* Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208; and Department of Biochemistry, Biozentrum, University of Basel, Basel, Switzerland CH-4056
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Abstract |
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G protein-coupled (GPC) receptors are
phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The -factor pheromone receptor (Ste2p)
of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding
-factor. Ubiquitination triggers Ste2p internalization
into the endocytic pathway. Here we demonstrate that
phosphorylation of Ste2p promotes downregulation by
positively regulating ubiquitination and internalization.
Serines and a lysine are essential elements of the Ste2p
SINNDAKSS internalization signal that can mediate
both constitutive and ligand-stimulated endocytosis.
The SINNDAKSS serines are required for receptor
phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence.
Mutants defective in yeast casein kinase I homologues
are unable to internalize
-factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these
kinases play a direct or indirect role in phosphorylating
the receptor. Finally, we provide evidence that the primary function of phosphorylation controlled by the
SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent
endocytosis is an important mechanism for the downregulation of GPC receptor activity.
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Introduction |
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CELL surface receptors coupled to heterotrimeric G proteins receive and transmit extracellular signals into the interior of the cell through the recognition and binding of specific ligands. Once cells receive and act upon a signal transmitted through receptor-ligand binding, they must return to a basal, unstimulated state for the appropriate regulation of growth and differentiation. Cells become desensitized to a signal and downregulate their response to it by a variety of mechanisms. Several components involved in initiating signal transduction are targets of downregulation, including the heterotrimeric G protein and the G protein-coupled (GPC)1 receptor itself. These components are generally downregulated by either modification and/or degradation.
Both G protein subunits and receptors become phosphorylated in response to ligand binding and this modification plays an important role in signal desensitization
(Cole and Reed, 1991; Lefkowitz, 1993
). Phosphorylation
of GPC receptors promotes desensitization by both uncoupling the receptor from its heterotrimeric G protein (for review see Dohlman et al., 1991
) and by facilitating
receptor internalization (Ferguson et al., 1995
; Naik et al.,
1997
; Pals-Rylaarsdam and Hosey, 1997
). However, although most G protein-coupled receptors undergo ligand-stimulated phosphorylation, the role of phosphorylation in
receptor desensitization varies. In addition, the signals
that stimulate GPC receptor internalization, and the fate
of the protein once it enters the cell, differ from receptor
to receptor.
The downregulation of the G protein-coupled -factor
receptor of Saccharomyces cerevisiae is triggered by a
novel internalization signal that requires modification of
the receptor tail with the polypeptide ubiquitin (Hicke and
Riezman, 1996
). The
-factor receptor (Ste2p), which is
expressed on the surface of a cells, stimulates the mating
response pathway upon binding the 13-amino acid pheromone secreted by
cells (for review see Bardwell et al.,
1994
). This receptor is constitutively internalized and degraded in the lysosome-like vacuole in the absence of
ligand, and its internalization rate is stimulated ~10-fold
in the presence of pheromone. Ligand-stimulated internalization also results in transport of the receptor to the vacuole; there is no evidence that Ste2p recycles from endosomes to the plasma membrane (Jenness et al., 1986
;
Singer and Riezman, 1990
; Schandel and Jenness, 1994
).
Ste2p is modified in two ways in response to
-factor binding: (a) its cytoplasmic tail becomes hyperphosphorylated
on serine and threonine residues, and (b) it is ubiquitinated on lysine residues (Reneke et al., 1988
; Hicke and
Riezman, 1996
).
Ubiquitination of the -factor receptor signals its ligand-
stimulated entry into the endocytic pathway. The role of
internalization signal is a new function for ubiquitin, a
highly conserved 76-amino acid polypeptide that has been
well characterized as a recognition tag for the degradation
of intracellular proteins by the multisubunit proteolytic
particle known as the 26S proteasome (for review see Ciechanover, 1994
; Hochstrasser, 1996
). Ubiquitin serves
as an internalization signal for multiple plasma membrane
proteins in yeast (Galan et al., 1996
; Roth and Davis, 1996
;
Kölling and Losko, 1997
), and ubiquitin-dependent internalization has also been shown to occur in mammalian
cells to promote the downregulation of a tyrosine kinase
signaling receptor (Strous et al., 1996
).
In previous work we observed that the mutation of
serine residues within a well-defined -factor receptor internalization signal, the SINNDAKSS sequence, abrogated the ability of this sequence to be ubiquitinated and
to promote receptor internalization (Hicke and Riezman,
1996
). In this paper we report that receptor phosphorylation
controlled by the SINNDAKSS serines promotes receptor internalization. Phosphorylation of the receptor cytoplasmic tail positively regulates ubiquitination at neighboring
lysines and these modifications are required for both constitutive and stimulated receptor internalization.
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Materials and Methods |
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Strains, Plasmids, Media, and Reagents
Mutations were introduced into the wild-type or 345Stop truncated form
of Ste2p as described (Rohrer et al., 1993) except Pfu DNA polymerase
(Stratagene, La Jolla, CA) was used. The sequence of the PCR-amplified
part of the resulting plasmids was determined to ensure that the expected
mutations had been introduced and not any others. Plasmids containing
the STE2 variants were introduced into the ura3 locus of strain RH3162
by single-step gene transplacement. The mutant Ste2 proteins were each
able to complement the ste2
mating defect of the parent strain. Two individual transformants of each mutant were assayed for their ability to internalize
-factor and in each case both transformants demonstrated similar
internalization kinetics.
The yck1 yck2-2 strain was generously provided by L. Robinson
(Louisiana State University Medical Center, New Orleans, LA) and was
crossed twice to our wild-type background to generate strains RH3589
(yck1
yck2-2) and RH3992 (YCK1 YCK2). The genotypes of all strains
used for the experiments described in this paper are listed in Table I.
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YPUAD rich medium and SD minimal medium have been described
(Zanolari et al., 1992; Hicke and Riezman, 1996
). SD-no phos buffer was
SD medium with KCl substituted for potassium phosphate and buffered
with 7 mM succinate, pH 5.8. SD-low phos buffer was SD-no phos buffer
with the addition of 50 mM KH2PO4.
