(Received for publication, September 7, 1995; and in revised form, November 6, 1995)
From the
Human muscarinic acetylcholine receptor m1 subtypes (m1
receptors) were expressed in and purified from insect Sf9 cells and
then subjected to phosphorylation by G protein-coupled receptor kinase
2 (GRK2) expressed in and purified from Sf9 cells and by protein kinase
C purified from rat brain (a mixture of ,
, and
types,
PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an
agonist-dependent or independent manner, respectively. G protein
subunits stimulated the phosphorylation by GRK2 but did not
affect the phosphorylation by PKC. The number of incorporated
phosphates was 4.6 and 2.8 mol/mol of receptor for phosphorylation by
GRK2 and PKC, respectively. The number of incorporated phosphates was
7.5 mol/mol receptor for phosphorylation by GRK2 followed by PKC, but
was 5.8 mol/mol of receptor for the phosphorylation by PKC followed by
GRK2. Major sites phosphorylated by GRK2 and PKC were located in the
third intracellular loop and the carboxyl-terminal tail, respectively.
These results indicate that GRK2 and PKC phosphorylate different sites
of m1 receptors and that the phosphorylation by PKC partially inhibits
the phosphorylation by GRK2, probably by affecting activation of GRK2
by agonist-bound receptors.
Muscarinic acetylcholine receptors consist of five subtypes. The
m1, m3, and m5 subtypes are linked to G family G proteins
and the m2 and m4 subtypes to G
/G
family G
proteins (for reviews, see (1, 2, 3) ).
Muscarinic receptors as well as other G protein-coupled receptors are
known to undergo desensitization following exposure to
agonists(4, 5, 6) . Desensitization of
receptors is generally believed to be mediated by receptor
phosphorylation. Homologous desensitization is thought to be linked to
agonist-dependent phosphorylation of receptors by G protein-coupled
receptor kinase (GRK) (
)and heterologous desensitization to
agonist-independent phosphorylation of receptors by second
messenger-activated protein kinases such as cAMP-dependent protein
kinase and protein kinase C (PKC).
GRK is a subfamily of serine and
threonine kinases and is characterized by phosphorylation of only the
stimulated forms of G protein-coupled receptors (for reviews, see (7, 8, 9) ). The GRK family includes
rhodopsin kinase (GRK1), -adrenergic receptor kinases 1 and 2
(GRK2, GRK3) and GRK4, GRK5, GRK6. GRK1 and GRK2/3 have been
characterized much more extensively than the other members. GRK1
phosphorylates rhodopsin in a light-dependent manner, and the light
dependence is at least partly due to the activation of GRK1 by
light-absorbed rhodopsin(10, 11) . GRK2/3 are
different from other GRKs in that GRK2/3 are activated by G protein
subunits and have longer carboxyl termini, which are the
sites that interact with
subunits(12, 13, 14, 15) . GRK2 is
synergistically activated by
subunits and mastoparan (16) and by
subunits and agonist-bound receptors (17) . Recently GRK2/3 have been reported to be activated by
phospholipids as well as
subunits, although there are some
discrepancies among authors(18, 19) . GRK2 was
originally isolated as a kinase that phosphorylates
-adrenergic
receptors in an agonist-dependent manner, but is now known to
phosphorylate different kinds of G protein-coupled receptors, including
muscarinic m2 (17, 20, 21, 22) and
m3(23) ,
2-adrenergic (
2A and
2B)(24) ,
and substance P (25) receptors. On the other hand,
1- (26) and
2C (24) -adrenergic receptors are
reported not to be phosphorylated by GRK2. It remains to be known how
the substrate specificity of GRK2 is determined.
Some contradictory results have been reported concerning the phosphorylation of m1 receptors. Muscarinic receptors purified from porcine brain, which contain m1 receptors as a major component, were phosphorylated in an agonist-dependent manner by a muscarinic receptor kinase that was purified from porcine brain and had properties similar to GRK2(27) . On the other hand, human m1 receptors expressed in and purified from insect Sf9 cells were not phosphorylated in an agonist-dependent manner by either the muscarinic receptor kinase or GRK2 under the same conditions where m2 receptors purified from SF9 cells were phosphorylated in an agonist-dependent manner by the muscarinic receptor kinase or GRK2(28) . Richardson et al.(29) also reported that human m2 receptors expressed in Sf9 cells were phosphorylated in an agonist-dependent manner by an endogenous kinase, but human m1 receptors were not phosphorylated under the same conditions.
