(Received for publication, August 7, 1995; and in revised form, September 28, 1995)
From the
The phenomenon of acute desensitization of G-protein-coupled
receptors has been associated with several events, including receptor
phosphorylation, loss of high affinity agonist binding,
receptor:G-protein uncoupling, and receptor internalization. However,
the biochemical events underlying these processes are not fully
understood, and their contributions to the loss of signaling remain
correlative. In addition, the nature of the kinases and the receptor
domains which are involved in modulation of activity have only begun to
be investigated. In order to directly measure the role of
G-protein-coupled receptor kinases (GRKs) in the desensitization of the
m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative
allele of GRK2 was used to inhibit receptor phosphorylation by
endogenous GRK activity in a human embryonic kidney cell line. The
dominant-negative GRK2 reduced agonist-dependent
phosphorylation of the m2 mAChR by
50% and prevented acute
desensitization of the receptor as measured by the ability of the m2
mAChR to attenuate adenylyl cyclase activity. In contrast, the
agonist-induced internalization of the m2 mAChR was unaffected by the
GRK2
construct. Further evidence linking receptor
phosphorylation to acute receptor desensitization was obtained when two
deletions of the third intracellular loop were made which created m2
mAChRs that did not become phosphorylated in an agonist-dependent
manner and did not desensitize. However, the mutant mAChRs retained the
ability to internalize. These data provide the first direct evidence
that GRK-mediated receptor phosphorylation is necessary for m2 mAChR
desensitization; the likely sites of in vivo phosphorylation
are in the central portion of the third intracellular loop (amino acids
282-323). These results also indicate that internalization of the
m2 receptor is not a key event in desensitization and is mediated by
mechanisms distinct from GRK phosphorylation of the receptor.
Agonist-dependent receptor phosphorylation has been demonstrated
in an increasing number of studies involving members of the
G-protein-coupled receptor (GPR) ()superfamily. These
phosphorylation events have been best studied with the visual GPR,
rhodopsin, and the
-adrenergic receptor
(
-AR), where rapid phosphorylation of these receptors
by a member of the G-protein-coupled receptor kinase (GRK) family
underlies one or more aspects of receptor
desensitization(1, 2) . In these models, GRK-mediated
receptor phosphorylation is believed to cause rapid desensitization by
allowing a member of the arrestin family of proteins to bind the
phosphorylated receptor(1, 2) . Arrestin binding
attenuates the ability of the receptors to modulate activity of
downstream effectors by preventing further receptor/G-protein coupling.
Upon agonist activation, many GPRs are also internalized, or
sequestered away from the cell surface into an altered cellular
environment where they are unable to bind hydrophilic ligands. The role
that internalization plays in modulation of receptor activity remains
largely undefined and may well differ for different types of GPRs. For
the -AR, increasing evidence points to receptor
internalization being independent of the loss of receptor signaling,
but essential for recycling of the desensitized
GPRs(3, 4, 5) . However, with other GPRs,
internalization may well play a pivotal role in the termination of GPR
signaling.
The muscarinic acetylcholine receptors (mAChR) have been
the subject of many studies regarding the basis of GPR
desensitization(6) . There are five subtypes of
mAChR(7, 8, 9, 10, 11, 12, 13, 14) which
couple to two distinct signaling pathways. The m1, m3, and m5 mAChR
activate phospholipase C, while the m2 and m4 subtypes attenuate
adenylyl cyclase activity and in some cell systems weakly activate
phospholipase
C(12, 13, 15, 16, 17, 18) .
The m2 subtype of mAChR has been the focus of several desensitization
studies designed to understand the molecular basis of desensitization.
In intact cells, agonists induce phosphorylation of receptors to a
stoichiometry of 3-5 mol of phosphate per mol of
receptor(19, 20, 21) . These ``in
vivo'' phosphorylated receptors are desensitized, as seen in
their decreased ability to activate G-proteins to either bind GTPS
or hydrolyze GTP(19) , or to elicit a negative inotropic
response(22) . The kinase(s) responsible for agonist-dependent
m2 mAChR phosphorylation in vivo remain unidentified. The m2
mAChR can be phosphorylated in vitro by members of the GRK
family in a manner similar to that observed in
vivo(23, 24, 25, 26) . At
present, six members (GRK1-GRK6) of the family are known.