Protein phosphatase 1 was obtained from Boehringer Mannheim
GmbH (Mannheim, Germany) and calf intestinal alkaline phosphatase (CIP) was from New England Biolabs Inc. (Beverly, MA). EXPRE35S35S
protein labeling mix was from New England Nuclear Life Science Products (Boston, MA), H332PO4 and Tran35SLabel were from ICN Pharmaceuticals Inc. (Irvine, CA), and H235SO4 was from Amersham Pharmacia
Biotech. Inc. (Piscataway, NJ). The purification of 35S-labeled -factor
(Singer and Riezman, 1990
; Dulic et al., 1991
) and of affinity-purified Ste2p
antiserum (Hicke and Riezman, 1996
) have been described previously.
-Factor Internalization Assays
All assays were performed as previously described (Dulic et al., 1991) on
strains that were propagated overnight in YPUAD. Briefly, cells were
grown at 24° or 30°C to a density of 0.5-2 × 107 cells/ml. Cells were harvested, washed in YPUAD, and then resuspended to 5 × 108 cells/ml in
ice-cold YPUAD. 35S-labeled
-factor was added and allowed to bind to
cells for 45 min on ice. Unbound pheromone was removed by centrifugation at 4°C, and cells were resuspended to 5 × 108 cells/ml in YPUAD prewarmed to 30°C. Aliquots of cells were withdrawn after different times,
washed in pH 1.0 buffer to remove surface-bound
-factor, or in pH 6.0 buffer, filtered, and then the amount of cell-associated radioactivity was
determined by scintillation counting. To assay internalization in the end4
and yck1
yck2-2 mutant strains, cells were harvested and washed as
above, then resuspended in 37°C YPUAD and incubated for 15 min at
37°C. 35S-labeled
-factor was added and the assay was performed as described above. A time course of internalization was generated for each strain by expressing the amount of internalized
-factor as a ratio of cpm
detected in pH 1.0-washed cells to that detected in pH 6.0-washed cells at
each time point.
Receptor Clearance Assays
The measurement of receptors cleared from the cell surface in the absence
of -factor was performed as described (Jenness and Spatrick, 1986
; Rohrer et al., 1993
) with the following modifications: cells were propagated
as described for internalization assays, collected by centrifugation, and
then resuspended in YPUAD to 5 × 106 cells/ml. After incubation for 5 min at 30°C, cycloheximide was added to 20 µg/ml to inhibit new receptor
synthesis and then incubation was continued at 30°C. To measure ligand-stimulated receptor clearance,
-factor was added to a final concentration
of 10
6 M. At different time points, aliquots of 5 × 107 cells were removed
and then filtered onto nitrocellulose disks. The filters were incubated in
YPUAD with 10 mM NaN3 and 10 mM NaF to remove
-factor that remained bound to cell surface receptors. The filters were removed and the cell
suspension was centrifuged, resuspended in YPUAD/10 mM NaN3 with 10 mM NaF, and then incubated with 35S-labeled
-factor alone or 35S-labeled
-factor plus 4 × 10
5 M unlabeled
-factor. Receptor clearance assays
were performed two or three times for each experiment. Although the rate
of clearance for each strain varied slightly from experiment to experiment, the differences in clearance rates between strains were reproducible.
-Factor Recovery Assays
-Factor recovery assays were performed in a manner similar to that described previously (Rohrer et al., 1993
). Briefly, cells were propagated
overnight in YPUAD at 24°C to ~107 cells/ml, diluted to 2 × 106 cells/ml
in YPUAD, and then incubated with 2.5 × 10
8 M synthetic
-factor at
30°C for 2 h to arrest cells in the G1 phase of the cell cycle. Cells arrest at
this phase of the cell cycle without buds. Cells were washed with medium
harvested from a MATa yeast culture that contained the secreted Bar1
protease. Bar1p degrades
-factor and this medium was used to remove
all pheromone bound to the arrested cells. Washed cells were resuspended in the same volume of YPUAD used for
-factor incubation and
then incubated at 30°C. To measure recovery from the pheromone-
induced G1 arrest, the number of budded cells in each culture was determined by counting cells in a hemacytometer at various times after resuspension.
Immunoblots and Immuneprecipitations
Immunoblots on end4, ste2
, and wild-type cell extracts to detect the
-factor receptor were carried out as described previously (Hicke and
Riezman, 1996
). The immunoblot performed on extracts of end4-1 and
yck1
yck2-2 cells was done as described except cells were preincubated
for 15 min at 37°C before harvesting the no
-factor sample. Incubation
was continued at 37°C after the addition of
-factor.
For the phosphatase treatment of radiolabeled -factor receptors, immuneprecipitations of receptors from cell extracts prepared before and after exposure to
-factor were performed as described (Hicke and Riezman, 1996
), except only a single round of precipitation was done, and the
washed immuneprecipitates were not immediately eluted from protein
A-Sepharose beads. For treatment with CIP, the precipitates were washed
twice in CIP buffer (50 mM Tris-HCl, pH 8.6, 1 mM MgCl2, 1 mM PMSF,
1 µg/ml each of pepstatin, antipain, and leupeptin), resuspended in 50 µl
of the same buffer, and then incubated with 30 U CIP for 1 h at 37°C. For
treatment with protein phosphatase 1, the beads were washed with protein
phosphatase 1 buffer (50 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, 5 mM DTT,
0.2 mM MnCl2, 200 µg/ml BSA, 1 mM PMSF, 1 µg/ml each of pepstatin,
antipain, and leupeptin), resuspended in 30 µl of the same buffer, and then incubated with 1 mU protein phosphatase 1 for 30 min at 30°C. After the phosphatase treatment, the precipitated proteins were eluted from the beads in
9 M urea, 40 mM Tris-HCl, pH 6.8, 0.1 mM EDTA, 5% SDS by heating
for 10 min at 37°C and then resolved by SDS-PAGE on 10 or 12.5% acrylamide gels. The proteins were visualized by exposure of the dried gel to
X-OMAT film (Eastman Kodak Co., Rochester, NY).
