(Received for publication, September 22, 1995; and in revised form, November 13, 1995)
From the
Phosphorylation of G-protein-linked receptors is thought to play
a central role in receptor regulation and desensitization. Unlike the
case of the extensively studied -adrenergic receptor/adenylate
cyclase pathway, in which receptor-specific phosphorylation is known to
be mediated by
-adrenergic receptor kinase (
-ARK), the
kinases responsible for phosphorylation of phospholipase C-linked
receptors have yet to be identified, although a role for
-ARK has
been implicated. This study describes the purification of a novel
40-kDa receptor kinase from porcine cerebellum that is able to
phosphorylate the phospholipase C-linked m3-muscarinic receptor in an
agonist-dependent manner. The assay for kinase activity was based on
the ability of the kinase to phosphorylate a bacterial fusion protein,
Ex-m3, containing amino acids Ser
-Leu
of the third intracellular loop of the m3-muscarinic receptor.
Purification of the muscarinic receptor kinase from a high speed
supernatant fraction of porcine cerebellum was achieved using the
following steps: (i) 30-60% ammonium sulfate cut and successive
chromatography on (ii) butyl-Sepharose (iii) Resource Q, (iv) Resource
S, and (v) heparin-Sepharose. The purified protein kinase represented
an
18,600-fold purification and was a single polypeptide with a
molecular weight of
40 kDa. Based on the chromatographic mobility,
molecular weight, and kinase inhibitor studies, the kinase, designated
MRK, was shown to be distinct from previously characterized second
messenger regulated protein kinases,
-ARK, and other members of
the G-protein-linked receptor kinase family. It therefore represents a
new class of receptor kinase.
Many cell surface neurotransmitter and hormone receptors respond
to agonist occupation by activation of phospholipase C. The subsequent
hydrolysis of the lipid substrate phosphatidylinositol 4,5-bisphosphate
releases the second messengers inositol 1,4,5-trisphosphate
(Ins(1,4,5)P) (
)and diacylglycerol (reviewed in (1) ). The second messenger response to a number of
phospholipase C-linked receptors, including the m3-muscarinic receptor,
is often complex, consisting of a burst of Ins(1,4,5)P
production reaching a peak within the first few seconds of
receptor stimulation followed by a lower sustained phase of
Ins(1,4,5)P
generation that is maintained for tens of
minutes to hours(2, 3, 4) . Similar patterns
of Ins(1,4,5)P
generation are seen in response to agonist
occupation of receptors for GRH(5) , angiotensin(6) ,
bombesin, and CCK(7) .
Little is known of the molecular
mechanisms underlying these complex second messenger responses,
although recent evidence suggests that some phospholipase C-linked
receptors undergo a rapid partial desensitization that results in
decreased phospholipase C activity within seconds of agonist
occupation(3, 4, 7, 8) . Consistent
with this notion are studies from our laboratory demonstrating that the
early peak phase of Ins(1,4,5)P production in response to
m3-muscarinic receptor stimulation can be desensitized by a short
pre-exposure to agonist, whereas the sustained phase of
Ins(1,4,5)P
production is resistant to desensitization (3) .
One possible mechanism regulating the
receptor/phospholipase C pathway is receptor phosphorylation. The
involvement of -adrenergic receptor phosphorylation in the
desensitization of the
-adrenergic/adenylate cyclase system has
been well documented (reviewed in (10) ). In this case the
agonist-occupied form of the
-adrenergic receptor is
phosphorylated by two kinases, protein kinase A and a receptor-specific
kinase termed
-adrenergic receptor kinase (
-ARK). The process
of receptor phosphorylation results in uncoupling of the
-adrenergic receptor from the G
-protein(10) .
It is now clear that in addition to
-adrenergic receptors other
G-protein linked receptors also exist as phosphoproteins. In
particular, phospholipase C/G
-coupled CCK(11) ,
m3-muscarinic(12) ,
-adrenergic(13) ,
platelet-activating factor(14) ,
thrombin(15, 16) , and neurokinin-2 receptors (17) have all been shown to exist as phosphoproteins in intact
cells, and the level of phosphorylation is enhanced following agonist
stimulation. The receptor-specific kinase(s) responsible for these
phosphorylation events have yet to be fully characterized, although
some authors have suggested that
-ARK may act as a general
G-protein-linked receptor kinase with a broad substrate
specificity(18) . Certainly,
-ARK has been implicated in
the phosphorylation of CCK receptors in pancreatic acinar cells (19) and recombinant thrombin receptors expressed in Xenopus oocytes and fibroblasts(15) . Furthermore,
substance P and m3-muscarinic receptors have been shown to act as in vitro substrates for
-ARK(18, 20) .
