(Received for publication, June 13, 1995; and in revised form, August 18, 1995)
From the
The -adrenergic receptor (
AR)
belongs to the large family of G protein-coupled receptors. Mutation of
tyrosine residue 326 to an alanine resulted in a
AR
mutant (
AR-Y326A) that was defective in its ability to
sequester and was less well coupled to adenylyl cyclase than the
wild-type
AR. However, this mutant receptor not only
desensitized in response to agonist stimulation but down-regulated
normally. In an attempt to understand the basis for the properties of
this mutant, we have examined the ability of this regulation-defective
mutant to undergo agonist-mediated phosphorylation. When expressed in
293 cells, the maximal response for phosphorylation of the
AR-Y326A mutant was impaired by 75%. Further
characterization of this phosphorylation, using either forskolin
stimulation or phosphorylation site-deficient
AR-Y326A
mutants, demonstrated that the
AR-Y326A mutant can be
phosphorylated by cAMP-dependent protein kinase (PKA) but does not
serve as a substrate for the
-adrenergic receptor kinase 1
(
ARK1). However, overexpression of
ARK1 led to the
agonist-dependent phosphorylation of the
AR-Y326A
mutant and rescue of its sequestration.
ARK1-mediated rescue of
AR-Y326A sequestration could be prevented by mutating
putative
ARK phosphorylation sites, but not PKA phosphorylation
sites. In addition, both sequestration and phosphorylation of the
wild-type
AR could be attenuated by overexpressing a
dominant-negative mutant of
ARK1 (C
ARK1-K220M).
These findings implicate a role for
ARK1-mediated phosphorylation
in facilitating wild-type
AR sequestration.
The exposure of the -adrenergic receptor
(
AR) (
)to catecholamines initiates its
biological response via coupling to the stimulatory G protein
(G
), which then mediates the stimulation of adenylyl
cyclase(1, 2) . However, this receptor-mediated
adenylyl cyclase response to agonist is followed by a rapid uncoupling
of the receptor from its effector system, termed desensitization. The
mechanisms of desensitization have been particularly well studied using
the
AR as a model system(2) . Several studies
have demonstrated that the functional uncoupling of the
AR from G
is the consequence of its
phosphorylation by one of two types of
kinases(3, 4, 5, 6, 7, 8, 9) .
Desensitization of agonist-occupied or activated
AR
involves phosphorylation by a growing family of G protein-coupled
receptor kinases, of which
-adrenergic receptor kinase (
ARK1)
is a member(3, 5, 9) . This phosphorylation
serves to promote the binding of
-arrestin to the receptor, which
when bound further uncouples the
receptor(10, 11, 12) . Moreover,
cAMP-dependent protein kinase (PKA) phosphorylation can desensitize the
AR in response to elevated intracellular cAMP
levels(6, 7, 8, 9) .
In addition
to functional uncoupling of the AR and G
,
agonist-mediated receptor internalization (sequestration) results in
spatial uncoupling, such that in response to agonist plasma membrane
receptors are removed to an intracellular compartment, probably into
endosomes(13) . This has led to speculation that sequestration
might represent a major mechanism of
AR
desensitization. However, a large body of experimental evidence
suggests that this is not the case, as both pharmacological
manipulations and mutant receptors have been used to demonstrate that
the
AR desensitizes in the absence of receptor
sequestration(4, 14, 15, 16, 17) .
In addition, receptor desensitization proceeds much faster than
sequestration(18, 19) . Thus, sequestration mostly
affects receptors that have already been uncoupled from G
.
This has led to the suggestion that receptor sequestration, rather than
playing a role in receptor desensitization, might play a more important
role in mediating the resensitization of desensitized receptors (14, 15, 16, 20) .
Sequestered
ARs are phosphorylated to a lesser extent than plasma
membrane-associated receptors, which prompted the proposal that
AR sequestration might be triggered by
phosphorylation(20) . However, further investigation of this
hypothesis using phosphorylation site-deficient
AR
mutants, as well as various truncated
ARs, led to the
conclusion that phosphorylation was not a prerequisite of
AR
sequestration(3, 9, 21, 22) .
Nonetheless, recent data have renewed interest in the role of
ARK
phosphorylation in receptor sequestration. Tsuga et al.(23) demonstrated that overexpression of
ARK1 could
facilitate m2 muscarinic acetylcholine receptor sequestration, whereas
overexpression of a dominant-negative
ARK1 could attenuate it.