The m1 receptor as well as the m2 and 2
receptors have a long third intracellular loop that contains sequences
similar to the putative phosphorylation sites in m2 (30) and
2 receptors(31) . In fact, a peptide corresponding to the
homologous sequence in m1 receptors can be phosphorylated by GRK2, and
the phosphorylation was markedly stimulated by G protein
subunits and mastoparan(6) . In addition, human m1 receptors
were found to be phosphorylated by GRK2 in an agonist-dependent manner
in the presence of
subunits and m2 or phosphorylation
site-deleted m2 receptors(6) . These results indicate that the
phosphorylation of m1 receptors by GRK2 is enhanced when GRK2 is
activated by
subunits and mastoparan or by
subunits and agonist-bound m2 receptors. This raises the question
whether m1 receptors are unable to activate GRK2 even in the presence
of agonist or the ability was impaired during the preparation of m1
receptors from Sf9 cells. We have extensively examined the purification
procedure of m1 receptors and found that m1 receptors can be
phosphorylated in an agonist-dependent manner by GRK2 after removing
unknown factor(s) that copurify with m1 receptors.
PKC are known to phosphorylate muscarinic receptors purified from porcine brain(32) , human m1 receptors expressed in and purified from Sf9 cells(33) , and muscarinic receptors purified from chick heart (34) . The present experiments were undertaken to determine whether GRK2 and PKC phosphorylation sites in human m1 receptors are independent or shared and whether the phosphorylation by one kinase affects the phosphorylation by another. We report here that different sites in m1 receptors are phosphorylated in an agonist-dependent and -independent manner by GRK2 and PKC, respectively, and that the phosphorylation by PKC reduces subsequent phosphorylation by GRK2.
A
standard assay tube for phosphorylation of m1 receptors by GRK2
contained the reconstituted vesicle (1 µl; 1.8-4.0 nM m1 receptors and 9-40 nM G in final
concentrations), 1 mM carbamylcholine or 10 µM atropine, 100 µM GTP, 1 or 10 µM [
-
P]ATP (2
10
cpm/tube, 1-10 cpm/fmol), and purified GRK2 (13
nM) in a medium of 20 mM Tris-HCl (pH 7.5), 5 mM MgCl
, 2 mM EDTA, 0.5 mM EGTA (total
volume, 40 µl). G
and GTP were added to supply G
protein
subunits and were substituted by
subunits
in some experiments. A standard assay tube for phosphorylation of m1
receptors by PKC contained the same components as the above except that
2 mM EDTA and 0.5 mM EGTA were replaced by 0.2 mM CaCl
and GRK2 was replaced by PKC (4.2 nM).
The phosphorylation reaction was carried out at 30 °C and was
terminated by addition of 20 µl of 5% sodium dodecyl sulfate (SDS)
solution containing medium for SDS-polyacrylamide gel electrophoresis
(SDS-PAGE), followed by autoradiography. The band of m1 receptors was
cut out and counted by the use of Cerenkov's effect.
Figure 1:
Effect of
polyethylene glycol (PEG) treatment on agonist-dependent
phosphorylation of m1 receptors by GRK2. Purified m1 receptors were
reconstituted with G protein G in a lipid mixture. The
reconstituted vesicles were treated with 50% PEG and centrifuged. The
pellet was recovered in the same volume as the original vesicle
suspension. The original reconstituted vesicle (the upper
figure) or the pellet after PEG treatment (the lower
figure) were subjected to phosphorylation by GRK2 in the presence
of 1 µM [
P]ATP (10 cpm/fmol), 0.1
mM GTP, and 1 mM carbamylcholine or 10 µM atropine at 30 °C for indicated time, followed by SDS-PAGE,
autoradiography, and counting of the m1 receptor band. The ordinate shows the amount of [
P]phosphate
incorporated into m1 receptors expressed as mol/mol. The amount of m1
receptors was estimated by the [
H]quinuclidinyl
benzylate binding activity and was 220 fmol/assay tube. This
preparation of m1 receptors contained a contaminant of approximately 25
kDa, which is phosphorylated by both GRK2 and PKC. The band is thought
to be a fragment of the m1 receptor, because it reacts with both
anti-i3 and anti-C tail (see Fig. 7). Detailed experimental
conditions are described under ``Experimental
Procedures.''