The two isotypes of -ARK (
-ARK-1 and -2) are members of a
family of protein kinases that includes rhodopsin kinase, IT-11, GRK-5,
and GRK-6, which are related on the basis of primary amino acid
sequence homology and are collectively termed the G-protein-linked
receptor kinase family (GRK) (reviewed in (21) ). The existence
of the GRK family suggests that G-protein-linked receptor
phosphorylation may be mediated by more than one receptor-specific
kinase. However, the cellular substrates for IT-11, GRK-5, and GRK-6
are unknown despite in vitro evidence that these kinases are
able to phosphorylate a number of G-protein-linked
receptors(22, 23) .
The possibility that a receptor
kinase(s) other than -ARK may be involved in the phosphorylation
of G-protein-linked receptors has been suggested by recent studies from
our laboratory on the phospholipase C-linked m3-muscarinic
receptor(12) . These studies have demonstrated that recombinant
human m3-muscarinic receptors expressed in CHO cells (CHO-m3 cells)
undergo agonist-mediated phosphorylation on serine. The time course for
receptor phosphorylation is very rapid, occurring within seconds of
agonist addition, and correlates with the rapid but partial
desensitization of the phosphoinositide response seen within the first
20 s of receptor stimulation(8, 9, 12) .
Initial characterization demonstrated the kinase to be distinct from
the known second messenger-regulated protein kinases, cAMP-dependent
protein kinase, cGMP-dependent protein kinase,
calcium/calmodulin-dependent protein kinase, and protein kinase
C(12) . Further characterization in a broken cell preparation
revealed that a membrane-associated kinase was able to phosphorylate
the m3-muscarinic receptor and that this kinase was not affected by
heparin or zinc at concentrations that inhibit
-ARK(24) .
These findings indicated that m3-muscarinic receptor phosphorylation
was mediated by a novel receptor kinase.
Described here is the purification from porcine cerebellum of a novel 40-kDa protein kinase that is able to phosphorylate the m3-muscarinic receptor in an agonist-dependent manner. The purification was based on the ability of the muscarinic receptor kinase to phosphorylate a bacterial fusion protein encoding a region of the third intracellular loop of the m3-muscarinic receptor (Ex-m3).
Figure 8:
Phosphorylation of truncated fusion
proteins by extracts from CHO-m3 cells and purified porcine brain MRK. A, diagrammatic representation of the fusion proteins. The filled bars in Ex-m3 denote the position of serine residues
and denotes the serine residues that are strong candidates for
the phosphoacceptor site(s) for MRK (see ``Discussion''). B, Coomassie Blue stain showing the relative positions of
equimolar amounts (
0.8 µM) of the fusion proteins on
a 12% SDS-PAGE gel. C, autoradiograph showing the
phosphorylation of the fusion proteins by purified porcine brain MRK. D, autoradiograph showing the phosphorylation of the fusion
proteins by partially purified CHO-m3 cytosolic kinase. E,
autoradiograph showing the phosphorylation of the fusion proteins by
membranes from CHO-m3 cells. F, autoradiograph showing the
phosphorylation of the fusion proteins by a partially purified kinase
extracted from CHO-m3 membranes by a high salt wash. For each of the
above experiments the fusion proteins were visualized on the gel by
Coomassie Blue staining to confirm equal loading and to confirm the
relative mobilities of the fusion proteins on the gel. Indicated are
the positions of prestained molecular weight standards. The results
shown are representative of two
experiments.
pEx345-427 was constructed using the
following PCR primers: 5` primer, CCCGGATCCCTGGAGAACTCCGCC; 3` primer,
CCGGAATTCAAGCTTGGAGAAGCTTTT. These primers were used to amplify a
region of the human m3-muscarinic receptor cDNA that encodes amino
acids Ser-Leu
. The primers were
designed to allow in-frame cloning into pGEX-2T via a BamHI
site at the 5` end and an EcoRI site at the 3` end.
pEx376-463 was synthesized using the same strategy, but in
this case the 5` PCR primer was CCCGGATCCACCATCCTCAACTCCACC, and the 3`
primer was CCCGAATTCCAGAGTGGCTTCCTTGAAG. This amplified a region of
human m3-muscarinic cDNA that encoded amino acids
Thr-Leu
, which was then cloned into
pGEX-2T.
pHind was constructed by digestion of pEx-m3 with the
restriction enzyme HindIII. This removed a section of cDNA
from the muscarinic region of pEx-m3 that encoded for amino acids
Leu
-Lys
, inclusive.
pH-V was
constructed by ligating two PCR reaction products into pGEX-2T in a
three-way ligation where pGEX-2T was digested with BamHI (5`)
and EcoRI (3`), PCR reaction product 1 was digested with BamHI (5`) and ApaI (3`), and PCR reaction product 2
was digested with ApaI (5`) and EcoRI (3`). PCR
primers used were (for product 1) 5` primer CCCGGATCCCTGGAGAACTCCGCC
and 3` primer GCTGGGCCCCGGAAGCTTGAGCAC and (for product 2) 5` primer
CAGGGGCCCGAGGAGGAGCTGGGG and 3` primer CCCGAATTCCAGAGTGGCTTCCTTGAAG.