Previously, we reported that mutation of tyrosine residue 326 to an
alanine residue in the seventh transmembrane domain of the
AR resulted in a sequestration-defective receptor
(
AR-Y326A)(15) . The
AR-Y326A
mutant, while unable to sequester, could desensitize, down-regulate,
and be phosphorylated in response to agonist and, although its coupling
was impaired, was able to maximally stimulate adenylyl cyclase in
membranes (15) . More importantly, this receptor mutant was
impaired in its ability to resensitize. This suggested that the
AR-Y326A mutant might provide an excellent tool for
directly testing if agonist-promoted receptor sequestration played a
role in receptor dephosphorylation. However, further examination of the
ability of the
AR-Y326A mutant to be phosphorylated
revealed that the
AR-Y326A mutant could not be used to
study receptor dephosphorylation since, while it was a substrate for
PKA phosphorylation, it did not serve as a substrate for
phosphorylation by G protein-coupled receptor kinases. Thus, the
previously described desensitization of this mutant (15) was
likely the consequence of PKA- rather than
ARK-dependent
mechanisms of desensitization. Interestingly though, we have used the
AR-Y326A mutant in conjunction with
ARK1
overexpression, as well as a dominant-negative
ARK1, to
demonstrate that
ARK phosphorylation can facilitate
AR sequestration.
Tsuga et
al.(23) , in a recent report, suggested that the ability
of ARK1 to facilitate m2 muscarinic acetylcholine receptor
sequestration implied a unique role of phosphorylation for this class
of G
-coupled receptor distinct from the
G
-coupled
AR. The present results
demonstrate a clear role for
ARK phosphorylation in the
facilitation of
AR sequestration, suggesting that
phosphorylation plays a broader role in agonist-promoted G
protein-coupled receptor sequestration than previously envisaged.
A point
mutation in bovine ARK1 (K220M) was generated by polymerase chain
reaction using a 5` primer encompassing an AccI (position 614)
5` of the lysine to be mutated. The codon at positions 658-660,
AAG (lysine 220), was mutated to ATG (methionine). A 3` primer located
3` to a second AccI site (position 941) was used in
conjunction with the 5` primer to generate the reading frame containing
both AccI sites. The DNA was cloned into pCR(TM)II
(Invitrogen) according to the specifications of the manufacturer.
Positive clones were isolated, and the mutation, as well as the
integrity of the coding sequence between both AccI sites, was
confirmed by dideoxy DNA sequencing. The AccI fragment was
isolated and replaced in C
ARK1 (26) cloned
in pBC. An MscI-RsrII cassette in pcDNA1/Amp C
ARK1 was replaced with the same cassette isolated from the
pBC construct to create C
ARK1-K220M in pcDNA1/Amp.
Figure 1:
Comparison of the whole cell
phosphorylation of wild-type ARs and
AR-Y326A mutants. 293 cells were transfected with
pcDNA1/Amp cDNA encoding
AR and
AR-Y326A and assayed for whole cell phosphorylation as
described under ``Experimental Procedures.'' A, time
course for the phosphorylation of wild-type
AR (725
± 261 fmol/mg of whole cell protein) and
AR-Y326A mutant (646 ± 175 fmol/mg of whole
cell protein) in response to stimulation with 10 µM ISO
(15 s to 20 min). The R values for the curve fits for
AR and
AR-Y326A mutant
phosphorylation time courses were 0.97 and 0.98, respectively. B, the comparison of agonist-induced (10 µM ISO)
phosphorylation with forskolin (FSK)-mediated (25 µM in the presence of 1 mM IBMX) phosphorylation of the
wild-type
AR (772 ± 457 fmol/mg of whole cell
protein) and
AR-Y326A mutants (763 ± 385
fmol/mg of whole cell protein). In each experiment, the basal
phosphorylation for each receptor type was subtracted from that seen in
the presence of agonist to give the agonist-stimulated increase in the
level of phosphorylation. This value, in turn, was compared with that
seen with wild-type
AR in the same experiment and
expressed as a percentage of the agonist-induced wild-type
AR phosphorylation, which was increased 5.7 ±
1.0-fold above basal, following 15-min stimulation with 10 µM ISO. The data from the autoradiographs were analyzed using a
Molecular Dynamics phosphorimaging system. Basal phosphorylation
(agonist-independent phosphorylation) of the
AR and
AR-Y326A mutant in A and B was
identical as quantitated using the Molecular Dynamics phosphorimaging
system. The data in each panel represent the mean ± S.D. (bars) values from four different experiments; where no error
bar is shown, the S.D. was smaller than the symbol. In B, the asterisk indicates p < 0.001 versus wild-type
AR phosphorylation in response to
agonist.