Figure 2:
Effect of various concentrations of
carbamylcholine and atropine on the phosphorylation of m1 receptors by
GRK2. Purified m1 receptors were reconstituted with G,
precipitated with PEG, and then subjected to phosphorylation by GRK2 as
described in the legend to Fig. 1, except that different
concentrations of carbamylcholine or different concentrations of
atropine with 1 mM carbamylcholine were used, and incubation
time was 60 min.
Figure 4:
Effect of G protein subunits on
the phosphorylation of m1 receptors by GRK2 and PKC. Purified m1
receptors were reconstituted in a lipid vesicle without G proteins,
precipitated with PEG, and then subjected to phosphorylation by GRK2 or
PKC in the presence or absence of 1 mM carbamylcholine and 66
nM G protein
subunits. Counts of
P in
the band of m1 receptors in SDS-PAGE were measured and those for m1
receptors phosphorylated in the presence of both carbamylcholine and
subunits were taken as 100%.
Figure 7: Digestion with a low concentration of trypsin of m1 receptors phosphorylated by GRK2 or PKC. PEG-treated m1 receptors were phosphorylated with GRK2 or PKC, then treated with 0.2 µg/ml trypsin at 30 °C for 5 min and transferred to PVM membranes. Subsequently membranes were incubated with anti-i3 or anti-C tail antibodies followed by immunostaining and autoradiography. Each lane contained 15 pmol of m1 receptors as original amount. A band of approximately 25 kDa is thought to be a fragment of the m1 receptor.
Figure 3:
Phosphorylation of m1 receptors by PKC.
Purified m1 receptors were reconstituted with G,
precipitated with PEG, and then subjected to phosphorylation by PKC in
the presence of 0.1 mM GTP and 1 mM carbamylcholine
or 10 µM atropine. Procedures for preparation of m1
receptors and phosphorylation assay were the same as described in the
legend to Fig. 1, except that 2 mM EDTA and 0.5 mM EGTA in the reaction medium were replaced by 0.2 mM CaCl
. In some experiments, phosphatidylserine and
diolein were also included in the incubation medium in addition to the
lipid mixture, but no significant difference was found in the level of
phosphorylation.
Figure 5:
Phosphorylation of m1 receptors by GRK2
and PKC. Purified m1 receptors were reconstituted with G,
precipitated with PEG, and then subjected to phosphorylation by GRK2
and then PKC (the upper figure) or by PKC and then GRK2 (the lower figure). The assay medium for GRK2 contained 10
µM [
P]ATP, 1 mM carbamylcholine, 2 mM EDTA, and 0.5 mM EGTA. To
this assay medium, 5 mM CaCl
was added together
with PKC (the upper figure). The assay medium for PKC
contained 10 µM [
P]ATP, 1 mM carbamylcholine, and 0.1 mM CaCl
. To this
medium, 2 mM EGTA and 1 mM EGTA were added together
with GRK2 (the lower figure). The sum of phosphorylation by
GRK2 alone and that by PKC alone is shown as GRK2 + PKC (calc.) in both figures.
Figure 6:
Effect of ATP concentrations on
phosphorylation of m1 receptors by GRK2 and PKC. Experimental
procedures were the same as described in the legend to Fig. 5,
except that m1 receptors were subjected to phosphorylation in the
presence of different concentrations of [P]ATP
(2.35 cpm/fmol) for 120 min with GRK2 alone (
) or PKC alone
(
) or for 60 min with GRK2 and then for another 60 min after
addition of PKC (GRK2 then PKC) (
) or for 60 min with PKC and then
for 60 min after addition of GRK2 (PKC then GRK2) (
). The sum
of phosphorylation by GRK2 alone and that by PKC alone is shown as
``Calc. (PKC + GRK2)''
(
).
Very recently, Tsu et al.(42) reported that GRK2 is phosphorylated by PKC and that
the phosphorylated GRK2 has a 31% lower K and 10%
higher V
for phosphorylation of rhodopsin. We
have also examined if the activity of GRK2 to phosphorylate m1
receptors is affected by preincubation of GRK2 with PKC, but have not
found significant increase of the activity of GRK2. It remains to be
determined if this is due to the difference in substrates or
experimental conditions.