The resulting construct encoded a truncated form of Ex-m3 where amino
acids His
-Val
, inclusive, were
deleted.
The above constructs were used to transform E. coli (DH5). Induction and purification of fusion proteins was the
same as that described for Ex-m3.
Membranes were prepared from CHO-m3 cells by
homogenization of the cells as above but this time in 15 ml of TE
buffer plus protease inhibitors. Membranes were collected by
centrifugation at 15,000 g for 10 min and resuspended
in kinase buffer at 1 mg protein/ml.
10 µl of either the membrane or cytosolic preparations were used in the assay for kinase activity.
The sample was then passed through a
6-ml Resource Q (Pharmacia) column. This resolved muscarinic receptor
kinase activity from casein kinase II that during the course of this
study was found to also phosphorylate the Ex-m3 fusion protein (see
``Results'' and ``Discussion''). The flow-through
from the Resource Q column was applied to a 1-ml Resource S (Pharmacia)
column. The Resource S column was then eluted using a linear gradient
of 0-0.5 M NaCl over 20 bed volumes (flow rate =
1 ml/min). 1-ml fractions were collected. The kinase activity eluted as
a single peak at 0.32 M NaCl. These fractions were
combined and passed through a 1-ml heparin-Sepharose column
equilibrated with 0.32 M NaCl. The column was eluted using a
linear gradient of 0.32-2.0 M NaCl. The kinase activity
eluted as a single peak at
0.87 M NaCl.
Membranes from the above preparation were either used
directly to test for muscarinic receptor kinase activity or extracted
with 15 ml of 1.5 M KCl/TE buffer for 3 h at 4 °C. The
extract was dialyzed against TE buffer and run through a 1-ml Resource
S column. The column was eluted as described above. The peak kinase
activity eluted at 0.32 M NaCl.
In experiments to test the ability of the fusion protein Ex-m3 to inhibit the m3-muscarinic receptor phosphorylation, Ex-m3 (3.5 µg) or a molar equivalent of glutathione S-transferase was added to the reaction mixture. At the end of the reaction Ex-m3 (3.5 µg) was added to control tubes, thereby ensuring that all tubes contained equal amounts of Ex-m3, since the fusion protein will compete with the receptor for the antibody in the immunoprecipitation. The reaction was then stopped in the way described above, and m3-muscarinic receptors were then solubilized and immunoprecipitated.
In experiments where
purified muscarinic receptor kinase was tested for its ability to
phosphorylate the intact m3-muscarinic receptor, an aliquot of the
kinase preparation (10 µl, 2.5 pmol) was added to the reaction
mixture. To control tubes a buffer blank was added (this gave a final
NaCl concentration of 87 mM). The reaction was then started
and terminated as described above.
The bacterial fusion protein Ex-m3, containing a region
of the third intracellular loop of the human m3-muscarinic receptor
(Ser-Leu
) acted as a substrate for a
kinase in cytosolic extracts from CHO-m3 cells (Fig. 1). There
was no phosphorylation of the bacterially expressed glutathione S-transferase (Fig. 1). Furthermore, following
digestion of phosphorylated Ex-m3 fusion protein with thrombin, a
process that releases the m3-muscarinic receptor region, the
P label was associated only with the m3-muscarinic region
and not the glutathione S-transferase portion of the fusion
protein (data not shown). A similar kinase activity was also identified
in membrane preparations from CHO-m3 cells, but the kinase activity was
30-fold lower than that observed in cytosolic extracts. Studies
described below indicate that the kinase associated with the membrane
fraction is likely to be the same as the cytosolic kinase.
Figure 1: Phosphorylation of Ex-m3 by a cytosolic extract from CHO-m3 cells. A high speed supernatant extract from CHO-m3 cells (10-50 µg of protein) was tested for kinase activity capable of phosphorylating the recombinant bacterial proteins, Ex-m3 (3.5 µg) and glutathione S-transferase (2.0 µg, GST). Shown is a Coomassie Blue stain of the purified bacterial proteins indicating their relative positions on a 12% SDS-PAGE gel and an autoradiograph showing the phosphorylated products. Indicated are the positions of prestained molecular weight standards. The results are representative of at least five experiments.