Parallel radiolabeling of strains with H332PO4 and Tran35SLabel was
performed as described previously (Zanolari et al., 1992) with the following exceptions: After incubation in SD-low phos buffer, cells were harvested, washed in SD-no phos, and then split into two aliquots. One aliquot was resuspended in 2 ml of SD-no phos and subsequently labeled
with 1 mCi H332PO4; the other aliquot was resuspended in 2 ml SD-low
phos and then radiolabeled with 100 µCi Tran35SLabel. Radiolabeling was
performed for 1 h and then each labeled sample was split into two equal
volumes. One volume was immediately transferred to a tube containing a
one-tenth vol of 10×Stop and then frozen in liquid N2. The other volume
was treated with 10
6 M synthetic
-factor for 10 min at 30°C and then collected in the same manner. After lysis of cells in each sample by mechanical agitation with glass beads in 100 µl of breaking buffer (9 M urea, 40 mM Tris-HCl, pH 6.8, 0.1 mM EDTA, 140 mM 2-mercaptoethanol), an
additional 100 µl of breaking buffer and SDS to 1% were added, and then
the extracts were heated to 37°C for 10 min. Immuneprecipitations of receptors from these extracts were performed as described. Radiolabeled
receptors were detected using a Storm 860 PhosphorImager (Molecular
Dynamics, Inc., Sunnyvale, CA).
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Results |
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Serines and Lysine Are Essential Components of the SINNDAKSS Internalization Signal Required for Both Constitutive and Ligand-stimulated Endocytosis
A truncated version of Ste2p (345Stop) that has lost approximately two-thirds of its cytoplasmic tail, yet is internalized rapidly in response to pheromone, carries a single
well-defined internalization signal (SINNDAKSS) that is
both necessary and sufficient for receptor endocytosis
(Rohrer et al., 1993). The sequence of the truncated receptor
tail is shown in Fig. 1. Ubiquitination of the SINNDAKSS
lysine (K337) is required to trigger 345Stop receptor internalization (Hicke and Riezman, 1996
). Mutation of single
serine residues within the SINNDAKSS sequence leads to small effects on the rate of receptor internalization (Rohrer et al., 1993
) and simultaneous mutation of all three
SINNDAKSS serines to alanine severely retards ligand-stimulated receptor internalization, indicating that these
residues serve a redundant function within the internalization sequence. These serines are required for internalization because they promote ubiquitination at K337 in the
SINNDAKSS sequence (Hicke and Riezman, 1996
). Like the wild-type receptor, the 345Stop receptor undergoes
slow constitutive endocytosis in the absence of pheromone.
To determine whether the serines and lysine within the
SINNDAKSS sequence also mediate constitutive internalization of the receptor, we assayed the ability of mutant receptors carrying the triple serine to alanine mutation 3S
A, 345Stop (S331A, S338A, S339A, 345Stop), or a
mutation of K337 to arginine (K337R, 345Stop), to be
cleared from the cell surface in the absence of
-factor.
The receptor clearance assay we used measured the number of
-factor binding sites that remained on the cell surface after cells were treated with cycloheximide to inhibit
new receptor synthesis. The 345Stop truncated receptor was constitutively internalized with a half-time of ~1 h. In
contrast, both the K337R and 3S
A variants of this receptor were not internalized constitutively (Fig. 2 A). These
data suggested that modification of the 345Stop receptor
tail with ubiquitin was required for constitutive internalization. Previously we demonstrated that ubiquitinated forms of Ste2p accumulated in response to
-factor stimulation in end4
mutant cells that cannot internalize proteins from the plasma membrane. To test whether Ste2p is
ubiquitinated in the absence of
-factor, we performed immunoblots on extracts prepared from wild-type and end4
cells expressing full-length receptor that had never been
exposed to pheromone. Fig. 2 B shows that higher molecular weight forms of Ste2p were detected in end4
cells.
These forms were previously shown to be ubiquitinated
Ste2p by demonstrating that they were diminished in cells
that lack ubiquitin-conjugating machinery and that they
specifically precipitated with antiubiquitin antiserum (Hicke
and Riezman, 1996
). Ubiquitinated Ste2p forms were absent in ste2
cells that did not express the receptor and
were observed at a very low level in wild-type cells. The
detection of ubiquitinated full-length Ste2p in unstimulated cells suggests that ubiquitination can signal constitutive internalization of the full-length
-factor receptor as
well as that of the 345Stop truncated receptor.
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Serines within the SINNDAKSS Internalization Signal Are Required for Constitutive Receptor Phosphorylation, Which Is a Prerequisite for Ligand-stimulated Hyperphosphorylation and Ubiquitination
Serine phosphorylation has been shown to positively regulate the ubiquitination of cytosolic proteins, such as the cyclins and the transcription factor inhibitor IB, which undergo stimulated or regulated modification with ubiquitin
(Chen et al., 1995
; Deshaies et al., 1995
; Yaglom et al.,
1995
). The
-factor receptor is known to be constitutively
phosphorylated and to become hyperphosphorylated in
response to
-factor binding (Reneke et al., 1988
). These
two observations led to the idea that the SINNDAKSS
serines may be required for receptor internalization because they are sites of phosphorylation that regulate ubiquitination at the SINNDAKSS lysine (Hicke and Riezman, 1996
). To test whether mutations in these serines
affect phosphorylation of the 345Stop truncated receptor,
we immuneprecipitated different variants of the 345Stop receptor before and after exposure to
-factor, and then
treated the immuneprecipitates with protein phosphatase
(Fig. 3). The mobility of the 345Stop truncated receptor
that had not been exposed to
-factor was increased upon
incubation with protein phosphatase 1 (Fig. 3, lanes 1 and 3),
an enzyme that dephosphorylates serine and threonine
residues. This indicated that the truncated receptor was constitutively phosphorylated. The dephosphorylated
345Stop receptor migrated as a doublet, as does the full-length receptor, perhaps due to heterogeneous glycosylation of the protein (Blumer et al., 1988
; Konopka et al.,
1988
; David et al., 1997
). Upon binding pheromone, several new species of the 345Stop receptor with decreased
mobility appeared (Fig. 3, lane 2). These new species were
due to serine/threonine hyperphosphorylation because the pheromone-stimulated receptor was reduced to the same
relative mobility as unstimulated receptor by incubation
with protein phosphatase (Fig. 3, lane 4). Like the 345Stop
receptor, the K337R, 345Stop receptor was phosphorylated constitutively and in response to
-factor (Fig. 3,
lanes 5-8). Since the K337R, 345Stop receptor is unable to
be ubiquitinated, this observation indicates that ubiquitination is not required for receptor phosphorylation. In contrast, the 3S
A, 345Stop receptor migrated with a
lower molecular weight than the 345Stop receptor in both
the absence and presence of
-factor (Fig. 3, lanes 9 and
10). In addition, the mobility of the 3S
A, 345Stop receptor did not shift upon incubation with phosphatase in either case (Fig. 3, lanes 11 and 12). These results indicated
that the 3S
A, 345Stop receptor was not phosphorylated constitutively or in response to pheromone binding.
