has been
suggested that the ability of PKA to desensitize the
-AR seemingly does not depend on receptor occupancy by
agonist and therefore mediates heterologous
desensitization(7, 11, 13, 32) . In
contrast, phosphorylation of
-AR by
-ARK depends
on receptor occupancy and occurs only in the presence of
-AR
agonists(5, 7, 8, 9) . Thus, in
contrast to PKA,
-ARK-mediated phosphorylation is generally
thought to be critical for homologous desensitization of the
-AR.
We examined activation and subsequent
desensitization of the -AR-G
-adenylyl
cyclase system in intact cells using an approach different than
previous studies (i.e. adenylyl cyclase enzyme assays in
membranes). This is of particular physiological importance since
adenylyl cyclase activity is regulated by a complex network of
interacting pathways (for review, see (33, 34, 35, 36, 37) ). For
example, adenylyl cyclase isoforms present in S49 cells, Type VI and
Type VII(29, 38) , are regulated by
Ca
, PKA, PKC, G
, and G
(28, 38, 39 and references therein). Furthermore, the ability to
detect receptor desensitization using adenylyl cyclase assays in
membranes is influenced by Mg
concentration(31, 40, 41) . Thus,
multiple modulators of adenylyl cyclase activity can be affected during
the preparation and treatment of membranes.
Assessment of forskolin
binding provides a means to examine the role of PKA in
-AR-mediated desensitization that circumvents the
problems associated with determining adenylyl cyclase activity in
membranes. The interaction of forskolin with adenylyl cyclase is
enhanced by the activation of
G
(19, 37, 42, 43) .
Therefore, the amount of [
H]forskolin associated
with cells following agonist treatment can be used as an index of
G
-adenylyl cyclase
interaction(19, 20, 21) . Using this assay,
we show here that WT and kin
S49 cells display very
different extents of receptor-mediated desensitization. That is,
whereas about 70% of isoproterenol-stimulated
[
H]forskolin binding sites desensitize in WT
cells, only about 30% undergo desensitization in the absence of PKA (i.e. kin
cells or in the presence of a PKA
inhibitor). This desensitization is dependent on receptor occupancy, as
evidenced by the inability of PGE
and other agents that
raise cAMP to mimic the effect of isoproterenol. Moreover, treatment of
intact WT cells with isoproterenol did not effect the amount of guanine
nucleotide-dependent forskolin binding following permeabilization of
cells. In agreement with [
H]forskolin binding
results, WT cells exhibited a greater extent of agonist-dependent
desensitization of cAMP production than kin
cells.
Overall, these results argue that PKA is important for homologous (i.e. agonist-specific) desensitization of the
-AR pathway in WT S49 cells.
Several lines of
evidence in both WT and kin S49 cells suggest that
PKA-mediated phosphorylation of
-AR is important for
heterologous
-AR desensitization and that
-ARK
mediates homologous desensitization. For example, membranes prepared
from WT cells treated with agents that increase cAMP levels (and hence
the activity of PKA) display decreased epinephrine-stimulated adenylyl
cyclase activity relative to membranes from untreated
cells(11) . Likewise, treatment of WT S49 cell membranes with
PKA in vitro inhibited epinephrine-stimulated adenylyl cyclase
activity (32) . However, there was no additional effect of PKA
treatment on adenylyl cyclase activity in membranes prepared from WT
cells previously treated with
-AR agonist. These results suggest
that activation of PKA either by
-AR agonists or by heterologous
pathways has a similar effect on
-AR desensitization
and that this desensitization can occur independent of receptor
occupancy. In contrast, treatment of WT or kin
cells
with isoproterenol reduced to the same extent both receptor affinity
for agonist and isoproterenol-stimulated adenylyl cyclase activity in
membranes prepared from treated
cells(5, 14, 44) . These effects were
dependent on the presence of
-AR agonist. Since
-AR-stimulated adenylyl cyclase activity was inhibited
equally in both WT and kin
cells, it was concluded
that homologous desensitization was mediated by a receptor-specific
kinase and not by PKA. Taken together, these results support the
conclusion that PKA and
-ARK mediate heterologous and homologous
-AR desensitization respectively.
Previous results,
however, have not conclusively demonstrated an important role for PKA
in homologous -AR desensitization. For example, while
Shih and Malbon show that isoproterenol treatment of several cell lines
decreased the subsequent cAMP response elicited following agonist
rechallenge and that the extent of inhibition was substantially
reversed in cells treated with PKA antisense cDNA, the requirement for
-AR occupancy on this desensitization was not
investigated(45) . In other studies, Liggett et al. demonstrated that cells expressing the wild-type
-AR displayed a rapid decrease in the rate of cAMP
accumulation after 2 min of agonist stimulation, whereas the increased
rate of cAMP production induced by agonist was prolonged in cells
expressing
-AR mutant constructs lacking consensus
sites for PKA phosphorylation. Different studies with
-AR constructs have suggested that mutation of either
the putative PKA or
-ARK consensus phosphorylation sites can
reduce
-AR agonist-mediated desensitization of adenylyl cyclase
activity(47) . In contrast, other studies have shown that
deletion of putative PKA phosphorylation sites in the
-AR inhibited heterologous but not homologous receptor
desensitization (13) . These seemingly contradictory
observations can be reconciled by noting that these mutated
-AR receptors contain multiple mutations in different
domains of the receptor which could indirectly affect receptor function
and desensitization(48) . Moreover, the extent of receptor
expression can substantially alter
-AR
desensitization(49) . We believe our data obtained using
[
H]forskolin binding assays are the first to
demonstrate a role for PKA in homologous
-AR
desensitization in a native cell.
Overall, the data presented here
indicate that assessment of [H]forskolin binding
provides a useful measure to examine desensitization of
-AR-induced responses in S49 cells. Results from
forskolin binding studies indicate that
-AR-induced PKA activation
promotes an uncoupling of the
-AR receptor and
G
, thereby reducing the responsiveness of this system. The
inability of cAMP analogs and forskolin to mimic this effect and the
ability of guanine nucleotide to stimulate
[
H]forskolin binding in permeabilized cells
pretreated with isoproterenol further suggest that PKA is important for
homologous desensitization of the
-AR-G
-adenylyl
cyclase system. Taken together with previous results, our data are
consistent with a model whereby the concerted phosphorylation of
receptor by both PKA and one or more forms of GRK is important for the
homologous desensitization of the
-AR system observed
in intact cells.