Although the
wild-type AR and
AR-Y326A mutant
phosphorylate at somewhat different rates, this could not account for
the large differences in the extent of phosphorylation observed for
these two receptors. The
AR serves as substrate for
phosphorylation by both PKA and
ARK(3, 4) .
Therefore, the
AR-Y326A mutant was tested for its
ability to be phosphorylated by these two types of kinases. This was
tested in two ways. First, wild-type
ARs and
AR-Y326A mutants, expressed in 293 cells, were
challenged for 15 min with either 10 µM ISO or 25
µM forskolin (in the presence of 1 mM IBMX) to
stimulate adenylyl cyclase and raise intracellular cAMP levels in the
presence or absence of receptor activation, respectively. These results
are illustrated in Fig. 1B and are normalized to
agonist-induced stimulation of wild-type
AR
phosphorylation. Under these conditions, the magnitude of agonist- and
forskolin-induced phosphorylation of the
Y326A mutant
was identical (26 ± 5.7% and 23 ± 7.6% of wild-type
AR phosphorylation, respectively). This was equivalent
to forskolin-induced phosphorylation of the wild-type
AR (21 ± 7.6%). Second, wild-type
AR and
AR-Y326A mutants were prepared
in which their putative sites of PKA and
ARK phosphorylation were
removed. The removal of the putative PKA and
ARK phosphorylation
sites in either the wild-type
AR or
AR-Y326A did not prevent the mobilization of an
adenylyl cyclase response by any of the phosphorylation site-deficient
mutants ((3) ; data not shown). The agonist
induced-phosphorylation of these phosphorylation site-deficient mutants
is illustrated in Fig. 2, A and B. In 293
cells, the wild-type
AR was phosphorylated
predominantly at
ARK phosphorylation sites (69.3 ± 15.3%)
rather than PKA phosphorylation sites (22 ± 7.9%). However, the
AR-Y326A served solely as a PKA substrate, as the
level of its phosphorylation was unchanged by the removal of
ARK
phosphorylation sites (20 ± 7.5% versus 17 ±
2.3%).
AR-Y326A mutant phosphorylation was essentially
inhibited upon the removal of its PKA phosphorylation sites (Fig. 2).
Figure 2:
Phosphorylation of wild-type
AR and
AR-Y326A phosphorylation
site-deficient mutants. 293 cells were transfected with pcDNA1/Amp cDNA
encoding wild-type (WT),
ARK phosphorylation
site-deficient (
ARK-) or PKA phosphorylation
site-deficient (PKA-)
AR and
AR-Y326A and assayed for whole cell phosphorylation as
described under ``Experimental Procedures.'' A,
autoradiograph from a representative experiment showing the whole cell
phosphorylation of each receptor following a 15-min incubation in the
absence(-) or presence (+) of 10 µM ISO.
Expression of each of the mutant receptors was equivalent in these
experiments, and each lane was loaded with equivalent amounts of
receptor protein as described under ``Experimental
procedures.'' B, The mean ± S.D. (bars)
for the quantitative analysis of four different experiments. In these
experiments, the data were normalized to the agonist-induced wild-type
AR phosphorylation which was increased 6.6 ±
1.2-fold above basal (see legend to Fig. 1). The expression
levels for each receptor (fmol/mg of whole cell protein) were as
follows
AR-WT = 1725 ± 455,
AR-
ARK
= 1729 ±
122,
AR-PKA
= 1995 ±
183,
AR-Y326A-WT = 1665 ± 268,
AR-Y326A-
ARK
= 1786
± 128 and
AR-Y326A-PKA
= 1879 ± 283. As seen in the autoradiograph there
was no difference in the basal phosphorylation for any of the
receptors. Asterisk, p < 0.05; double
asterisks, p < 0.0005 versus agonist-stimulated wild-type
AR phosphorylation.
, p < 0.01 versus agonist-stimulated
wild-type
AR-Y326A
phosphorylation.