By treatment of m1 receptors
phosphorylated by GRK2 with higher concentrations of trypsin, molecular
sizes of P-labeled bands decreased from 38 to 13 and
finally to 3 and 2 kDa (Fig. 8). All of these phosphorylated
bands were immunoprecipitated with anti-i3 but not with anti-C tail
antibodies. Bands with 3 and 2 kDa were resistant to treatment with
higher concentrations of trypsin. The recovery of radioactivity in the
3- and 2-kDa bands obtained by treatment of the receptor with 50
µg/ml trypsin was 50-70% of the initial amount in the intact
receptor. This result indicates that these bands contain the major
phosphorylation sites, although they may not be the sole
phosphorylation sites. In contrast, when m1 receptors phosphorylated by
PKC were treated with higher concentrations of trypsin, the 14-kDa
peptide with phosphorylation sites by PKC was broken down to smaller
fragments, and no
P-labeled bands were detected on
SDS-PAGE (data not shown). Bands immunoprecipitated with anti-C tail
antibodies also became undectable following treatment with higher
concentrations of trypsin under conditions where bands precipitated
with anti-i3 antibodies could still be detected. These results indicate
that major PKC phosphorylation sites are located in the
carboxyl-terminal portion, which is easily broken down by treatment
with trypsin, and that major GRK2 phosphorylation sites are in peptides
which are resistant to trypsin treatment and contain at least part of
the third intracellular loop.
Figure 8: Digestion with high concentrations of trypsin of m1 receptors phosphorylated by GRK2. PEG-treated m1 receptors (3.6 pmol as original amount in total volume of 300 µl) were phosphorylated by GRK2, then treated with indicated concentrations of trypsin for 5 min at 30 °C. An aliquot of the reaction mixture (33 µl) was directly subjected to SDS-PAGE followed by autoradiography. Digitonin (0.1% in a final concentration) and then anti-i3 or anti-C tail antibodies (10 µl of anti-serum) were added to a portion of the reaction mixture (100 µl). After incubation at 4 °C overnight, Pansorbin was added and the suspension was centrifuged. The pellet was subjected to SDS-PAGE followed by autoradiography.
In the present paper we have shown that human m1 receptors are phosphorylated in an agonist-dependent manner by GRK2 and independent manner by PKC. The number of phosphorylation sites were estimated to be 4-5 for phosphorylation by GRK2 and 2-3 for phosphorylation by PKC. These phosphorylation sites appear to be different from each other, because the sum of sites phosphorylated by GRK2 and PKC was not significantly different from the number of sites phosphorylated following sequential phosphorylation by GRK2 and PKC. This conclusion was further supported by the finding that major phosphorylation bands obtained by trypsin treatment of m1 receptors phosphorylated by GRK2 are different from those obtained by the same treatment of m1 receptors phosphorylated by PKC. This conclusion is consistent with the fact that the consensus sequence for phosphorylation by PKC should include basic amino acids around serine and threonine residues (43) but phosphorylation sites by GRK2 should be franked by acidic amino acids rather than basic amino acids(44) .
Major phosphorylation sites by PKC have been
located in a sequence within 14 kDa from the carboxyl-terminal segment,
consistent with previous results using muscarinic receptors purified
from porcine brain (37) . The 14-kDa peptide is thought to
contain residues 333-460 based on the assumption that the peptide
ends at the carboxyl terminus, and the average molecular mass of each
residue is 110 Da. The 14-kDa peptide labeled with P was
sensitive to trypsin treatment and was broken down to small peptides
undectable following SDS-PAGE, consistent with the fact that there are
numerous lysine and arginine residues in the carboxyl-terminal portion.
There are two serine and two threonine residues in the
carboxyl-terminal tail (R*DT
FR*,
K*R*PGS
VHR*T
D-S
R* QC-OH) and
one serine and two threonine residues in the carboxyl-terminal portion
of the third intracellular loop (K*R*PT
R*K*,
K*R*K*T
FS
LVK*EK*K*K*), which are franked by
basic amino acids and are therefore good candidates for PKC
phosphorylation sites. In fact, peptides corresponding to the
carboxyl-terminal tail (sequence 435-460) and to the
carboxyl-terminal part of the third intracellular loop (sequence
346-365) were phosphorylated by protein kinase C, although a
peptide corresponding to the sequence 422-434 was not
phosphorylated(6) . Thus, Thr
, Ser
,
Ser
, Thr
, and Ser
are likely
candidates for sites phosphorylated by PKC.