Rhodopsin kinase (GRK1) was the first to be identified on the basis of
its ability to phosphorylate rhodopsin in a light-dependent
manner(27) , while
ARK1 (GRK2) was subsequently identified
as a kinase that phosphorylated the
-AR in an
agonist-dependent manner(28) . Some members of this family
recognize the m2 mAChR in vitro in a manner that is
exquisitely dependent on agonist occupancy. GRK2 and GRK3 (
ARK2)
are able to phosphorylate the activated m2 mAChR to a high
stoichiometry(23, 24, 25, 26) .
Other members of the GRK family, GRK5 and GRK6, also phosphorylate the
m2 mAChR in an agonist-dependent manner in vitro, but at a low
rate and extent(25, 29, 30) . However, the
role(s) of these kinases in regulating receptors in intact cells
remains largely unexplored, in part because no selective inhibitors of
these kinases are available for in vivo work. In this study,
we report studies using a dominant-negative allele of GRK2
(GRK2
) and receptor mutants that provide new insight
into events underlying receptor desensitization in intact cells.
Figure 1:
Effects of
GRK2 on the agonist induced phosphorylation of m2 mAChR
in tsA201 cells. tsA201 cells were transiently transfected with cDNA
encoding the m2 mAChR alone or cotransfected with cDNA encoding
GRK2
. A is a representative immunoblot showing
levels of endogenous GRK2 (lane 1) and overexpressed
GRK2
(lane 2) detected with an anti-GRK2
antibody. B and C, 60 h post-transfection, cells were
labeled with
P
for 4 h and then incubated in
the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 1 mM carbachol for 15 min. The
membrane-bound receptors were solubilized, purified by
immunoprecipitation, and processed by SDS-PAGE (8% gel) followed by
immunoblot and PhosphorImager analysis. A representative immunoblot (Panel B) and phosphorimage (Panel C) are shown. D, the data from the PhosphorImager analysis were normalized
to the amount of protein loaded per lane as assessed from a
densitometric analysis of the immunoblot for each experiment. Data from
four to seven independent experiments were quantified. Open
bars, control cells; shaded bars, cells cotransfected
with GRK2
. Basal levels of receptor phosphorylation
were normalized to 0. Carbachol-induced phosphorylation levels were
normalized to 100%; this corresponded to a stoichiometry of 3-5
mol of phosphate per mol of receptor. The amount of phosphorylation
measured in carbachol-treated cells coexpressing GRK2
(2.0 ± 0.2-fold over basal levels, n = 4)
was significantly different (*p < 0.05) from the
phosphorylation measured in carbachol treated control cells (2.9
± 0.3-fold over basal levels, n =
7).
Figure 2:
Effects
of GRK2 on m2 mAChR coupling to adenylyl cyclase and
desensitization. Cells transiently transfected with cDNAs encoding the
m2 mAChR and LHR without (A) or with GRK2
(B) were assessed for cAMP levels following various drug
treatments. Basal cAMP levels were normalized to 0%; LH-induced levels
were defined as 100%. Cells were pretreated for 20 min with medium
alone (open bars) or for 20 min with 1 mM carbachol (shaded bars) to measure m2 mAChR signaling through adenylyl
cyclase and the desensitization of this response. Data shown are the
means ± S.E. for three independent experiments performed in
triplicate. (*Significantly different from the inhibition of adenylyl
cyclase activity measured in naive cells, p <
0.01.)
In cells that coexpressed
GRK2, m2 mAChR desensitization was dramatically
changed. Cells pretreated with medium alone showed a similar pattern of
m2 mAChR signaling as observed in control cells, in that carbachol
caused a significant reduction in the LH-induced increase in cAMP to 69
± 5% of LH levels. Note that a small but significant reduction
in the magnitude of m2 mAChR-mediated cAMP attenuation in cells
expressing GRK2
was observed, although the response was
still readily measured and significantly different than the levels of
cAMP observed with LH treatment alone. It is possible that
GRK2
prevents some G
-mediated attenuation of
cyclase activity in these cells, as expression of all or part of this
protein has been shown to prevent G
effects in other
systems(38, 44) . Nonetheless, in contrast to cells
transfected with m2 mAChR alone, cells coexpressing GRK2
that were pretreated with carbachol for 20 min did not
desensitize and retained their ability to attenuate the cAMP induction
caused by LH treatment (78 ± 1% of LH levels, Fig. 2B). This was not significantly different from the
response observed in naive cells. Taken together, the ability of
GRK2
to ablate the phosphorylation and desensitization
of the m2 mAChR data suggests that m2 mAChR phosphorylation by GRK2, or
another closely related kinase, is required for desensitization of the
m2 mAChR cyclase response. In addition, these data are the first direct
demonstration of a requirement for agonistdependent phosphorylation in
the acute desensitization of the m2 mAChR.
