(Received for publication, October 25, 1994; and in revised form, December 13, 1994)
From the
During short term agonist exposure, the
-adrenergic receptor (
AR) undergoes
rapid functional desensitization caused by phosphorylation of the
receptor by the
-adrenergic receptor kinase (
ARK). This
signal quenching is similar in nature to that found with a number of
G-protein coupled receptors in which agonist-promoted desensitization
is due to
ARK phosphorylation; like these other receptors, the
precise molecular determinants of the receptor required for
ARK
phosphorylation are not known.
To delineate such a motif in the
human AR (
C10), we constructed six
mutated receptors consisting of deletions or substitutions of
Ser-296-299 in the EESSSS sequence of the third intracellular
loop of the receptor. These were expressed in Chinese hamster ovary and
COS-7 cells, and agonist-promoted desensitization and receptor
phosphorylation were assessed. Deletion of the EESSSS sequence and
substitution of alanine for all four serines resulted in a total loss
of phosphorylation and desensitization. Mutant receptors that retained
two of the original serines (AASS and SSAA) underwent agonist-promoted
phosphorylation of 55 ± 7% and 57 ± 8% of the
phosphorylation found for wild type
C10. Additional
substitution mutants (SSSA and SAAA) underwent 77 ± 1% and 27
± 4% of wild type phosphorylation, respectively. Thus,
substitution of alanine for each additional serine decreased overall
phosphorylation as compared with wild type
C10 by
25%, which is consistent with all 4 serines being phosphorylated.
Mutated receptors that only partially phosphorylated (as compared with
wild type) failed to undergo agonistpromoted desensitization.
Thus,
ARK-mediated phosphorylation of
C10 occurs at
Ser-296-299 in the third intracellular loop, and this represents
the critical step in rapid agonistpromoted desensitization. A number of
other G-protein coupled receptors that undergo desensitization have a
sequence motif similar to that which we have found for
ARK-mediated phosphorylation of
C10, suggesting
that these receptors may also be substrates for
ARK.
-Adrenergic receptors (
AR) (
)are present in a large variety of central and peripheral
tissues and are known to mediate such diverse physiological functions
as platelet aggregation, inhibition of lipolysis and insulin secretion,
autoregulation of neurotransmitter release, and modulation of vascular
tone(1) . As members of the superfamily of G-protein coupled
receptor family,
AR share a number of conserved
structural features including an extracellular amino terminus, an
intracellular carboxyl terminus, and seven transmembrane-spanning
domains linked by three extracellular loops and three intracellular
loops(2) . Studies that have used chimeric or mutated forms of
G-protein coupled receptors have demonstrated the involvement of the
transmembrane domains in ligand binding (3) and the cytosolic
domains in G-protein coupling (3) and regulatory processes such
as receptor sequestration, down-regulation, and
phosphorylation(4) .
Receptor phosphorylation by a variety
of kinases is one mechanism by which the phenomenon of desensitization,
or refractoriness, of the stimulated receptor-mediated response
develops during conditions of prolonged agonist exposure. There are
essentially two classes of kinases which are involved in the
phosphorylation of G-protein coupled receptors: those that are
activated by second messenger production (e.g. cAMP-dependent
protein kinases and protein kinase C) and those that are second
messenger-independent (e.g. G-protein coupled receptor
kinases). G-protein coupled receptor kinases phosphorylate the
agonist-occupied form of G-protein coupled receptors at serine and
threonine residues in the intracellular domains, typically the third
intracellular loop or carboxyl terminus(5) . One such kinase,
the -adrenergic receptor kinase (
)or
ARK, although
originally named for its first defined role in the phosphorylation and
desensitization of the
AR(8) , has been shown
to phosphorylate a number of G-protein coupled receptors including the
muscarinic m
receptor(9) , the substance P
receptor(10) , the adenosine A
receptor(11) , the thrombin receptor(12) , and
germane to this report, the cloned human
AR,
C10(14, 15, 16) .