Figure 3:
Effect of the overexpression of ARK1
on
AR and
AR-Y326A mutant
phosphorylation. 293 cells were transfected with 0, 1, 2.5, or 10
µg of
ARK1 cDNA in a pcDNA1 expression vector along with
pcDNA1/Amp cDNA either coding for wild-type
AR (1237
± 142 fmol/mg of whole cell protein) or
AR-Y326A (2071 ± 250 fmol/mg of whole cell
protein) and assayed for whole cell phosphorylation as described under
``Experimental Procedures.'' A, immunoblot of the
overexpression of
ARK1 protein with increasing amounts of
transfected
ARK1/2 cDNA. The far right lane shows the
appropriate migration of 10 ng of purified
ARK1. Two major
molecular weight species were detected with
ARK1/2 antibody, when
ARK1 was overexpressed using the pcDNA1/Amp expression vector. The lower band migrates at the same molecular weight as the
purified
ARK1. The upper band is presumed to represent
improperly processed
ARK1 resulting from its overexpression in 293
cells. Nontransfected 293 cells express both
ARK1 and
ARK2
but cannot be easily visualized in the immunoblot without saturating
the lanes containing samples of cells overexpressing
ARK1. B, autoradiograph showing a representative whole cell
phosphorylation of wild-type
AR and
AR-Y326A following coexpression with increasing
amounts of
ARK1 following incubation for 15 min in the
absence(-) or presence (+) of 10 µM ISO. Each
lane was loaded with equivalent amounts of wild-type and Y326A mutant
receptor protein as described under ``Experimental
procedures.'' The major species of the
AR
expressed in 293 cells is a glycoprotein of a molecular mass ranging
from 56 to 85 kDa. Immunoblots with biotinylated 12CA5 identified each
of the major phosphorylated bands (data not shown). C, the
mean ± S.D. (bars) of the quantitative analysis of
three different experiments. In these experiments, the data were
normalized to the agonist-induced wild-type
AR
phosphorylation, which was increased 4.2 ± 1.4-fold above basal
(see legend to Fig. 1). The basal phosphorylation of either
receptor was unaffected by the coexpression of increasing amounts of
ARK1. p < 0.05 (asterisk) or p <
0.001 (double asterisks) versus control
agonist-stimulated
AR
phosphorylation.
Since the
AR-Y326A mutant phosphorylation deficit could be
reversed by overexpressing
ARK1, we reasoned that agonist-promoted
sequestration might also be influenced by the overexpression of
ARK1. Under control conditions, 42 ± 3.1% of wild-type
ARs and 5 ± 1.9% of
AR-Y326A
mutants sequestered in response to 10 µM ISO stimulation (Fig. 4). However, as shown in Fig. 4, the increase in
ARK1 expression (Fig. 3A) produced a progressive
rescue of
AR-Y326A mutant sequestration essentially
back to wild-type
AR levels. Overexpression of
ARK1 had no effect on the sequestration of the wild-type
AR.
Figure 4:
Effect of the overexpression of ARK1
on
AR and
AR-Y326A mutant
sequestration. 293 cells were transfected with 0, 1, 2.5, or 10 µg
of
ARK1 cDNA in a pcDNA1 expression vector along with pcDNA1/Amp
cDNA either coding for wild-type
AR (967 ± 85
fmol/mg of whole cell protein) or
AR-Y326A mutant (812
± 94 fmol/mg of whole cell protein) and assayed for
agonist-promoted sequestration as described under ``Experimental
Procedures.'' Basal sequestration of both the
AR
and
AR-Y326A mutant were unaffected by coexpression
with increasing amounts of
ARK1. Basally sequestered receptors
represented 27 ± 4% and 31 ± 2.4% of total cellular
AR and
AR-Y326A, respectively, in
these experiments. The data represents the mean ± S.E. (bars) for three different experiments. p < 0.01 (asterisk) or p < 0.0001 (double
asterisks) versus control agonist-promoted
AR sequestration. p < 0.005 (
) or
p < 0.0005 (
) versus control agonist-promoted
AR-Y326A mutant
sequestration.
In addition to 293 cells, the ability of the
AR-Y326A mutant to sequester was also tested in CHO,
CHW, and COS7 cells. In each case, the sequestration of the
AR-Y326A mutant was impaired to an equivalent or
greater extent than reported here for its sequestration in 293 cells,
indicating that its inability to sequester in response to agonist was
not the consequence of the cellular environment in which it was tested
(data not shown).