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To test the role of the SINNDAKSS serines in the full-length Ste2p cytoplasmic tail, which contains a total of fifteen serines and nineteen threonines, we mutated these
serines in wild-type Ste2p and then tested the ability of the
3S A receptor (S331A, S338A, S339A) to be internalized
in the absence and presence of
-factor. Fig. 4, A and B
shows assays that measured the ability of wild-type receptors and 3S
A receptors to be cleared from the cell surface in the absence and presence of pheromone. The 3S
A mutations reduced the rate of constitutive internalization of the full-length receptor approximately threefold
(Fig. 4 A). These mutations reduced the rate of ligand-stimulated receptor removal from the cell surface to a
small extent (Fig. 4 B). A direct assay of
-factor ligand internalization confirmed the slower rate of stimulated endocytosis by the 3S
A receptor (Fig. 4 C). Receptor
clearance assays measure the number of receptors present
at the cell surface that have not been internalized, in addition to those that may have been returned to the cell surface by recycling. The wild-type
-factor receptor is not recycled after internalization (Jenness and Spatrick, 1986
;
Schandel and Jenness, 1994
); however, it was possible that
a mutation in the receptor may divert it into a putative recycling pathway. The almost complete clearance of the
3S
A receptor from the cell surface in the presence of
-factor (Fig. 4 B) suggested that these mutations did not
induce the receptor to recycle but impaired its ability to be
internalized. In addition, the 3S
A receptor that was constitutively cleared from the cell surface was degraded (data not shown) and not accumulated intact in the cell as
would be expected for a receptor on a recycling pathway.
Therefore, the SINNDAKSS serines are important regulators of internalization within the wild-type tail, primarily
in the absence of ligand. There may be additional serines
distal to amino acid 345 that also serve this function.
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Negatively Charged Amino Acids Partially Substitute for Serines in the SINNDAKSS Internalization Signal
The inability of the 3S A, 345Stop mutant to be phosphorylated suggested that the three SINNDAKSS serines
are sites of phosphorylation that positively regulate ubiquitination and internalization. To determine whether negatively charged amino acids that might mimic phosphorylated serines substitute for the SINNDAKSS serines in regulating internalization, we mutated these serines to aspartate or glutamate (refer to Fig. 1). Fig. 5 A shows
-factor internalization assays on cells expressing the 3S
D,
345Stop, 3S
E, 345Stop, or 3S
A, 345Stop mutants. The
mutations to aspartate or glutamate partially restored the
ability of the 345Stop receptor to be internalized in response to pheromone binding. We then assayed the ability of the 3S
E, 345Stop mutant to be internalized in the absence of pheromone. Fig. 5 B indicates that this mutation
did not restore constitutive internalization. The same result was observed for the 3S
D, 345Stop receptor (data
not shown). To determine why the substitution of glutamate for serine in the SINNDAKSS sequence restored
stimulated endocytosis, but not constitutive endocytosis, we analyzed the phosphorylation state of the 3S
E,
345Stop receptor by treating radiolabeled immuneprecipitated receptors with phosphatase. Fig. 6 A shows that, unlike the 345Stop receptor, the unstimulated 3S
E, 345Stop
receptor migrated with a similar, though not identical, mobility before and after incubation with alkaline phosphatase, indicating that this receptor was not constitutively
phosphorylated to the same extent as the 345Stop receptor
(Fig. 6 A, compare lanes 1 and 5). After treatment with
-factor, higher molecular weight forms of the 3S
E,
345Stop receptor appeared (Fig. 6 A, lane 2). The size of
these higher molecular weight forms of 3S
E, 345Stop
receptor decreased upon treatment with phosphatase (Fig.
6 A, lane 8), indicating that these forms were phosphorylated.
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A more quantitative measurement of the relative phosphorylation state of the 3S E, 345Stop, 3S
A, 345Stop,
and 345Stop receptors was obtained by radiolabeling cells
expressing the different receptors with H332PO4 followed
by immuneprecipitation of the phosphorylated receptors. To compare the levels of receptors expressed in the different strains, identical aliquots of each culture were also radiolabeled with 35[S]methionine. Fig. 6 B shows the 32P-labeled
and 35S-labeled receptors isolated before and after treatment with pheromone. The 345Stop receptor was labeled
strongly with radioactive phosphate in the presence and
absence of
-factor (Fig. 6 B, lanes 3 and 4). The 3S
A,
345Stop receptor was not labeled significantly with phosphate (Fig. 6 B, lanes 5 and 6), confirming that this receptor
underwent neither constitutive or stimulated phosphorylation. The 3S
E, 345Stop receptor was not phosphorylated as heavily as the 345Stop receptor, although phosphorylation increased upon binding of
-factor (Fig. 6 B,
lanes 1 and 2). The level of 3S
E, 345Stop receptor expression, however, as measured by the amount of 35S-labeled
receptor precipitated from each strain, appeared to be
lower that than of the other receptors. To quantify the relative levels of phosphorylation, the amount of 32P-labeled
receptor detected was normalized to the level of 35S-labeled
receptor precipitated from the same strain (Fig. 6 C). These
data indicate that the 3S
E, 345Stop receptor was constitutively phosphorylated approximately twofold less than
the 345Stop receptor, and underwent ligand-stimulated
phosphorylation to a level that was ~75% of that of the
stimulated 345Stop receptor. These observations suggest
that constitutive phosphorylation, or negatively charged amino acids, in the SINNDAKSS sequence are required
for phosphorylation at non-SINNDAKSS serines. The
stimulated phosphorylation that occurs at non-SINNDAKSS
serines in the 3S
E mutant can explain the ability of this
receptor to undergo ligand-stimulated internalization.