We have previously demonstrated agonist-driven m3-muscarinic receptor phosphorylation in membranes from CHO-m3 cells, suggesting that the muscarinic receptor kinase is, at least in part, associated with the plasma membrane(24) . In order to confirm that the Ex-m3 fusion protein was acting as a pseudosubstrate for the m3-muscarinic receptor kinase, experiments were conducted to investigate the ability of Ex-m3 to block agonist sensitive muscarinic receptor phosphorylation in membranes from CHO-m3 cells. Fig. 2shows that Ex-m3 was able to inhibit agonist-induced m3-muscarinic receptor phosphorylation in CHO-m3 membranes, whereas glutathione S-transferase had no effect. Note also, that in addition to blocking m3-muscarinic receptor phosphorylation, the Ex-m3 fusion protein was itself phosphorylated (Fig. 2).
Figure 2:
Inhibition of agonist-driven
phosphorylation of m3-muscarinic receptors in membranes from CHO-m3
cells. Membranes from CHO-m3 cells (50 µg of protein, 0.1 pmol
of m3-receptor) were challenged with or without 1 mM carbachol
in the presence of 100 µM [
-
P]ATP (1-4 cpm/fmol of ATP)
for 10 min at 32 °C. The receptors were then solubilized,
immunoprecipitated, and resolved by 8% SDS-PAGE. The effect of Ex-m3
(3.5 µg) or a molar equivalent of glutathione S-transferase (2.0 µg, GST) on agonist-driven
m3-muscarinic receptor phosphorylation was determined. Note: in the
lane where Ex-m3 was added, the fusion protein as well as the receptor
was immunoprecipitated. Indicated are the positions of prestained
molecular weight standards. The results are representative of two
experiments.
The chromatography steps involved in
purification of the muscarinic receptor kinase are illustrated in Fig. 3and summarized in Table 1. Cerebella from 15 pigs
(180 g) were homogenized in 1 liter of TE buffer before
centrifugation to prepare a cytosolic S200 fraction (volume, 750 ml;
protein, 5.2 g). Following a 30-60% ammonium sulfate cut and
dialysis against TE buffer containing 1 M (NH
)
SO
the sample (0.94 g in
120 ml) was fractionated on a 70-ml butyl-Sepharose column. A number of
minor peaks of kinase activity eluted in the gradient, but the main
kinase activity eluted as a single peak at
0.54 M (NH
)
SO
(Fig. 3A).
Figure 3: Purification of MRK from porcine cerebellum. 30-60% ammonium sulfate cut of a high speed supernatant preparation from 15 pig cerebella was dialyzed against TE buffer and applied to a 70-ml butyl-Sepharose column. A, the elution profile of kinase activity and proteins from the butyl-Sepharose column. The autoradiograph shows the peak phosphorylation of Ex-m3 fusion protein. The peak fractions were pooled, dialyzed against TE buffer, and passed through a 6-ml Resource Q column before being resolved on a Resource S column. B, the elution profile of kinase activity and proteins from the Resource S column. C, an autoradiograph showing phosphorylation of Ex-m3 by fractions from the Resource S elution against a silver stain of 20-µl aliquots of each of the fractions. Shown is the position of a 40-kDa polypeptide, the elution of which correlates with the kinase activity. The peak fractions from the Resource S elution were pooled and applied to a 1-ml heparin-Sepharose column. D, autoradiograph showing Ex-m3 kinase activity of fractions eluting from the heparin-Sepharose column against a silver stain of 90-µl trichloroacetic acid-precipitated aliquots of each of the fractions. The position of a 40-kDa polypeptide that co-elutes with the kinase activity is shown. The results shown are from a single purification protocol that was repeated at least four times.
The sample was then run through a
6-ml Resource Q anion exchange column. Casein kinase II, which
co-purifies with the muscarinic receptor kinase activity up to this
stage, binds to this column, whereas muscarinic receptor kinase with
its alkali pI passes through the column. The flow-through from the
Resource Q column was applied to a 1-ml Resource S cation exchange
column. Ex-m3 phosphorylation activity eluted from this column as a
single peak at 0.32 M NaCl (Fig. 3B). Fig. 3C shows the elution profile of kinase activity
and the silver stain of 20-µl aliquots of each of the fractions.
The kinase activity co-elutes with a protein of
40 kDa. In the
final chromatography step, utilizing a 1-ml heparin-Sepharose column,
the elution profile of kinase activity again correlated precisely with
the elution of the 40-kDa protein (Fig. 3D).
Furthermore, silver staining indicated that fraction 12 from the
heparin-Sepharose elution, containing the peak of kinase activity,
contains only the 40-kDa polypeptide. The kinase was, therefore,
considered homogeneous and designated MRK (muscarinic receptor kinase).