For these experiments, geranylgeranylated
(C isoprenylated) versions of
ARK1 or
ARK1-K220M
(C
ARK1) in pcDNA1/Amp were used. Previously, Kong et al.(29) demonstrated that
ARK1-K220R did not
inhibit
AR sequestration. However, this
dominant-negative
ARK1 mutant appeared to be impaired in its
interaction with G protein
subunits(29) .
Isoprenylation of
ARK1 directly targets the cytosolic kinase to
the membrane, without the need for coupling of the receptor to
G
, resulting in the subsequent dissociation of
G
which then mediates translocation of
ARK to
the plasma membrane(5, 26, 30) . This
suggested that a C
isoprenylated dominant-negative
ARK1 might be more effective at inhibiting
AR
phosphorylation and sequestration. When tested, C
BARK1,
like wild-type
ARK1, could rescue both wild-type
AR-Y326A mutant sequestration (compare Fig. 4and Fig. 5) and phosphorylation (data not shown).
C
ARK1 was used to test the rescue of phosphorylation
site-deficient
AR and
AR-Y326A
mutants. Removal of PKA phosphorylation sites had no effect on the
sequestration of the wild-type
AR, but removal of
ARK phosphorylation sites reduced agonist-induced sequestration by
50%, again hinting toward a potential role for phosphorylation in the
process. Overexpression of C
ARK1 was unable to
rescue the sequestration of the
ARK phosphorylation-deficient
AR. In the absence of C
ARK1
overexpression, all of the
AR-Y326A mutants were
impaired in their ability to sequester. However, overexpression of
C
ARK1 rescued the sequestration of the wild-type
AR-Y326A and PKA phosphorylation site-deficient
AR-Y326A mutants, but was unable to rescue the
sequestration of the
ARK phosphorylation site-deficient
AR-Y326A mutant.
Figure 5:
Effect of C
ARK1 on the
sequestration of wild-type
AR and
AR-Y326A phosphorylation site-deficient mutants. 293
cells were transfected with 1 µg of pcDNA1/Amp C
ARK1 cDNA along with pcDNA1/Amp cDNA encoding one of the
following: wild-type (WT),
ARK phosphorylation
site-deficient (
ARK-), or PKA phosphorylation
site-deficient (PKA-)
AR and
AR-Y326A and assayed for agonist-promoted
sequestration as described under ``Experimental Procedures.''
The basal sequestration of each of the receptors was equivalent and was
not affected by coexpression of 1 µg of C
ARK1
cDNA. Basally sequestered receptors represented 30 ± 1.8% of
total cellular receptors in these experiments. The expression levels
for each receptor (fmol/mg of whole cell protein) were as follows
AR-WT = 1102 ± 173,
AR-
ARK
= 1108 ±
243,
AR-PKA
= 1440 ±
252,
AR-Y326A-WT = 757 ± 112,
AR-Y326A-
ARK
= 795
± 161 and
AR-Y326A-PKA
= 951 ± 161. The data represent the mean ±
S.E. (bars) for three to five different experiments. p < 0.05 (asterisk) or p < 0.005 (double
asterisks) versus agonist-promoted wild-type
AR sequestration. p < 0.05 (
) or p < 0.005 (
) versus agonist-promoted
wild-type
AR-Y326A mutant
sequestration.
A kinase mutant with a methionine
residue substituted for lysine 220 in the catalytic domain of C
ARK1 (C
ARK1-K220M) was overexpressed
using a pcDNA1/Amp expression vector (Fig. 6A) and
tested for its ability to inhibit agonist-induced wild-type
AR phosphorylation (Fig. 6B) and
sequestration (Fig. 7). Agonist-induced phosphorylation of the
AR was inhibited significantly following
cotransfection with either 1 µg (31 ± 11%, p <
0.01) or 2.5 µg (40 ± 6.8%, p < 0.001) of
dominant-negative kinase DNA, but
AR-Y326A mutant
phosphorylation was unaffected (Fig. 6C).