Figure 3:
Time
course of agonist induced m2 mAChR internalization. Cells were
transfected with cDNAs encoding the m2 mAChR alone (open
bars), or cotransfected with GRK2 (shaded
bars) or with excess wild-type GRK2 (black bars). The
percentage of surface receptors present after various times of exposure
to agonist was measured in whole cell binding assays using the
hydrophilic ligand [
H]NMS, as described under
``Experimental Procedures.'' Results shown are the means
± S.E. of three or four independent experiments performed in
triplicate.
Figure 4:
Deletion mutagenesis and analysis of
phosphorylation of mutant mAChRs. A, diagram of the deleted
residues in m21 (underlined, bottom row) and
m2
2 (entire sequence shown). Serine and threonine residues are black; the KT3 tag is shaded gray. Stably or
transiently transfected cells expressing the various forms of m2 mAChRs
were labeled with
P
and then incubated with or
without carbachol for 15 min. Receptors were immunoprecipitated and
analyzed by immunoblot (B) using monoclonal 1D1 tissue culture
supernatant and PhosphorImager (C). Lanes 1 and 2, full-length m2 mAChR; lanes 3 and 4,
m2
1 mAChR. The arrows mark the position of the
full-length wild-type receptor (black arrow) and
m2
1-mAChR (gray arrow). The radioactive bands evident in lanes 3 and 4 are nonspecific proteins that
consistently coimmunoprecipitated with both the wild-type and mutant
receptors. The shape and mobility distinguishes them from either form
of the m2 mAChR. Data shown are representative of five independent
experiments.
Both deletion mutants bound the
antagonist [H]QNB and the agonist carbachol with
affinities similar to the full-length receptor, as analyzed by
Scatchard analysis and agonist-competition curves in whole cell binding
studies (data not shown). All three forms of the m2 mAChR were tagged
with a modified KT3 epitope. These tagged receptors also bound ligands
appropriately (data not shown). Stable HEK cell lines expressing these
receptors were prepared and used to measure agonist dependent
phosphorylation, desensitization, and internalization. Like the
transiently expressed m2 mAChR, the stably expressed full-length
receptor was phosphorylated in an agonist-dependent manner (Fig. 4, B and C). However, neither the
m2
1 deletion mutant (Fig. 4, B and C),
nor the m2
2 mutant (data not shown) became phosphorylated over
basal levels following exposure to carbachol. Because the smaller
deletion was as deficient in agonist-induced phosphorylation as the
larger deletion, we conclude that the likely sites for
agonist-dependent phosphorylation are located in the i3 loop within
residues 282-323.
Figure 5:
Analysis of m21 coupling to adenylyl
cyclase and desensitization. Stable cell lines expressing full-length
m2 mAChR (A) or m2
1 mAChR (B and C)
were pretreated with medium alone (open bars) or 1 mM carbachol (shaded bars) for 20 min (A and B) or 60 min (C), and the ability of the receptors to
attenuate
-AR-induced increases in adenylyl cyclase activity was
measured. Data shown are the means ± S.E. for three independent
experiments performed in triplicate (A and B) or one
representative experiment performed twice (C). (*Significantly
different from the inhibition of adenylyl cyclase activity measured in
naive cells, p < 0.01.)