Using -ARK-specific antibodies (a kind gift from Dr. R. J.
Lefkowitz, Howard Hughes Medical Institute, Duke University, Durham,
NC) it was established that
-ARK co-purified with the muscarinic
receptor kinase on the butyl-Sepharose column.
-ARK, reported
previously to have an alkali pI(25) , bound to the Resource S
column and eluted at
0.175 mM salt (data not shown). It
was therefore possible to resolve
-ARK from MRK (eluting at
0.32 M NaCl) on the Resource S column. Furthermore,
-ARK immunoreactivity corresponded to fractions 6, 7, and 8 on the
Resource S elution; however, no Ex-m3 phosphorylation activity was
observed in these fractions, demonstrating that
-ARK was not able
to phosphorylate the Ex-m3 fusion protein (see Fig. 3B).
Figure 4:
Autophosphorylation of the muscarinic
receptor kinase. An aliquot (20 µl, 20 ng of kinase) of the
heparin-Sepharose-purified kinase preparation (MRK) was incubated in
kinase buffer containing 100 µM [
-
P]ATP (1-4 cpm/fmol of ATP)
for 30 min at 37 °C. The reaction was stopped by trichloroacetic
acid precipitation of the proteins. The protein pellet was dissolved in
SDS-loading buffer and resolved on a 12% SDS-PAGE gel. Indicated are
the positions of prestained molecular mass standards. The results are
representative of three experiments.
Figure 5:
Phosphorylation of the m3-muscarinic
receptor in CHO-m3 cells. Membranes from CHO-m3 cells (50 µg of
protein, 0.1 pmol of receptor) made up in kinase buffer containing
100 µM [
-
P]ATP (1-4
cpm/fmol of ATP) were incubated in the presence or absence of 1 mM carbachol and with or without the antagonist atropine for 10 min
at 32 °C. To these preparations either a buffer control or purified
brain MRK (10 µl,
2.5 pmol) was added. The reaction was
stopped by adding solubilization buffer. The receptors were then
immunoprecipitated and resolved on an 8% SDS-PAGE gel. The
autoradiograph shown is representative of three experiments. Indicated
are the positions of prestained molecular weight
standards.
In control experiments using phosphorylated and nonphosphorylated Ex-m3 (phosphorylated using MRK) the m3-muscarinic receptor antiserum 332 was able to immunoprecipitate both forms of Ex-m3 equally well (data not shown). Thus, despite being raised against the region of the third intracellular loop that contains the phosphoacceptor sites for MRK, the binding of the m3-muscarinic receptor antiserum appears not to be affected by phosphorylation of these sites.
Figure 6: Effect of protein kinase inhibitors on muscarinic receptor kinase activity. The effect of heparin (1 µg/ml), zinc chloride (100 µM), RO-318220 (0.1 µM), H-89 (5 µM), and trifluoperazine (50 µM) on MRK (10 µl of Resource S-purified kinase) phosphorylation of Ex-m3 (3.5 µg) was tested. Indicated are the positions of prestained molecular weight standards. The results are representative of three experiments.
Phosphoamino acid analysis demonstrated that purified MRK phosphorylated Ex-m3 at serine (data not shown).
Kinetic analysis of MRK-mediated phosphorylation of Ex-m3 revealed a V of 1.15 ± 0.3 nmol phosphate
incorporated per min per mg of kinase and a K
of
0.4 ± 0.11 µM (n = 4; Fig. 7).
Figure 7:
Kinetic analysis of MRK phosphorylation of
Ex-m3. Various concentrations of Ex-m3 (0.6-0.03 µM)
were incubated with 50 ng of Resource S-purified MRK in kinase
buffer containing 50 µM [
-
P]ATP (0.4-1.0 cpm/fmol of
ATP) for 15 min. The reaction was terminated, and fusion proteins were
isolated as described in the text. Ex-m3 fusion protein was then
resolved by 12% SDS-PAGE, the gel was stained, and fusion proteins were
excised and counted. V
= 1.15 ±
0.3 nmol of phosphate incorporated per min per mg of kinase, and K
= 0.4 ± 0.11 µM (n = 4). The results shown are the mean ±
S.E. of four experiments.
Fig. 8C shows the pattern of phosphorylation of the various fusion
proteins by purified brain MRK. Glutathione S-transferase was
not phosphorylated, whereas Ex-m3 was the best substrate. Hind,
Ex345-427, and
H-V were phosphorylated by 23, 44, and 29%
the level of Ex-m3, respectively, whereas Ex376-463 was the
poorest substrate, showing only 2% of the phosphorylation seen for
Ex-m3 (as determined by densitometric analysis).