-Factor Receptor Modification and Internalization
Requires Yeast Casein Kinase I Activity
The kinase(s) that phosphorylates the -factor receptor
has eluded identification. However, Panek and co-workers
have recently demonstrated that constitutive internalization of the a-factor receptor (Ste3p), which is also phosphorylated and ubiquitinated (Roth and Davis, 1996
), is
blocked in a mutant that lacks activity of the yeast casein
kinase I homologues (Yck1p and Yck2p) (Panek et al.,
1997
). To determine whether mutations in the YCK genes also affect Ste2p internalization, we assayed the ability of a mutant that lacks the YCK1 gene and carries a temperature-sensitive allele of the YCK2 gene, yck2-2, to internalize
-factor. Fig. 7 A shows pheromone internalization assays performed on wild-type and yck1
yck2-2 cells after
incubation at 24° or 37°C. Wild-type cells internalized
-factor rapidly and to a similar extent at both temperatures.
yck1
yck2-2 cells internalized
-factor slowly at 24°C and
not at all at 37°C, similar to the behavior of end4 cells that
are defective in endocytosis.
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We then tested whether the Yck kinases are required to
phosphorylate and ubiquitinate Ste2p. Fig. 7 B shows an
immunoblot of extracts prepared from yck1 yck2-2 and
end4 cells before and after the addition of
-factor at 37°C.
Phosphorylated and ubiquitinated forms of the stimulated
receptor accumulated in end4 mutants that modify the receptor but are unable to internalize and degrade the protein (Hicke and Riezman, 1996
) (Fig. 7 B, lane 4). In contrast, modified forms of the stimulated receptor were not
observed in the yck1
yck2-2 mutant even though the receptor was not internalized. The mobility of the receptor
expressed in this mutant was affected very little by the
binding of
-factor (Fig. 7 B, lanes 1 and 2), indicating that
the receptor was phosphorylated to only a small extent. To
confirm that receptor phosphorylation was deficient in the
yck1
yck2-2 mutant, we immuneprecipitated receptors
from end4 and yck1
yck2-2 mutants incubated at the
nonpermissive temperature and then treated the precipitated receptors with protein phosphatase I. Fig. 7 C shows
that the mobility of receptors expressed in end4 cells before exposure to
-factor increased upon phosphatase treatment, indicating that the receptor was constitutively
phosphorylated (Fig. 7 C, lanes 5 and 7). In response to
-factor binding the mobility of the receptor decreased
(Fig. 7 C, lane 6). This mobility shift was due to phosphorylation because treatment of stimulated receptor with
phosphatase increased its mobility (Fig. 7 C, lane 8). The
receptor precipitated from yck1
yck2-2 cells was not constitutively phosphorylated (Fig. 7 C, lanes 1 and 3) because there was very little mobility shift upon treatment of unstimulated receptor with phosphatase. The mobility of the
receptor shifted slightly upon pheromone binding to
-factor at the nonpermissive temperature (Fig. 7 C, lane 2),
and this shift was reversed by incubation with phosphatase
(lane 4). Thus, there was some increase in receptor phosphorylation in response to ligand binding, but to a much
smaller extent than in end4 cells. These data demonstrate that receptor phosphorylation is severely compromised in
yck1
yck2-2 cells. The Yck proteins are required for
phosphorylation and ubiquitination of the wild-type
-factor receptor and its internalization, demonstrating that in
the absence of phosphorylation, the receptor is not internalized even when it carries an intact SINNDAKSS sequence.
The Primary Function of SINNDAKSS-dependent Phosphorylation in Signal Downregulation Is to Promote Receptor Internalization
The phosphorylation of GPC receptors has been proposed
to function in the activation and downregulation of signal
response. Because -factor receptor lacking its cytoplasmic tail is capable of transducing signal, the phosphorylation of receptor tail serines is not required for signal transmission (Konopka et al., 1988
; Reneke et al., 1988
). Instead,
we have presented evidence demonstrating that the phosphorylation of serines within the Ste2p cytoplasmic tail is
required for receptor internalization. It has been previously shown that internalization of Ste2p is a primary
mechanism by which cells recover from
-factor stimulation. Cells expressing the K337R, 345Stop receptor, which
is unable to be endocytosed, recover from
-factor-induced
growth arrest much more slowly than cells expressing the
345Stop receptor. However, these cells do eventually recover (Fig. 8; Rohrer et al., 1993
). It is possible that phosphorylation of the SINNDAKSS serines functions in signal downregulation not only by stimulating receptor
internalization but by other phosphorylation-dependent
mechanisms (for example see Chen and Konopka, 1996
).
To test whether the SINNDAKSS serines promote signal
downregulation by mechanisms other than receptor internalization, we analyzed the ability of cells expressing the 3S
A, 345Stop receptor, which is internalization defective, to recover from the cell cycle arrest induced by exposing MATa cells to
-factor pheromone. We incubated cultures of cells expressing different receptors with
-factor
to activate the growth arrest that occurs in cells stimulated
with pheromone.
-Factor was then washed away from the
cells and then the number of budded cells in the culture
was counted at different times after wash-out as an assay
of recovery from growth arrest. Fig. 8 shows that cells expressing the 345Stop receptor were fully recovered
(>95% of budded cells) 1 h after
-factor had been removed. Cells expressing the K337R, 345Stop receptor recovered very slowly. They were ~70% budded after 4 h.
Expression of the 3S
A, 345Stop receptor also allowed
cells to recover slowly (Fig. 8). Expression of a 3S
A,
K337R, 345Stop receptor, which is unable to be phosphorylated or ubiquitinated and is not internalized at all, resulted in recovery with the same kinetics as that observed
for the K337R, 345Stop receptor (data not shown). These
data indicated that the SINNDAKSS serines were not required for the recovery that occurred independent of receptor internalization observed with the K337R mutant.