We next tested the deletion mutants for their
ability to signal and desensitize. When cells expressing m21 (0.2
pmol/mg of protein) were challenged with carbachol and isoproterenol
for the cAMP assay, cAMP levels were 31 ± 8% of those seen with
isoproterenol alone (Fig. 5B). Thus, this deletion
mutant receptor was able to attenuate adenylyl cyclase to an extent
similar to wild-type receptors. However, the mutant receptor did not
desensitize; in m2
1 cells pretreated with carbachol for 20 min the
levels of cAMP were measured as 36 ± 6% (Fig. 5B) of isoproterenol levels. Thus, there was no
significant change in the ability of the m2
1 mAChR to signal
through adenylyl cyclase following carbachol pretreatment. Similarly,
the m2
2 mAChR showed no desensitization of its ability to
attenuate adenylyl cyclase following 20 min of carbachol pretreatment
(data not shown). When the carbachol pretreatment was extended to 1 h,
the m2
1 receptor showed only a small, statistically insignificant
reduction in its ability to attenuate adenylyl cyclase activity (Fig. 5C). Taken together with the results showing that
these receptors did not undergo agonist-induced receptor
phosphorylation, these data lend further credence to a direct role for
receptor phosphorylation in desensitization, and indicate that the
sites of GRK-mediated phosphorylation are most likely contained in the
i3 loop.
Figure 6:
Time course of agonist induced
internalization of m21 and m2
2 mAChR. Stable cell lines
expressing the wild-type (
-
), m2
1
(
-
), or m2
2 (
- - - - -
)
receptors were used to analyze receptor internalization. The percent of
surface receptor present after various times of exposure to agonist was
measured in whole cell binding assays using the hydrophilic ligand
[
H]NMS. Data shown are the means ± S.E. of
three to five independent experiments performed in
triplicate.
The data presented here lend new insights into several
unresolved questions regarding the molecular events involved in the
desensitization of the m2 mAChR. What kinase(s) mediate phosphorylation
of the receptors? Is agonist-induced receptor phosphorylation directly
responsible for receptor:effector uncoupling? What role does
internalization of the m2 mAChR play in acute loss of receptor
signaling? What domains of the receptor are involved in these
processes? The two approaches explored here, one using
GRK2, and the other utilizing the deletion mutants,
demonstrated that a reduction or loss of agonist-dependent
phosphorylation resulted in a loss of desensitization as well. It is
interesting that overexpression of GRK2
reduced levels
of receptor phosphorylation only by half but completely abolished
receptor desensitization. Several plausible scenarios may explain this
observation. First, the receptor may be phosphorylated at several
sites, of which only one or two may be critical for desensitization.
Another scenario might be that another kinase might be responsible for
the remainder of the phosphorylation, and these other phosphorylated
sites do not participate in the desensitization of adenylyl cyclase
coupling. Finally, half of the population of receptors may be fully
phosphorylated in cells coexpressing GRK2
, leaving the
other half unphosphorylated and available to signal and attenuate
adenylyl cyclase activity without becoming desensitized. Regardless of
the mechanism, it is clear from both the studies with GRK2
and the deletion mutants that phosphorylation mediated by a GRK
is necessary for m2 mAChR desensitization in vivo.
The
observation that GRK-mediated receptor phosphorylation is reduced by
coexpression of GRK2 is in agreement with several other
studies. This allele of GRK2 was shown to reduce agonist-dependent
phosphorylation of the
-AR in vitro and to
decrease
-AR desensitization in vivo, while
not affecting receptor internalization(38) . A slightly
different allele of GRK2, GRK2
, was shown to reduce
agonist-dependent m2 mAChR phosphorylation in COS7 and BHK
cells(48) . This study suggested that overexpression of
wild-type GRK2 could decrease the concentration of agonist required for
m2 mAChR internalization. However, overexpression of the
dominant-negative allele had only minimal effects on receptor
internalization, and the question of receptor:effector coupling was not
addressed(48) . More studies will be needed to fully understand
the role of receptor internalization in receptor regulation; perhaps
cell-specific mechanisms play a role in the differential regulation of
internalization.
The data presented here also suggest that m2 mAChR
desensitization is a distinct event from receptor internalization. It
has been observed that the rate of receptor desensitization is faster
than the rate of receptor
internalization(22, 47, 48, 49) .
This observation, along with data from the -AR system,
led us to postulate that receptor internalization is indeed independent
of receptor desensitization. The data presented here show that, under
conditions where receptor desensitization is blocked, receptor
internalization continues to occur. We conclude from this observation
that receptor internalization is not a key event in eliciting a loss of
signaling ability and, as discussed earlier, may occur by mechanisms
distinct from GRK-mediated, agonist-dependent phosphorylation of the m2
mAChR. While much more remains to be done to elucidate the events which
cause receptor internalization, it is possible that conformational
changes in the receptor, analogous to those which activate G-proteins,
allow the activated receptor to interact with the internalization
machinery. Another tenable proposal for mechanisms of internalization
might include second-messenger production for the activation of
endocytosis pathways(50, 51) .