Studies
from our laboratory (15, 17, 18) and
others(14, 16, 19) strongly support a direct
role of ARK-mediated phosphorylation in agonist-promoted
desensitization of
C10. However, as with other
G-protein coupled receptors that are phosphorylated by
ARK, the
precise serine or threonine residues in
C10 which are
phosphorylated are not known. This is largely because of the lack of a
defined consensus sequence for
ARK-mediated phosphorylation. For
the most part, attempts to delineate discrete residues within a
receptor which are phosphorylated by
ARK have been limited to the
prototypical substrate, the
AR. However, despite
extensive deletion (20) and substitution (20, 21, 22, 23) mutagenesis
studies, the sites for
ARK-mediated phosphorylation for the
AR remain unidentified. Moreover, in an examination of
the intracellular portions of the receptors that have been definitively
shown to be phosphorylated by
ARK, there are typically numerous
serine and threonine residues and a low amino acid sequence homology
between receptors.
Previously, we have shown that removal of 75
amino acids(257-332), including nine serine and threonine
residues, in the middle of the third intracellular loop of
C10 ablates the rapid component of agonist-promoted
desensitization and receptor phosphorylation(15) , indicating
that the sites for
ARKmediated phosphorylation are found within
this region. Of the nine serine and threonine residues, four serines
occur consecutively and are preceded by two glutamic acid residues
(EESSSS). Using a series of synthetic peptides in a reconstituted
phospholipid vesicle system with purified
ARK, Onorato et al.(19) showed that
ARK preferentially phosphorylates
serines preceded by amino-terminal acidic residues, with the most
preferred sequence being EXS. Consequently, we considered that
these aforementioned four consecutive serines (Ser-296-299) might
serve as sites for
ARK-mediated phosphorylation of
C10. To explore this possibility, six mutant
C10 receptors lacking all or some combination of the
four serines were constructed using site-directed mutagenesis,
recombinantly expressed in clonal cell lines, and assessed for their
ability to undergo the processes of agonist-promoted desensitization
and
ARK-mediated phosphorylation.
To ascertain the role of Ser-296-299 in the
agonist-promoted desensitization and ARK-mediated phosphorylation
of
C10, we initially constructed a deletion mutant
(denoted as DEL 293-304) lacking 12 amino acids, including the
four serines and preceding glutamic acids (Fig. 1). This mutant
failed to undergo agonist-promoted desensitization (see below), and so
we focused our efforts on the four serines within this region with five
additional mutants having direct substitution of the potentially
phosphorylated serines (Ser-296, 297, 298, and 299). For one of these
substitution mutants, all four serines in this region were replaced by
alanine residues (denoted as receptor AAAA). Two other mutants had two
consecutive serines of the four serines replaced by alanine residues
(denoted as receptors AASS and SSAA) (Fig. 1). Mutant and wild
type
C10 were transfected into CHO cells, and clones
stably expressing receptors at 3100 ± 200 fmol/mg were utilized
for pharmacological characterization and agonist-promoted
desensitization studies.
Figure 1:
Schematic for
deletion and substitution of potential ARK phosphorylation sites
in
C10. Shown is the proposed membrane topology of
C10 with potential
ARK phosphorylation sites in
the third intracellular loop. Roman numerals indicate
transmembrane domains I-VII. Circles with single
letters represent the primary amino acid sequence of the third
intracellular loop with serines and threonines indicated by darkened circles. Asterisks indicate Ser-296, 297,
298, and 299. The six mutants constructed include a deletion mutant
lacking amino acids 293-304 (DEL 293-304) and five
substitution mutants with various substitutions of Ser-296-299
with alanines (AAAA, AASS, SSAA, SSSA, and
SAAA).
Prior to functional studies, the
pharmacological characteristics of the mutant and wild type
C10 were assessed. As summarized in Table 1, all
receptors bound [
H]yohimbine with high affinity
and displayed the ability to form the agonist-receptor-G-protein high
affinity complex in [
H]yohimbine competition
studies with epinephrine, with
60-70% of the receptors
occupying the high affinity state. Both high and low affinity binding
constants of the mutant receptors for epinephrine were similar to those
of the wild type
C10, such that similar occupancy
would be observed during epinephrine exposure of 100 µM (Table 1). This high affinity binding was sensitive to
guanine nucleotide, as curves for both mutant and wild type receptors
were monophasic and were best fit to one-site models when 100
µM GTP was included in the assay (Table 1). In
addition (see below), the mutated
ARs functionally
coupled to G
as determined in adenylyl cyclase assays.