The recovery of cells expressing the 3S
A receptor was
only slightly faster than the recovery of cells expressing
the K337R mutant. Internalization of the 3S
A receptor
was also slightly faster than that of K337R (Hicke and
Riezman, 1996
). The 3S
E mutations, which partially restored ligand-stimulated receptor endocytosis, also largely
restored recovery from
-factor arrest (Fig. 8). The effect
of mutations in the SINNDAKSS serines on
-factor recovery mirror their effects on
-factor internalization, supporting the contention that the primary, if not the only,
function of the SINNDAKSS serines is to downregulate
the
-factor signal by internalization of Ste2p through internalization into the endocytic pathway.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this paper we present evidence that phosphorylation of
the G protein-coupled -factor receptor positively regulates receptor internalization by promoting the ubiquitination of lysine residues within the receptor cytoplasmic tail.
We showed previously that mutation of the three serines
within the SINNDAKSS internalization signal in a truncated receptor abolishes the ability of the receptor to be
ubiquitinated and internalized (Hicke and Riezman,
1996
). Now we demonstrate that these three serine residues
are required for constitutive phosphorylation and that this,
in turn, is required for the truncated receptor to undergo
ligand-induced hyperphosphorylation. The SINNDAKSS
serines are required for both constitutive and ligand-stimulated internalization of the truncated receptor.
Phosphorylation of serine residues, rather than just their
presence, is required for receptor ubiquitination and endocytosis because the yck1 yck2-2 mutant, which is severely
compromised in its ability to phosphorylate the
-factor
receptor, cannot ubiquitinate or internalize wild-type receptor even though it carries an intact SINNDAKSS sequence. This conclusion is also supported by the analysis
of a receptor in which the SINNDAKSS serines have been
replaced with negatively charged glutamates. Glutamate
replacements of the serines restores ligand-stimulated phosphorylation to a level of ~75% of that of the 345Stop receptor. The replacements also partially rescue ligand-stimulated internalization of the receptor, presumably by
facilitating ligand-induced phosphorylation on residues
outside the SINNDAKSS sequence. Although we have
not directly demonstrated that the SINNDAKSS serines
are sites of phosphorylation, it is likely that one or more of
these residues are constitutively phosphorylated because
negatively charged amino acids substitute for their function in promoting ligand-stimulated phosphorylation. The
3S
E, 345Stop mutant was constitutively phosphorylated
on non-SINNDAKSS serines at a low level, but it was not
constitutively internalized. The constitutive phosphorylation level of this mutant may be below the threshold level
required to promote internalization, or the 3S
E mutations may shift constitutive phosphorylation to residues
that are not normally modified and may not function efficiently to promote receptor internalization.
The SINNDAKSS serines regulate internalization not only in the 345Stop truncated receptor but also in the wild-type tail, most likely through a similar mechanism. Mutation of the three SINNDAKSS serines to alanines in the full-length receptor leads to a defect in constitutive internalization even though the receptor tail carries numerous serine (15) and threonine (19) residues, and can be ubiquitinated at many of its eight tail lysines.
We propose that the following series of events controls
internalization of the -factor receptor from the plasma
membrane into the cell. The receptor is constitutively
phosphorylated on serine residues. Constitutive phosphorylation and ubiquitination mediate the slow constitutive
uptake of the receptor. Upon stimulation, the receptor becomes hyperphosphorylated and modified further with
ubiquitin. Phosphorylation precedes and regulates ubiquitination at the SINNDAKSS lysine because ubiquitination at this lysine does not occur in a mutant lacking the
SINNDAKSS serines. In contrast, phosphorylation is normal in a mutant lacking the ubiquitination site. Our results
demonstrate that constitutive and stimulated internalization are mediated not by different signals but by modulating the level of the same signal, phosphorylation-dependent
ubiquitination. It is not known how receptor phosphorylation effects an increase in receptor ubiquitination. Phosphorylated serines may recruit ubiquitination machinery
to the receptor by providing favorable sites of interaction
with ubiquitin-conjugating enzymes or a ubiquitin protein
ligase.
Ligand-stimulated phosphorylation may occur at sites
that become available for modification due to the conformation change in the receptor induced by pheromone
binding (Bukusoglu and Jenness, 1996). Negative charge
in the SINNDAKSS sequence, provided either by constitutively phosphorylated serines or by glutamate, may be
required for this conformational change. Alternatively,
negative charge in the SINNDAKSS sequence may facilitate recognition of the receptor by its kinase. Kinases that
phosphorylate Ste2p have not been identified. S. cerevisiae
does not carry homologues of the GPC receptor kinases
(GRKs) that phosphorylate many mammalian GPC receptors, and the Npr1 kinase that appears to be involved in
the ubiquitin-dependent downregulation of the general
amino acid permease (Vandenbol et al., 1990
; Hein et al.,
1995
) is not required for
-factor receptor endocytosis
(our unpublished data). However, we have shown that
mutants lacking casein kinase I activity do not constitutively phosphorylate the receptor. Since constitutive phosphorylation is required for both constitutive and ligand- induced internalization, this can explain the inability of the yck1
yck2-2 mutant to internalize
-factor. The Yck kinases may directly phosphorylate the receptor or they may
phosphorylate another protein that regulates receptor
phosphorylation. The Yck proteins may also phosphorylate other components of the endocytic machinery (Panek
et al., 1997
).
Although several functions have been proposed for
GPC receptor phosphorylation, we show here that phosphorylation at or near the membrane proximal SINNDAKSS
sequence of the Ste2p tail functions to promote receptor
internalization. The SINNDAKSS serines probably do not
promote downregulation of Ste2p by mechanisms other
than receptor internalization because the 3S A, 345Stop
mutant recovers from pheromone stimulation with the
same slow kinetics as the internalization-defective K337R,
345Stop mutant. Our results do not rule out that phosphorylation of sites more distal, in the last quarter of the receptor tail, may function in an alternate mechanism of receptor downregulation (Chen and Konopka, 1996
).