Finally, the sites
of phosphorylation and domains responsible for desensitization of the
m2 mAChR remain to be defined. The data presented here narrow down the
search for phosphorylation sites to serine and threonine residues in
amino acids 282-323. However, it should be noted that such a
large deletion may have disturbed secondary structural elements
necessary for receptor phosphorylation, GRK activation, or arrestin
binding. Thus, it is possible that the phosphorylated residues lie
outside this deleted region. One concern is that receptors may activate
the GRKs (46) and that the lack of phosphorylation observed
here might be due to loss of a kinase activation domain rather than the
loss of actual phosphorylation sites. However, a larger deletion of the
i3 loop (amino acids 233-380) did not affect the ability of the
m2 mAChR to increase activity of GRK2 toward a peptide substrate in
vitro(46) , strongly suggesting that the m21 mutant
should also be able to activate GRKs. Deletion of residues
282-323 did not alter the ability of the m2 mAChR to attenuate
adenylyl cyclase activity but abolished desensitization of this
response. This suggests that site-directed mutagenesis of individual
residues in this region may well lead to fruitful discovery of the
sites of receptor phosphorylation and desensitization in vivo.
Data obtained from experiments using point mutants may indicate if the
m2 mAChR requires full phosphorylation for receptor uncoupling from
adenylyl cyclase or if, like other GPRs such as rhodopsin, preferential
phosphorylation of one or two specific residues is sufficient for
receptor desensitization(52) . Interestingly, a recent study of
the
-AR showed that elimination of one or more of the
four agonist-induced phosphorylation sites abolished desensitization,
while internalization was unaffected even when phosphorylation was
completely abolished by mutating all four phosphorylated residues to
alanine(53) .
It is likely that GRK activity is involved in
desensitization of a wide variety of GPRs, as many different receptors
have now been shown to be agonist-dependent substrates for purified
GRKs in vitro. These receptors include but are not limited to
the -AR(28) , the
-AR (54) , and the substance P receptor(55) . In intact
cells, coexpression of GRK2 augmented desensitization of the thrombin
receptor expressed in Xenopus oocytes(56) , and
antibodies against GRK3 (
ARK2) inhibited desensitization of
olfactory receptors in olfactory cilia (57, 58) .
Different regions are reported to be involved in mediation of
internalization in various GPRs. Studies of the angiotensin II receptor (59) , the -AR(60) , the parathyroid
hormone receptor(61) , and the
-adrenergic
receptor (62) indicate that the C termini of these proteins
are involved in internalization while the i3 loop of several subtypes
of mAChRs are the likely regions for internalization control of these
receptors(47, 63, 64) . Deletions of the C
terminus of the m1 mAChR had no effect on receptor internalization,
while mutations in the i3 loop reduced internalization(63) . In
addition, deletion of the i3 loop of the m4 mAChR slowed the rate of
internalization initially, but by 1 h internalization was equivalent to
that seen in wild-type receptor(64) . A slight reduction in m2
mAChR internalization was observed when several serine/threonine
residues within the region deleted in our studies were mutated to
alanine(47) . A partial loss of internalization suggests that
more than one domain of a GPR may be involved in internalization and/or
that more than one internalization pathway exists. Investigation into a
role of other domains of the m2 mAChR may yield more information as to
the structural basis for m2 mAChR internalization.
In summary, we have shown that agonist-dependent phosphorylation of the m2 mAChR in intact cells is at least partially mediated by a GRK and is absolutely required for desensitization of the G-protein-mediated attenuation of adenylyl cyclase activity, while GRK activity is not needed for receptor internalization. The sites of GRK-mediated phosphorylation of the m2 mAChR are likely found in amino acids 282-323; phosphorylation in this domain appears to be required for desensitization but not for internalization, although deletion of this domain reduces the rate and extent of receptor internalization. Future studies will determine the exact residues which are phosphorylated to give a more complete picture of the structural basis of m2 mAChR desensitization.