For studies of agonist-promoted desensitization, wild type
C10 (
C10), the deletion mutant (DEL
293-304), and the substitution mutants (AAAA, AASS, and SSAA)
were exposed to 100 µM epinephrine for 30 min, and the
effects of agonist pretreatment on
AR-mediated
inhibition of adenylyl cyclase activity were assessed in the membranes.
As shown in Fig. 2, wild type
C10-mediated
inhibition of adenylyl cyclase activity underwent desensitization
following a 30-min exposure to agonist. This desensitization was
manifested as a
5-fold increase in the EC
for
epinephrine-mediated inhibition (0.26 ± 0.03 µMversus 1.25 ± 0.12 µM, p < 0.002, Table 2) with no change in the maximal
inhibition (44 ± 2% versus 48 ± 2% decrease in
forskolin-stimulated cyclase activity, p = NS, Table 2). In contrast, all of the mutant receptors failed to
undergo agonist-promoted desensitization, with no significant change in
the EC
or maximal inhibition of adenylyl cyclase activity
( Fig. 2and Table 2).
Figure 2:
Effects of substitution or deletion of Ser
296-299 on agonist-promoted desensitization of
C10. CHO cells expressing wild type
C10 (WT
C10) and the mutants
DEL 293-304, AAAA, AASS, and SSAA were exposed to 100 µM epinephrine for 30 min, membranes prepared, and adenylyl cyclase
activities determined in the presence of 1.0 µM forskolin
and the indicated concentrations of epinephrine (EPI). Data
are presented as the percent of forskolin-stimulated activity.
Desensitization was manifested as an increase in the EC
for epinephrine-mediated inhibition for wild type
C10 and was not detected in any of the mutated
receptors. Results shown represent the mean ± S.E. of four
individual experiments performed.
To establish whether the loss of
desensitization was, in fact, due to a loss of phosphorylation, whole
cell phosphorylation studies were carried out using wild type
C10 and the three substitution mutants AAAA, AASS, and
SSAA. These were carried out using COS-7 cells transiently expressing
ARK and mutant or wild type
C10. Cotransfection
with
ARK was utilized for several reasons. First, it has been
reported recently by Kurose and Lefkowitz (16) that in Western
blotting experiments with antibodies specific for
ARK and
ARK2,
ARK is by far the most predominant of the two
ARK
isoforms expressed in CHO cells. Thus, phosphorylation studies carried
out with
ARK would more likely correlate with the functional
studies of agonist-promoted desensitization which were performed in CHO
cells. Second, thus far definitive phosphorylation of purified,
reconstituted
C10 has only been demonstrated using
ARK(14) . Finally, since these receptors were each
overexpressed, it seemed prudent also to overexpress the kinase to
ensure its availability for receptor phosphorylation.
For these
studies, transfected COS-7 cells were preincubated with P
and then exposed to either vehicle alone or
vehicle plus 10 µM UK-14304. Mutant and wild type
C10 were then purified and electrophoresed through 10%
discontinuous SDS-polyacrylamide gels. Incorporation of
P
under basal and agonist-treated conditions
for mutant and wild type
C10 was quantitated using a
PhosphorImager. Comparisons of the extent of agonist-promoted
phosphorylation between mutant and wild type
C10 were
made using the net phosphorylation, i.e. after subtraction of
basal phosphorylation. As shown in Fig. 3, all receptors
migrated at a molecular mass of
78 kDa. Wild type
C10 clearly underwent agonist-promoted phosphorylation
with a 3-4-fold increase in receptor phosphorylation during
agonist exposure as compared with basal. In contrast, the complete
substitution mutant AAAA displayed a virtual complete loss in the
extent of agonist-promoted phosphorylation, which amounted to only 9
± 2% of the phosphorylation found with wild type
C10 (n = 4, p < 0.001, Fig. 3). The partial substitution mutants AASS and SSAA, which
also did not undergo agonist-promoted desensitization, were
phosphorylated 55 ± 7% and 57 ± 8% as compared with the
wild type
C10, respectively (n = 4, p < 0.002, Fig. 3). Studies performed in the same
manner using CHO cells stably expressing the complete substitution
mutant AAAA and wild type
C10 yielded similar results
wherein the wild type
C10 readily underwent
agonist-promoted phosphorylation (
4-fold), while the mutant AAAA
did not (-0.5 ± 1.6% net phosphorylation as compared with
the wild type
C10, n = 3, p < 0.001).