Phosphorylation has been shown to regulate the ubiquitination of cytosolic proteins that are targeted for degradation by the proteasome. Serine phosphorylation positively regulates the ubiquitination of cyclins that undergo
regulated degradation at specific stages of the cell cycle
(Deshaies et al., 1995; Yaglom et al., 1995
). In addition,
the relationship between serine phosphorylation and the
ubiquitination and degradation of the transcription factor inhibitor, I
B, is well documented. I
B is a cytosolic protein that undergoes regulated degradation by the ubiquitin-proteasome pathway in response to a number of extracellular signals (Chen et al., 1995
; Roff et al., 1996
).
These signals induce the phosphorylation of specific serine
residues, which is required for the protein to be ubiquitinated at neighboring lysines (Brown et al., 1995
). Like the
cytosolic I
B, Ste2p also undergoes phosphorylation- dependent ubiquitination, indicating that this may be a
general mechanism for triggering the ubiquitination of both
cytosolic and membrane proteins that undergo regulated
ubiquitin-dependent destruction.
Many, if not all, eukaryotic GPC receptors are phosphorylated on serine residues in response to binding of their
cognate ligand. Phosphorylation of the 2 adrenergic receptor by kinases of the GRK family promotes its interaction with arrestin, which is required for internalization of
the protein (Ferguson et al., 1995
, 1996
; Goodman et al.,
1996
; Ménard et al., 1996
). Yeast do not have an obvious
arrestin homologue and we show here that phosphorylation of the
-factor receptor promotes internalization by
positively regulating receptor ubiquitination. The phosphorylation of some mammalian receptors appears to play
no role in their internalization (Holtmann et al., 1996
; Oppermann et al., 1996
). Thus, ligand-induced phosphorylation of GPC receptors is likely to have different functions from receptor to receptor, and the type and location of
phosphate modification may specify the mechanism(s) by
which a receptor is downregulated. Although the ubiquitin-dependent internalization of mammalian GPC receptors has not been described, a number of receptor tyrosine kinases undergo ligand-stimulated ubiquitination that may mediate stimulated internalization (Mori et al.,
1992
; Miyazawa et al., 1994
; Yee et al., 1994
; Galcheva-Gargova et al., 1995
; Strous et al., 1996
). A subset of mammalian GPC receptors may also be regulated by phosphorylation-dependent ubiquitination.
![]() |
Footnotes |
---|
Received for publication 27 June 1997 and in revised form 5 March 1998.
H. Riezman was funded by the Swiss National Science Foundation and the Canton Basel Stadt. L. Hicke was funded by a New Investigator Award in the Basic Pharmacological Sciences from the Burroughs Wellcome Foundation, a Lurie Cancer Center American Cancer Society Institutional Research grant (IRG-93-037-04-IRG), and the National Institutes of Health (1 R01 DK53257-01).We are grateful to L. Robinson for the yck1 yck2-2 strain and for communicating helpful discussion. We thank M. Geli (Biozentrum, University
of Basel, Basel, Switzerland) and A. Matouschek (Northwestern University, Evanston, IL) for improving the manuscript with their critical comments.
![]() |
Note Added in Proof |
---|
While this manuscript was under review, Marchal et al. (Marchal, C., R. Haguenauer-Tsapis, and D. Urban-Grimal. 1998. Mol. Cell. Biol. 18:314-321) described experiments suggesting that serine residues within a PEST-like sequence of the yeast uracil permease are required for the phosphorylation and ubiquitin-dependent internalization of the permease.
![]() |
Abbreviations used in this paper |
---|
CIP, calf intestinal alkaline phosphatase; GPC, G protein-coupled.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bardwell, L., J.G. Cook, C.J. Inouye, and J. Thorner. 1994. Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae. Dev. Biol. 166: 363-379 |
2. |
Blumer, K.J.,
J.E. Reneke, and
J. Thorner.
1988.
The STE2 gene product is the
ligand-binding component of the ![]() |
3. |
Brown, K.,
S. Gerstberger,
L. Carlson,
G. Franzoso, and
U. Siebenlist.
1995.
Control of I![]() ![]() |
4. |
Bukusoglu, G., and
D.D. Jenness.
1996.
Agonist-specific conformational changes
in the yeast ![]() |
5. |
Chen, Q., and
J.B. Konopka.
1996.
Regulation of the G-protein-coupled ![]() |
6. |
Chen, Z.,
J. Hagler,
V.J. Palombella,
F. Melandri,
D. Scherer,
D. Ballard, and
T. Maniatis.
1995.
Signal-induced site-specific phosphorylation targets I![]() ![]() |
7. | Ciechanover, A.. 1994. The ubiquitin-proteasome proteolytic pathway. Cell. 79: 13-21 |
8. |
Cole, G.M., and
S.I. Reed.
1991.
Pheromone-induced phosphorylation of a G
protein ![]() |
9. |
David, N.E.,
M. Gee,
B. Andersen,
F. Naider,
J. Thorner, and
R.C. Stevens.
1997.
Expression and purification of the Saccharomyces cerevisiae ![]() |
10. | Deshaies, R.J., V. Chau, and M. Kirschner. 1995. Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway. EMBO (Eur. Mol. Biol. Organ.) J. 14: 303-312 [Abstract]. |
11. | Dohlman, H.G., J. Thorner, M.G. Caron, and R.J. Lefkowitz. 1991. Model systems for the study of seven-transmembrane-segment receptors. Annu. Rev. Biochem. 60: 653-688 |
12. | Dulic, V., M. Egerton, I. Elguindi, S. Raths, B. Singer, and H. Riezman. 1991. Yeast endocytosis assays. Methods Enzymol. 194: 697-710 |
13. |
Ferguson, S.S.G.,
L. Ménard,
L.S. Barak,
W.J. Koch,
A.M. Colapietro, and
M.G. Caron.
1995.
Role of phosphorylation in agonist-promoted beta2-adrenergic receptor sequestration: Rescue of a sequestration-defective mutant receptor by beta-ARK1.
J. Biol. Chem.
270:
24782-24789
|
14. |
Ferguson, S.S.G.,
W.E. Downey III,
A.-M. Colapietro,
L.S. Barak,
L. Ménard, and
M.G. Caron.