Cotransfection with larger amounts of DNA did not further enhance the
inhibition of wild-type
AR phosphorylation by the
dominant-negative kinase. Shown in Fig. 7is a dose-response
curve for agonist-promoted wild-type
AR sequestration
in either the presence or absence of 1 µg of cotransfected C
ARK1-K220M cDNA. Overexpression of C
ARK1-K220M significantly reduced the maximal response for
sequestration, 25 ± 5.8% (p < 0.05). Expression of
the dominant-negative kinase had no effect on the EC
for
sequestration. The EC
values in the presence and absence
of C
ARK1-K220M were 11 ± 2.9 nM and
12 ± 1.7 nM, respectively. Under similar conditions,
AR-Y326A mutant sequestration was unaffected by
C
ARK1-K220M, which is presumably consistent with the
inability of C
ARK1-K220M to rescue phosphorylation
of the mutant receptor (data not shown).
Figure 6:
The effect of C
ARK1-K220M overexpression on the phosphorylation of wild-type
AR and
AR-Y326A mutant. 293 cells
were transfected with 0, 1, or 2.5 µg of pcDNA1/Amp C
ARK1-K220M cDNA along with pcDNA1/Amp cDNA coding for
wild-type
AR (1409 ± 104 fmol/mg of whole cell
protein) or
AR-Y326A (994 ± 164 fmol/mg of
whole cell protein) and assayed for whole cell phosphorylation as
described under ``Experimental Procedures.'' A,
immunoblot of the overexpression of C
ARK1-K220M
protein with increasing amounts of transfected cDNA. Two major
molecular weight species were also detected with
ARK1/2 antibody,
when C
ARK1-K220M was overexpressed using the
pcDNA1/Amp expression vector (see Fig. 3legend). B,
autoradiograph showing a representative whole cell phosphorylation of
wild-type
AR following coexpression with increasing
amounts of C
ARK1-K220M incubated for 15 min in the
absence(-) or presence (+) of 10 µM ISO. Each
lane was loaded with equivalent amounts of receptor protein as
described under ``Experimental procedures.'' C, the
mean ± S.D. (bars) of the quantitative analysis of four
different experiments. In these experiments, the data were normalized
to the agonist-induced wild-type
AR phosphorylation
which was increased 5.1 ± 1.9-fold over basal (see legend to Fig. 1). The basal phosphorylation of either receptor was
unaffected by the coexpression of increasing amounts of C
ARK1-K220M. p < 0.01 (asterisk) or p < 0.001 (double asterisks) versus control agonist-stimulated
AR
phosphorylation.
Figure 7:
Dose response for the inhibition of
wild-type AR sequestration by C
ARK1-K220M. 293 cells were transfected with pcDNA1/Amp cDNA either
coding wild-type
AR with (1435 ± 319 fmol/mg of
whole cell protein) or without (1345 ± 150 fmol/mg of whole cell
protein) 1 µg of pcDNA1/Amp C
ARK1-K220M cDNA and
assayed for agonist-promoted sequestration as described under
``Experimental Procedures'' except that sequestration was
measured following 30-min stimulation with 10
to
10
M ISO. The basal sequestration of the
AR in these experiments in the presence or absence
C
ARK1-K220M cDNA was 33 ± 1.2% and 30
± 0.8%, respectively. The data represent the mean ± S.E. (bars) for three different experiments. The R values
for the curve fits in the presence and absence of C
ARK1-K220M were 0.96 and 0.98,
respectively.
The present experiments clearly demonstrate a role for
ARK-mediated phosphorylation in facilitating
AR
sequestration. This idea is supported by three observations. First,
ARK1, when overexpressed, rescues both the phosphorylation and the
sequestration of the
AR-Y326A mutant which was
defective in its ability to be phosphorylated by
ARK and to
sequester in response to agonist stimulation. Second, overexpressed
ARK1 can rescue the sequestration of PKA phosphorylation
site-deficient, but not
ARK phosphorylation site-deficient,
AR-Y326A mutants. Third, sequestration of the
wild-type
AR can be attenuated by overexpressing a
dominant-negative
ARK1, which also diminishes agonist-induced
phosphorylation of the receptor to a similar extent. These results are
in agreement with the work of Tsuga et al.(23) where
they describe the ability of
ARK1 to augment m2 muscarinic
acetylcholine receptor sequestration.