Figure 3:
Agonist-promoted phosphorylation of wild
type C10 and mutant receptors AAAA, AASS, and SSAA.
COS-7 cells transiently coexpressing
ARK with wild type
C10 (WT
C10) and the mutant
receptors AAAA, AASS, and SSAA were incubated for 2 h with 0.3 Ci/ml
P
and then exposed to either vehicle or 10
µM UK-14304 for 15 min at 37 °C. Wild type and mutant
C10 were purified as described under
``Experimental Procedures'' and subjected to 10%
discontinuous SDS-polyacrylamide gel electrophoresis. Dried gels were
quantitated using a PhosphorImager and exposed to x-ray film. Shown is
a representative experiment of four
performed.
Based on the complete loss of agonist-promoted
desensitization and phosphorylation found with the mutant AAAA, we
concluded that agonist-promoted phosphorylation occurs solely within
this cluster of four serine residues and not at other serines and
threonines within the receptor. However, since the mutants AASS and
SSAA were partially phosphorylated to an equivalent extent (50%),
the precise residues of Ser-296-299 which are phosphorylated
remained unclear. Since AASS and SSAA, with two of the four serines
remaining, underwent
50% of the agonist-promoted phosphorylation
as seen with the wild type
C10, we presumed that the
total number of serines that are phosphorylated in wild type
C10 would be either four or two. To address these
possibilities, we constructed two additional mutants, denoted as
receptors SAAA and SSSA, and performed whole cell phosphorylation
experiments in parallel with the wild type
C10 and the
SSAA mutant.
The mutants SSSA, SSAA, and SAAA and wild type
C10 were transiently coexpressed with
ARK in
COS-7 cells, and whole cell phosphorylation studies carried out as
before. Comparison of
P incorporation revealed an
agonist-promoted phosphorylation of SSSA, SSAA, and SAAA of 77 ±
1%, 49 ± 6%, and 27 ± 4% of that found with the wild type
C10, respectively (p < 0.001 compared with
wild type
C10, n = 3, Fig. 4).
Thus, as summarized in Fig. 5, substitution of alanine for each
additional serine decreased overall phosphorylation compared with wild
type
C10 by
25%. These data, together with the
absence of agonist-promoted phosphorylation found with the mutant AAAA
and
50% agonist-promoted phosphorylation found with the mutant
AASS relative to wild type
C10, are consistent with
all four serines, Ser-296-299, being phosphorylated in the wild
type
C10.
Figure 4:
Agonist-promoted phosphorylation of wild
type C10 and the mutant receptors SSSA, SSAA, and
SAAA. Whole cell phosphorylation studies using COS-7 cells transiently
coexpressing
ARK with wild type
C10 (WT
C10) and the mutant receptors SSSA, SSAA, and
SAAA were carried out as described for the experiments in Fig. 3. Shown is a representative experiment of three
performed.
Figure 5:
Summary of agonist-promoted
phosphorylation for mutant and wild type C10. Data
from whole cell phosphorylation experiments described in Fig. 3and Fig. 4using wild type
C10 (WT
C10) and the mutants SSSA, SSAA, AASS,
SAAA, and AAAA are summarized. For each receptor, the extent of
agonist-promoted phosphorylation over basal (net phosphorylation) is
expressed as a percentage of the net phosphorylation obtained with wild
type
C10. Results shown are the mean ± S.E.
from four to seven experiments performed. All mutant receptors
displayed significantly less agonist-promoted desensitization as
compared with wild type
C10 (p < 0.002).