1996.
Role of ![]() |
15. |
Galan, J.M.,
V. Moreau,
B. André,
C. Volland, and
R. Haguenauer-Tsapis.
1996.
Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase
is required for endocytosis of the yeast uracil permease.
J. Biol. Chem.
271:
10946-10952
|
16. | Galcheva-Gargova, Z., S.J. Theroux, and R.J. Davis. 1995. The epidermal growth factor receptor is covalently linked to ubiquitin. Oncogene. 11: 2649-2655 |
17. | Goodman, Jr., O.B., J.G. Krupnick, F. Santini, V.V. Gurevich, R.B. Penn, A.W.
Gagnon, J.H. Keen, and J.L. Benovic. 1996. ![]() ![]() |
18. | Hein, C., J.-Y. Springael, C. Volland, R. Haguenauer-Tsapis, and B. André. 1995. NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol. Microbiol. 18: 77-87 |
19. | Hicke, L., and H. Riezman. 1996. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell. 84: 277-287 |
20. | Hochstrasser, M.. 1996. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30: 405-439 |
21. |
Holtmann, M.H.,
B.F. Roettger,
D.I. Pinon, and
L.J. Miller.
1996.
Role of receptor phosphorylation in desensitization and internalization of the secretin
receptor.
J. Biol. Chem.
271:
23566-23571
|
22. |
Jenness, D.D., and
P. Spatrick.
1986.
Down regulation of the ![]() |
23. |
Jenness, D.D.,
A.C. Burkholder, and
L.H. Hartwell.
1986.
Binding of ![]() |
24. |
Kölling, R., and
S. Losko.
1997.
The linker region of the ABC-transporter Ste6
mediates ubiquitination and fast turnover of the protein.
EMBO (Eur. Mol.
Biol. Organ.) J.
16:
2251-2261
|
25. |
Konopka, J.B.,
D.D. Jenness, and
L.H. Hartwell.
1988.
The C-terminus of the S. cerevisiae ![]() |
26. | Lefkowitz, R.J.. 1993. G protein-coupled receptor kinases. Cell. 74: 409-412 |
27. | Ménard, L., S.S. Ferguson, L.S. Barak, L. Bertrand, R.T. Premont, A.M. Colapietro, R.J. Lefkowitz, and M.G. Caron. 1996. Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration. Biochemistry. 35: 4155-4160 |
28. |
Miyazawa, K.,
K. Toyama,
A. Gotoh,
P.C. Hendrie,
C. Mantel, and
H.E. Broxmeyer.
1994.
Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells.
Blood.
83:
137-145
|
29. |
Mori, S.,
C.-H. Heldin, and
L. Claesson-Welsh.
1992.
Ligand-induced polyubiquitination of the platelet-derived growth factor ![]() |
30. |
Naik, N.,
E. Giannini,
L. Brouchon, and
F. Boulay.
1997.
Internalization and recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated internalization is modulated by phosphorylation of the C-terminal domain.
J. Cell Sci.
110:
2381-2390
|
31. |
Oppermann, M.,
N.J. Freedman,
R.W. Alexander, and
R.J. Lefkowitz.
1996.
Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C.
J. Biol. Chem.
271:
13266-13272
|
32. |
Pals-Rylaarsdam, R., and
M. Hosey.
1997.
Two homologous phosphorylation
domains differentially contribute to desensitization and internalization of
the m2 muscarinic acetylcholine receptor.
J. Biol. Chem.
272:
14152-14158
|
33. |
Panek, H.,
J. Stepp,
H. Engle,
K. Marks,
P. Tan,
S. Lemmon, and
L. Robinson.
1997.
Suppressors of YCK-encoded yeast casein kinase 1 deficiency define
the four subunits of a novel clathrin AP-like complex.
EMBO (Eur. Mol.
Biol. Organ.) J.
16:
4194-4204
|
34. |
Reneke, J.E.,
K.J. Blumer,
W.E. Courchesne, and
J. Thorner.
1988.
The carboxy-terminal segment of the yeast ![]() |
35. |
Roff, M.,
J. Thompson,
M.S. Rodriguez,
J.-M. Jacque,
F. Baleux,
A.-M. Seisdedos, and
R.T. Hay.
1996.
Role of I![]() ![]() ![]() |
36. |
Rohrer, J.,
H. Bénédetti,
B. Zanolari, and
H. Riezman.
1993.
Identification of a
novel sequence mediating regulated endocytosis of the G protein-coupled
![]() |
37. | Roth, A.F., and N.G. Davis. 1996. Ubiquitination of the a-factor receptor. J. Cell Biol. 134: 661-674 [Abstract]. |
38. |
Schandel, K.A., and
D.D. Jenness.
1994.
Direct evidence for ligand-induced internalization of the yeast ![]() |
39. |
Singer, B., and
H. Riezman.
1990.
Detection of an intermediate compartment
involved in transport of ![]() |
40. | Strous, G., P. van Kerkhof, R. Govers, A. Ciechanover, and A.L. Schwartz. 1996. The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO (Eur. Mol. Biol. Organ.) J. 15: 3806-3812 [Abstract]. |
41. | Terrell, J., S. Shih, R. Dunn, and L. Hicke. 1998. A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol. Cell. 1: 193-202 . |
42. | Vandenbol, M., J.C. Jauniaux, and M. Grenson. 1990. The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue. Mol. Gen. Genet. 222: 393-399 |
43. | Yaglom, J., M.H.K. Linskens, S. Sadis, D.M. Rubin, B. Futcher, and D. Finley. 1995. p34Cdc28-mediated control of Cln3 cyclin degradation. Mol. Cell. Biol. 15: 731-741 [Abstract]. |
44. |
Yee, N.S.,
C.M. Hsiau,
H. Serve,
K. Vosseller, and
P. Besmer.
1994.
Mechanism
of down-regulation of c-kit receptor: roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C.
J. Biol. Chem.
269:
31991-31998
|
45. | Zanolari, B., S. Raths, B. Singer-Krüger, and H. Riezman. 1992. Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction. Cell. 71: 755-763 |