Major GRK2
phosphorylation sites have been recovered in 3- and 2-kDa bands, which
interact with anti-i3 and are resistant to treatment with trypsin. It
is interesting to note that the amino-terminal half of the third
intracellular loop contains fewer basic and many more acidic amino acid
residues than the carboxyl-terminal half. Serine and threonine residues
in the amino-terminal half of the third intracellular loop are not
flanked by basic amino acid residues, in contrast with serine and
threonine residues in the carboxyl-terminal tail, the carboxyl-terminal
half of the third intracellular loop, and the second intracellular
loop. In particular, serine and threonine residues in the sequence from
275 to 303, K*EE
E
E
E
D
E
GS
ME
S
LT
S
S
E
GE
E
D
GS
E
VVIK*, are most likely to be GRK2
phosphorylation sites, because 1) the sequence contains basic amino
acid residues only at both ends and the corresponding peptide with a
molecular mass of 3 kDa is expected to be resistant to trypsin
treatment, 2) a peptide corresponding to the sequence 279-293 was
phosphorylated in vitro by GRK2(6) , and 3) sequence
286-291 fits to a consensus sequence for phosphorylation in
vivo by GRK2, which was proposed from studies on
2-adrenergic
receptors(31) . In addition, the replacement by alanine of
serine and threonine residues in the sequence 284-291 is reported
to cause the attenuation of sequestration of m1 receptors (45) . The kinase involved in the sequestration of m1 receptors
has not been identified, but may be GRK2 or related kinases, because
the phosphorylation by GRK2 of serine and threonine residues in the
third intracellular region of m2 receptors is known to facilitate their
sequestration(22) . These results taken together suggest that
the primary GRK2 phosphorylation sites reside in the 276-303
amino acid sequence of the third intracellular loop, although this does
not exclude the possibility that the serine and threonine residues in
the other parts of intracellular loops might be phosphorylated by GRK2.
The number of sites phosphorylated by GRK2 was estimated to be 2.9
when m1 receptors were subjected to phosphorylation by GRK2 following
phosphorylation by PKC, a value significantly lower than the value of
4.6 obtained following phosphorylation with GRK2 alone. This result
indicates that the phosphorylation of m1 receptors by GRK2 is inhibited
by prior phosphorylation by PKC. GRK2 is known to be synergistically
activated by G protein subunits and mastoparan or related
peptides (16) and by
subunits and agonist-bound
receptors(17) . Peptides corresponding to the second
intracellular loop, the carboxyl-terminal end of the third
intracellular loop and the carboxyl-terminal tail of m2 receptors
activated GRK2(16) . GRK2 was also strongly activated by a
peptide corresponding to the carboxyl-terminal tail of m1 receptors and
weakly activated by a peptide corresponding to the carboxyl-terminal
end of the third intracellular loop of m1 receptors. (
)All
these peptides contain many basic amino acid residues. It is tempting
to speculate that the phosphorylation by PKC of serine and threonine
residues in the carboxyl-terminal tail or in the carboxyl-terminal end
of the third intracellular loop of m1 receptors reduces the ability of
the receptor to interact with and activate GRK2. Another possibility is
that the interaction between
subunits and m1 receptors is
impaired by phosphorylation of m1 receptors by PKC. The
carboxyl-terminal tail of rhodopsin has been reported to be the site
for interaction with
subunits(46) , although no
direct evidence is available for m1 receptors.
The agonist-dependent phosphorylation of m1 receptors was first detected when m1 receptors reconstituted into lipid vesicles were precipitated with PEG prior to the phosphorylation reaction. The PEG treatment reproducibly caused both a decrease in the agonist-independent phosphorylation and an increase in the agonist-dependent phosphorylation. One of the simplest explanation is that inhibitory and/or stimulatory factors were removed upon precipitation with PEG. The putative factors are not likely to be residual detergents or agonists, because the concentrations of these compounds in the reaction mixture are too low to affect the phosphorylation. We have detected a band of 3 kDa in the m1 preparations, which was well phosphorylated by GRK2, and the amount of the band was markedly reduced with the precipitation procedure with PEG. Such a band was not detected in the m2 preparation, which are phosphorylated by GRK2 in an agonist-dependent manner even in the absence of the precipitation procedure. The nature of the band remains to be identified.
In the present paper, we have shown that m1 receptors undergo the phosphorylation by GRK2 and PKC, which may provide the molecular basis of homologous and heterologous desensitization, respectively. A novel type of cross-talk between phosphorylation of m1 receptors by PKC and GRK2 was suggested from the finding that the phosphorylation of m1 receptors by GRK2 is attenuated by the preceding phosphorylation with PKC.