A muscarinic
receptor kinase activity with properties indistinguishable from brain
MRK could be isolated from CHO-m3 cell cytoplasm. A high speed
supernatant fraction of CHO-m3 cells was passed through a Resource S
column from which a kinase able to phosphorylate Ex-m3 was eluted as a
single peak at 0.32 M NaCl. This is the same point at
which brain MRK elutes from the Resource S column. Testing the
partially purified CHO-m3 cell cytosolic kinase preparation against the
fusion protein substrates, it was clear that in addition to possessing
chromatographic properties equivalent to brain MRK, the CHO-m3
cytosolic kinase also phosphorylated the complement of fusion proteins
with the same specificity as that seen for the brain kinase (Fig. 8D). This suggests that the brain MRK and the
kinase responsible for Ex-m3 phosphorylation in CHO-m3 cell cytoplasm
are likely to be homologous or very closely related.
Crude membrane
preparations of CHO-m3 cells also possess the ability to phosphorylate
the Ex-m3 fusion protein (Fig. 8E). However, the
substrate specificity of this kinase was quite different from that seen
for the CHO-m3 cytosolic kinase and for the brain MRK. In order to test
whether the difference in substrate specificity may be due to the lipid
environment, CHO-m3 membranes were washed with 1.5 M KCl for 3
h. This procedure removed agonist-mediated m3-muscarinic receptor
kinase activity from the membrane, indicating that the kinase was not
an integral membrane protein (data not shown). Following high salt
treament, membranes no longer contain the ability to phosphorylate the
Ex-m3 fusion protein. The extracted proteins were resolved on a
Resource S column. On elution of this column a single peak of kinase
activity was detected at 0.32 M NaCl. This kinase
activity showed a similar preference for fusion protein substrates as
observed for brain kinase and CHO-m3 cytosolic kinase (Fig. 8F). Therefore, on the basis of substrate
specificity and chromatographic behavior it appears that the muscarinic
receptor kinase activity extracted from CHO-m3 membranes is similar or
identical to that observed in CHO-m3 cell cytosol and to that of brain
MRK.
The present study describes the purification from porcine
cerebellum of a 40-kDa protein kinase that phosphorylates the
agonist-occupied form of the phospholipase C-linked m3-muscarinic
receptor. Based on its molecular weight, protein kinase inhibitor
studies, and chromatographic mobilities, the kinase purified here,
termed MRK, was shown to be distinct from second messenger-regulated
protein kinases, -ARK and other members of the GRK family and
therefore represents a new class of receptor kinase.
Purification of
MRK was based on an assay in which the bacterial fusion protein (Ex-m3)
containing amino acids Ser-Leu
of the
third intracellular loop of the human m3-muscarinic receptor was
phosphorylated by the same kinase as that responsible for m3-muscarinic
receptor phosphorylation. Evidence that Ex-m3 acted as a
pseudosubstrate for the m3-muscarinic receptor kinase came from studies
on CHO-m3 cell membranes. Previously we described agonist-sensitive
phosphorylation of m3-muscarinic receptors in membrane preparations
from CHO-m3 cells indicating that the muscarinic receptor kinase was,
at least in part, associated with the plasma membrane(24) . In
this study we demonstrate the ability of Ex-m3 to inhibit
agonist-mediated phosphorylation of intact m3-muscarinic receptors in
membrane preparations. Furthermore, in these experiments the Ex-m3
fusion protein was itself phosphorylated, suggesting that Ex-m3 blocked
the action of the m3-muscarinic receptor kinase by acting as a
pseudosubstrate.
During the course of this study it was found that
Ex-m3 was also a substrate for casein kinase II. Indeed, using the
Ex-m3 phosphorylation assay the three subunits of casein kinase II were
purified to homogeneity and identified by amino acid sequencing. ()Although casein kinase II was able to weakly phosphorylate
the m3-muscarinic receptor in membrane preparations, this was not
agonist-dependent.
These data clearly suggest that casein
kinase II is not involved in agonist-mediated phosphorylation of the
m3-muscarinic receptor in intact cells.
Tissue distribution studies
revealed muscarinic receptor kinase activity to be rich in cortex and
cerebellum, although the activity in cerebellum was 10-fold
greater. It was, therefore, decided to purify this activity from a
cytosolic fraction of porcine cerebellum. The elution profile of Ex-m3
phosphorylation activity in the final two column steps in the
purification protocol precisely correlated with the elution of a single
polypeptide at
40 kDa. Fraction 12 from the heparin-Sepharose
column (the final column step) contained the peak of kinase activity,
and the only detectable protein present, as determined by silver stain,
was the 40-kDa polypeptide. The kinase preparation was therefore
considered to be homogeneous at this stage, and this represented
18,600-fold purification. The fact that the 40-kDa polypeptide
contained kinase activity was established by demonstrating that in
common with many protein kinases (e.g. Refs. 25 and 30) the
40-kDa protein was able to undergo autophosphorylation.