The agonist-promoted phosphorylation obtained with the mutant AAAA was
not significantly greater than basal phosphorylation (p = NS).
Receptor sequestration (internalization)
has been thought to play little, if any, role in short term
agonist-promoted desensitization of C10(17) .
Nevertheless, since desensitization was so dramatically affected by
these mutations, we assessed agonist-promoted sequestration in the
mutant AAAA using a [
H]yohimbine binding assay as
described previously(17) . For wild type
C10
and the mutant AAAA, receptor distribution in untreated cells was
similar with 8.5 ± 2.2% versus 13.3 ± 0.3%
sequestered, respectively (n = 3, p =
NS). Following exposure to 100 µM epinephrine for 30 min,
both wild type
C10 and the mutant AAAA underwent
agonist-promoted sequestration to a similar extent with 23.0 ±
1.7% versus 26.3 ± 3.3% sequestered, respectively (n = 3, p = NS). From these data it
appears that the lack of desensitization found in the mutants results
from a loss of receptor phosphorylation and not sequestration.
Agonist-promoted desensitization of AR has
been described in a transfected Chinese hamster fibroblast cell line
(CHW cells)(15) , transfected CHO
cells(17, 18) , NG108-15 cells(30) , HT-29
cells(31) , brain(32) , vascular smooth
muscle(33) , and vas deferens (34) . Studies from our
laboratory have demonstrated that agonist-promoted desensitization of
C10-mediated inhibition of adenylyl cyclase activity
occurs rapidly and that this short term component is due solely to
receptor phosphorylation (15, 16, 17) . There
are multiple lines of evidence which strongly implicate
ARK as the
phosphorylating kinase.
Prior to the cloning of the
AR, Benovic and colleagues (14) demonstrated
that purified platelet
AR (which is
C10) in an in vitro reconstituted
phospholipid vesicle system is a specific substrate for
ARK and is
phosphorylated in an agonist-dependent manner to a degree comparable to
that of the
AR. Using a similar system, Onorato et
al.(19) reported that the synthetic peptide
LEESSSSDHAERPPG, based on a stretch of sequence present in the third
intracellular loop of the
C10, is also a substrate for
ARK. Subsequently, we have found that agonist-promoted
desensitization of recombinantly expressed
C10 occurs
with a rapid onset (minutes), requires exposure to saturating
concentrations of agonist(15, 17, 18) , and
is selectively blocked by the
ARK inhibitor heparin(15) .
Short term agonist-promoted desensitization of
C10 is
accompanied by receptor phosphorylation (15) and a loss of high
affinity agonist binding of the receptor(17) , which is
indicative of receptor-G-protein uncoupling. Loss of agonist-promoted
phosphorylation in a mutated
C10 lacking a serine- and
threonine-rich 75-amino acid portion in the third intracellular loop
resulted in a loss of short term desensitization(15) . Taken
together, the above provide convincing evidence that
ARK-mediated
phosphorylation is the principal mechanism of short term
agonist-promoted desensitization of
C10.
Despite
the above, a precise sequence for ARK-mediated phosphorylation of
the
C10, or any other G-protein coupled receptor, had
not been delineated. We approached this by first deleting 12 amino
acids in the third intracellular loop of
C10, which
removed the EESSSS sequence but left the other 12 serine and threonine
residues in the loop (Fig. 1). This receptor failed to undergo
agonist-promoted desensitization, establishing that the determinants of
this process were confined to this region. We then systematically
substituted the serines within this sequence with alanines, and in a
series of five mutated receptors, assessed agonist-promoted receptor
phosphorylation and desensitization. The AAAA substitution mutant
failed to phosphorylate and to desensitize. Substitutions with alanines
leaving one, two, or three serines in place resulted in mutant
receptors that phosphorylated
25,
50, and
75% of the
wild type receptor, respectively, which is consistent with each of the
four serines being phosphorylated (Fig. 5). In functional
desensitization studies, we found that agonist-promoted desensitization
was completely ablated in the AASS and SSAA mutants (Fig. 2),
despite the fact that they were partially (
50%) phosphorylated as
compared with wild type
C10. There are two
possibilities for this. It may be that the conformational change
induced by phosphorylation of
C10 resulting in
receptor-G-protein uncoupling requires four phosphoserines. Partial
phosphorylation, then, would not result in partial desensitization
since the conformational change induced by such partial phosphorylation
is not sufficient to induce uncoupling. It may also be that we are not
able to detect small degrees of desensitization in the current assay
system. The latter possibility is less likely, since we have
reproducibly detected as little as 2-fold changes in the EC
for
C2-mediated inhibition of adenylyl cyclase
activity in an identical assay(17) .