The conclusion that ARK1
phosphorylation is involved in
AR sequestration
evolved from experiments testing the role of sequestration in receptor
dephosphorylation using the
AR-Y326A mutant. Upon
investigation, we found that the phosphorylation of the receptor mutant
was reduced and that it served predominantly as a substrate for
PKA-mediated phosphorylation, when expressed in 293 cells. This result
indicated that the
AR-Y326A mutant could not be used
to study receptor dephosphorylation. Nonetheless, these results
suggested that mutation of tyrosine residue 326 to an alanine not only
inhibits the ability of the
AR to sequester but also
abolished its ability to act as a substrate for
ARK
phosphorylation. Therefore, the desensitization of the
AR-Y326A mutant previously reported (15) was
likely the consequence of PKA- rather than
ARK-mediated
phosphorylation of the receptor, since both mechanisms have been
demonstrated to effectively desensitize the wild-type
AR(3, 4) .
Historically, most
studies have found that receptor phosphorylation was not essential for
AR sequestration. In particular, Hausdorff et al.(3) demonstrated that neither
ARK nor PKA
phosphorylation sites were required for sequestration of
AR stably expressed by CHW cells. In addition,
truncation of the carboxyl tail of the hamster
AR,
which removes its putative
ARK phosphorylation sites, resulted in
normal sequestration when expressed in mouse L cells(22) ,
although further truncation of this receptor did result in some
impairment of sequestration(31) . Finally, Lohse et
al.(4) , using permeabilized A431 cells, demonstrated that
sequestration was unaffected by inhibitors of either PKA and
ARK
phosphorylation. Indeed, in the present study,
ARs
lacking putative PKA and
ARK phosphorylation sites, when expressed
in 293 cells, also sequestered in response to agonist exposure,
although the
ARs lacking putative
ARK
phosphorylation sites were somewhat impaired in their sequestration
(50% of control). The observed impairment in the sequestration of
ARs lacking putative
ARK phosphorylation sites
might be related to the fact that we have used transient transfections
in the present study to examine their sequestration, whereas Hausdorff et al.(3) selected permanently transfected clonal
cell lines which might vary in their sequestration properties depending
upon the clone selected. Nonetheless, these mutant receptors do
sequester in response to agonist stimulation in 293 cells in the
absence of
ARK-mediated phosphorylation which is consistent with
previously described work. We have tested the ability of the
AR-Y326A mutant to sequester in four different cell
lines (CHO, 293, CHW, and COS7) and in each case the mutant was
impaired in its ability to sequester. This suggests that the inability
of this receptor to sequester and be phosphorylated by
ARK is
likely an intrinsic property of the
AR-Y326A mutant
receptor rather than the consequence of the cell type in which it has
been expressed
It is likely that the intrinsic properties of the
AR-Y326A mutant have allowed us to uncover a
previously unappreciated role for phosphorylation in the
AR sequestration process. The results suggest that
phosphorylation of the
AR by
ARK is facilitory
rather than required for sequestration.
ARK1 phosphorylation of
the
AR-Y326A mutant effectively rescues its complete
lack of sequestration, whereas removal of the
ARK phosphorylation
sites in the wild-type
AR only reduces sequestration
by 50% in 293 cells. This indicates that the basis for the
sequestration impairment of the
AR-Y326A mutant goes
beyond a simple lack of phosphorylation. We suggest that mutation of
tyrosine residue 326 to an alanine alters the ability of the
agonist-occupied receptor to achieve and/or maintain a conformational
state required for receptor phosphorylation by
ARK as well as
agonist-promoted sequestration. In fact, a smaller proportion of
AR-Y326A mutant receptors exhibit high-affinity
agonist binding(15) . The isomerization of the receptor from
its low- to high-affinity state (R
R*) might
serve to trigger such a change in receptor conformation. Initiation of
AR sequestration requires the occupancy of the
receptor with agonist; antagonist occupancy is not sufficient. Previous
results with a cyc
variant line of S49 lymphoma
cells, which lack functional G
, provide support for the
idea that agonist occupancy is sufficient for both homologous
desensitization (
ARK-dependent phosphorylation) and sequestration
in the absence of coupling to adenylyl cyclase(32) . Certainly,
constitutively active receptors which achieve R* in the
absence of agonist occupancy are both constitutively phosphorylated and
desensitized(33) . Thus, an impairment in the active
conformation of the
AR-Y326A mutant may explain a lack
of phosphorylation by the normal endogenous complement of G
protein-coupled receptor kinase in 293 cells, but that overexpression
of
ARK1 in these cells can overcome this deficit. In fact, Ungerer et al.(34) have suggested that in the heart
ARK
might be the limiting component in
-adrenergic receptor
desensitization. This might explain why overexpression of
ARK
leads to increased phosphorylation of the wild-type
AR
in several cell lines.