Reconstitution of the 40-kDa protein kinase with membranes prepared from CHO-m3 cells was used to determine if the purified brain kinase was able to phosphorylate the intact m3-muscarinic receptor. Previous studies from our laboratory have established the presence of an endogenous kinase able to mediate agonist sensitive phosphorylation of m3-muscarinic receptors in membranes from CHO-m3 cells(24) . These findings were confirmed in the present study, where, in the absence of exogenous kinase, agonist-sensitive phosphorylation of the m3-muscarinic receptor was observed in membranes prepared from CHO-m3 cells. However, the addition of the purified brain kinase preparation, although not significantly effecting basal phosphorylation, increased agonist-mediated phosphorylation of the m3-muscarinic receptor by 2-3-fold over that seen in the absence of exogenous kinase. This effect was completely reversed by the muscarinic antagonist atropine. These results confirm that the purified 40-kDa protein kinase was an m3-muscarinic receptor kinase. The kinase was therefore termed MRK (muscarinic receptor kinase).
The ability of the purified brain MRK to drive agonist-dependent phosphorylation of the m3-muscarinic receptor was one of two important criteria we set for establishing that the kinase purified by our method was the kinase responsible for m3-muscarinic receptor phosphorylation in intact cells. The other criterion was to establish that MRK was homologous or closely related to the kinase found in CHO-m3 cells. Since the only system in which m3-muscarinic receptor phosphorylation in intact cells has been demonstrated to date is in CHO-m3 cells, we reasoned that these cells must contain the relevant kinase and therefore would provide an important ``positive control'' for comparison with a purified kinase. Muscarinic receptor kinase activity could be detected in both membrane and cytosolic fractions from CHO-m3 cells. The kinase present in the cytosol of CHO-m3 cells possessed identical chromatographic properties as the purified brain MRK and showed the same substrate preference for truncated fusion proteins as MRK. However, the kinase associated with the membranes of CHO-m3 cells appeared to have a different substrate specificity than that of MRK. To test whether this was due to the membrane environment of the kinase, proteins were stripped from the plasma membrane using a high salt buffer. Ex-m3 phosphorylation activity contained within this extract could be resolved on a Resource S column and showed identical chromatographic properties and substrate specificity as that of CHO-m3 cytosolic kinase and brain MRK. It seems likely, therefore, that purified brain MRK and the muscarinic receptor kinase activity extracted from CHO-m3 membranes and present in CHO-m3 cell cytosol are either homologous or very closely related.
Recent studies have indicated that in addition to
phosphorylating adenylate cyclase-linked receptors (e.g.(31) ) -ARK is also able to phosphorylate the
agonist-occupied forms of a number of phospholipase C-linked receptors.
For example, heparin inhibition studies have suggested that
phospholipase C-linked CCK receptors are phosphorylated, at least in
part, by a ``
-ARK-like'' kinase in pancreatic acinar
cells(19) . Co-expression of thrombin receptors with
-ARK-2 in Xenopus oocytes blocks thrombin-mediated
inositol phosphate/calcium signaling(15) . Furthermore, in
reconstituted systems purified
-ARK has been demonstrated to
phosphorylate the partially purified substance P receptor (18) and recombinant m3-muscarinic receptors contained in
urea-treated Sf9 cell membranes(20) . There seems little doubt,
therefore, that
-ARK has a relatively broad substrate specificity
and has the potential to phosphorylate a number of phospholipase
C-linked receptors including m3-muscarinic receptors.
The kinase
identified in this study, however, is clearly distinct from -ARK
and offers an alternative mechanism for agonist-mediated m3-muscarinic
receptor phosphorylation. First, the molecular mass of MRK (
40
kDa) is significantly less than that of
-ARK 1 and 2 (
80 kDa)
and also less than the molecular masses of other members of the GRK
family that fall within the range of 53-67.7 kDa (see (32) ). Furthermore, immunoblot studies using an anti-
-ARK
antiserum revealed that
-ARK was resolved from MRK at the Resource
S ion exchange chromatography step. These data also demonstrated that
-ARK is unable to phosphorylate the Ex-m3 fusion protein.