As discussed earlier,
one limitation in the identification of sites for ARK-mediated
phosphorylation of G-protein coupled receptors is the lack of a defined
consensus sequence. Based on in vitro
ARK phosphorylation
studies using synthetic peptides(19) , and our current work, we
propose that EESSSS in
C10 represents such a sequence.
It may be that similar sequences in other G-protein coupled receptors
are recognized by
ARK. In this regard, as shown in Table 3,
we have examined the third intracellular loop and carboxyl-terminal
portions of members of several representative classes of the G-protein
coupled receptor family for amino acid sequences similar to the EESSSS
of
C10 as potential sites for
ARK-mediated
phosphorylation.
Notably, several of the G-protein coupled receptors
that contain such sequences including the
AR(6, 8) , the muscarinic m
receptor(9) , the thrombin receptor(12) , the
substance P receptor(10) , and the
AR (35) have been shown to be substrates for
ARK. Moreover,
for the
AR (20) and the thrombin
receptor(12) , removal of most of the carboxyl terminus or
substitution of serines and threonines in the carboxyl terminus ablates
ARK-mediated desensitization. Several other receptors in Table 3, including the opossum
AR(36) ,
the D
(37, 38, 39) and D
(40, 41) dopamine receptors, even though
ARK-mediated phosphorylation has not been definitively
demonstrated, display a rapid stage of agonist-promoted
desensitization, which in some cases has been shown to be accompanied
by receptor phosphorylation. Conversely, there are several receptors
known to be phosphorylated by
ARK which do not contain an
analogous sequence to EESSSS, including the adenosine A
receptor(11) , and the
AR subtype,
C2(16) . In this respect it is important to
note that the preference by
ARK for acidic residues immediately
adjacent to the phosphorylated moiety was established using peptides of
12 amino acids which lack the complex structure of an intact
receptor. So, it may be that within the intracellular portions of a
receptor there are residues separate from potential phosphorylation
sites in primary sequence, but nevertheless within the context of
overall receptor conformation, which provide an acidic milieu for
ARK. For instance, the third intracellular loop of
C2 contains a large stretch of 16 consecutive glutamic
acid residues as well as numerous serine and threonine residues. Also,
the human
AR (
C4) which has the
sequence ESS in the third intracellular loop, fails to undergo both
agonist-promoted desensitization (16, 17, 18) and
ARK-mediated
phosphorylation(16) . However, this sequence is not in the
analogous position to the EESSSS of
C10, and there is
marked diversity in the amino acid sequences of the third intracellular
loops of these two receptors Thus, although we have now defined a
precise receptor sequence of amino acids that undergo phosphorylation
by
ARK in intact cells, and similar sequences are present in a
number of G-protein coupled receptors, the location of such a sequence
and the surrounding milieu are also important variables when
considering whether a given receptor is a candidate for
ARK-mediated phosphorylation.
In summary, from the present
studies two conclusions may be drawn. First, the precise sites for
ARK-mediated phosphorylation of
C10 are the four
consecutive serines Ser-296-299 in the third intracellular loop.
And second, even partial phosphorylation of the receptor, as seen with
the mutants AASS and SSAA, does not result in detectable
agonist-promoted desensitization. These four amino acids provide the
structural basis for the primary mechanism of short term
agonist-promoted desensitization of
C10, and they
represent the first discrete motif for
ARK-mediated
phosphorylation to be identified in a G-protein coupled receptor.