The observation that ARs
lacking putative
ARK phosphorylation sites can sequester in
response to agonist stimulation clearly indicates that
ARK-mediated phosphorylation neither serves as the signal
initiating the sequestration process nor is it an absolute requirement.
Instead, we hypothesize that
ARK phosphorylation either stabilizes
the conformation of the receptor or promotes the interaction of the
receptor with some as yet unidentified cellular element that mediates
AR internalization, even in the absence of
ARK
phosphorylation. Since arrestins appear to be required for
desensitization, and phosphorylation leads to increased affinity of
rhodopsin,
AR, and m2 muscarinic receptor for members
of the arrestin
family(11, 35, 36, 37, 38) ,
it is tempting to speculate that arrestins might also play a role in
sequestration as they have also been shown to interact with
agonist-occupied nonphosphorylated receptor, albeit less
effectively(35, 36, 37, 38) .
The
dominant-negative ARK1-K220M, while able to inhibit both the
phosphorylation and sequestration of
AR to equivalent
extents in 293 cells, was not overwhelmingly effective at inhibiting
either process, 31 ± 11% and 25 ± 6%, respectively.
However, a modest effect of
ARK1-K220M on the sequestration of the
wild-type
AR might be expected, since complete
blockade of
ARK phosphorylation should lead at most to a 50%
decrease in sequestration (see Fig. 5,
ARK phosphorylation
site-deficient
AR mutant). In two recent
studies(23, 29) , dominant-negative
ARKs have
been tested for their ability to affect the sequestration and
phosphorylation of
AR and m2 muscarinic acetylcholine
receptors. Kong et al.(29) reported that
ARK-K220R, while able to inhibit in vitro
AR phosphorylation, was impaired in its
G
targeting and had no effect on sequestration.
However, Tsuga et al.(23) demonstrated that
ARK1-K220W could inhibit both the phosphorylation and
sequestration of the m2 muscarinic acetylcholine receptor subtype, but
this was dependent upon the level of endogenously expressed kinase. In
the present study, a similar result was obtained for C
ARK1-K220M, which attenuated both whole cell phosphorylation
and sequestration of the
AR. The apparent discordance
in the efficacy of a particular dominant-negative
ARK mutant to
inhibit sequestration might be dependent on the nature of the residue
substituted for lysine 220 or the cellular background in which they are
tested. In the work by Tsuga et al.(23) , the authors
concluded that
ARK-facilitated sequestration was unique to the
G
-coupled m2 muscarinic receptor, since putative
ARK
phosphorylation sites are likely found in the third intracellular loop
of this receptor rather than in the short cytoplasmic
tail(39) . Our findings indicate that, in contrast to what is
described in the literature for the
AR(3, 4, 21, 22) ,
a role for
ARK phosphorylation in facilitating receptor
sequestration might be more general.
Other studies also support the
idea that phosphorylation might be important for sequestration of the
AR. For example, the
AR does not
sequester or phosphorylate in response to agonist stimulation, yet,
replacing its carboxyl tail with the tail of the
AR
rescues both sequestration and phosphorylation of the chimeric
/
AR(40) . In addition, the
sequestration of other G protein-coupled receptors, such as the
angiotensin II, neurotensin, and
-adrenergic
receptors, as well as the receptor for parathyroid hormone and
parathyroid hormone-related protein, are inhibited by the truncation of
their carboxyl
tails(41, 42, 43, 44) . However, the
possibility exists that specific sequestration motifs might exist in
the tails of these receptors. Interestingly though, a regulatory
sequence identified in the tail of the receptor for parathyroid hormone
and parathyroid hormone-related protein contains several serine and
threonine residues that might act as potential
ARK phosphorylation
sites(42) .
In summary, the present studies demonstrate a
clear role for ARK1-mediated phosphorylation in the facilitation
of
AR sequestration. It will be of interest to
determine whether this property is unique to
ARK1 or if
phosphorylation by other members of the G protein-coupled receptor
kinase family can promote
AR sequestration as well. We
suggest that
ARK phosphorylation facilitates, rather than
initiates, sequestration as a
ARK phosphorylation site-deficient
AR mutant can sequester, albeit not normally.
ARK
phosphorylation has now been shown to facilitate the sequestration of
two different G protein-coupled receptors, indicating that
phosphorylation plays a broader role in agonist-promoted receptor
sequestration than originally envisaged.