Protein kinase inhibitor studies further distinguished MRK from
-ARK and previously characterized second messenger-regulated
protein kinases. The protein kinase inhibitors RO-318220 (protein
kinase C; (26) ), trifluoperazine
(Ca
/calmodulin-dependent protein kinase; (27) ), and H-89 (cGMP-dependent protein kinase and
cAMP-dependent protein kinase; (28) ) at concentrations at
least 10-fold above their reported IC
values had little
effect on MRK activity. Furthermore, zinc at a concentration reported
to inhibit purified
-ARK activity by >90% (25) had no
inhibitory effect on MRK activity. In contrast, heparin at 1 µg/ml
completely blocked MRK activity. Heparin has been reported to be a
relatively potent but nonselective protein kinase inhibitor acting on a
number of kinases including members of the GRK family:
-ARK(29) , GRK5, and GRK6(23) , in addition to
casein kinase II (33) and low density lipoprotein receptor
kinase(34) . The finding that heparin is also a potent
inhibitor of MRK raises a question about the suitability of this
reagent in studies aimed at characterization of protein kinases
responsible for G-protein-linked receptor phosphorylation (e.g.(19) ).
Interestingly, analysis of the kinetic
parameters of MRK phosphorylation of Ex-m3 demonstrate that Ex-m3 is a
good substrate for MRK with a K of 0.4 µM and V
of 1.15 nmol of phosphate
incorporated/min/mg. The K
for MRK phosphorylation
of Ex-m3 is similar to that of
-ARK phosphorylation of the
purified
-adrenoreceptor (K
= 0.25
µM; (25) ) and suggests that the K
that MRK shows for Ex-m3 may be approaching that for the intact
m3-muscarinic receptor. Unfortunately, purified m3-muscarinic receptors
are not presently available to test the kinetic properties of MRK
against the intact receptor substrate.
The mechanism of activation
of MRK is at present unclear. The presence of MRK in the cytoplasmic
and membrane fractions of CHO-m3 cells suggests that a translocation
process where MRK migrates to a membrane site on agonist stimulation in
a manner similar to that described for -ARK (35) may be in
operation. Initial studies have demonstrated that there is an increase
in MRK activity in the membrane fraction following agonist
stimulation;
however, without antibodies to MRK it is
difficult to discern between translocation of the kinase and an
increase in the kinase activity. Dose responses curves constructed for
m3-muscarinic receptor phosphorylation in intact CHO-m3 cells have,
however, suggested that the mechanism of m3-muscarinic receptor
phosphorylation is dependent on a small amplification step downstream
of receptor activation(36) . Experiments are currently in
progress to further elucidate this amplification process and to
investigate the role translocation may play.
It is interesting to
note that the cerebellum is enriched in MRK since this tissue is known
to contain only a small population of m3-muscarinic receptors (for
review see (37) ). This suggests that MRK may have a broader
substrate specificity than just the m3-muscarinic receptor. In support
of this assertion are recent studies from our laboratory showing that
the m1-muscarinic receptor is phosphorylated in an agonist-dependent
manner by a kinase with similar properties to MRK(38) . Further
studies using a range of recombinant G-protein-linked receptors are
presently underway to investigate the substrate specificity of MRK.
Particular attention is being directed toward the metabotropic
glutamate 1 receptor, which is abundantly expressed in Purkinje
cells of the cerebellum and has recently been reported to undergo
agonist-driven phosphorylation in transfected BHK cells, although in
this system a role for protein kinase C has been
implicated(39) .
The use of truncated fusion proteins of
Ex-m3 suggested that at least one site of MRK phosphorylation is
contained in the region of the third intracellular loop of the
m3-muscarinic receptor between Ser and Thr
,
since truncation of this region in Ex-m3 resulted in a fusion protein
(Ex376-463) that was very poorly phosphorylated. This region
contains seven potential serine phosphoacceptor sites, denoted with
∇ in Fig. 8A, which include the SASS motif
identified recently as being important for internalization of the
m3-muscarinic receptor(40) . The possible involvement of
phosphorylation in internalization of the receptor would conform with
the generalized view that G-protein-linked receptor phosphorylation is
associated with a diminution of receptor responsiveness.
Agonist-sensitive phosphorylation of the phospholipase C-linked
-adrenergic(13) ,
thrombin(15, 16) , platelet-activating
factor(14) , and neurokinin-2 receptors (17) are all
associated with desensitization of receptor responses. In the case of
the m3-muscarinic receptor, agonist-induced inositol phosphate
responses undergo partial desensitization within seconds of agonist
addition(4, 9) . The rapid onset of this
desensitization event correlates with the rapid time course of
m3-muscarinic receptor phosphorylation(12) , suggesting that
the two processes may be linked (8) . We are presently in the
process of determining the exact MRK phosphorylation sites on the
m3-muscarinic receptor with a view to making point mutations that will
render the receptor unable to be phosphorylated. Such a receptor will
be an invaluable tool in dissecting the role this novel receptor kinase
plays in regulation of transmembrane signaling.