(Received for publication, December 5, 1995; and in revised form, January 22, 1996)
From the
As the -adrenergic receptor
(
AR) is resistant to short term agonist-promoted
desensitization and sequestration, chimeric
/
receptors were generated to
identify the molecular determinants responsible for these regulatory
processes in the
AR. By exchanging single or multiple
intracellular domains of the
AR for the corresponding
regions of the
AR, we show that specific domains can
be identified as additive determinants for desensitization, while
sequestration is more dependent on global structural conformation. The
carboxyl-terminal tail, the third and the second intracellular loops of
the
AR provided additive contributions to the
desensitization observed upon short term agonist stimulation. The
second intracellular loop plays a role which is as important as that of
third cytoplasmic loop and carboxyl-terminal tail which had previously
been identified as the major determinants of agonist-promoted
desensitization. Additive contributions of the cytoplasmic domains of
the
AR were also observed for agonist-promoted
sequestration. The substitution of the first and second intracellular
loops and the carboxyl tail were associated with a
-like sequestration phenotype. However, in contrast to
what is observed for desensitization the co-substitution of the third
cytoplasmic loop with any of the other domains completely suppressed
sequestration. These results suggest that sequestration depends not
only on appropriate interactions of multiple molecular determinants
within the cytoplasmic region of the
AR but also on
conformational determinants that may influence their orientation.
Cellular responses to -adrenergic receptor (
AR) (
)stimulation are initiated by functional coupling of the
receptor with the stimulatory GTP-binding protein G
which
in turn activates adenylyl cyclase thus promoting a rise in
intracellular cAMP concentration. This signal transduction pathway is
tightly controlled by regulatory processes which, on the one hand,
prevent hormonal overload (desensitization) and, on the other hand,
reset the signaling pathways for further hormonal stimuli
(resensitization).
Rapid desensitization of the AR,
which occurs as early as a few minutes following the initiation of the
stimuli, results from the uncoupling of the receptor from
G
. Phosphorylation of the
AR by
cAMP-dependent protein kinase (protein kinase A) and
-adrenergic
receptor kinase (
ARK) is known to play a central role in this
process(1, 2, 3) . Although several
phosphorylation sites involved in this uncoupling process have been
unambiguously identified(4, 5) , the existence of
other important site(s) has never been ruled out.
Recent studies have suggested that agonist-promoted sequestration is a resensitization mechanism limiting the effects of short term desensitization. Indeed, blocking sequestration was found to significantly delay resensitization which normally occurs upon termination of receptor activation(6, 7) . According to the proposed model, phosphorylated receptors are sequestered in a subcellular compartment where they are dephosphorylated and become available for recycling to a fully functional conformation to the plasma membrane.
Although
significant efforts have been made, an unequivocal identification of
the molecular determinants triggering the sequestration process has yet
to be achieved. Previous studies using site-directed mutagenesis have
suggested the existence of several motifs located in various
cytoplasmic domains of the AR (8, 9, 10) , but a coherent functional
connection between these molecular determinants is lacking.
Previous
studies have also shown that the AR does not readily
undergo rapid agonist-promoted desensitization and
sequestration(11, 12, 13) . This resistance
to rapid regulation and the high level of sequence identity between the
AR and the
AR makes the latter an
excellent model to investigate the molecular determinants of
desensitization and sequestration. Indeed, an alternative to
site-directed mutagenesis in examining the molecular determinants of
desensitization is the construction of chimeric receptors. This
approach has the advantage of searching for the addition of regulatory
phenotypes rather than their loss. Construction of chimeric receptors
in which specific domains of the
AR have replaced
their counterparts within the
AR has already been
successfully used to study their contribution to desensitization.
Substitutions of the carboxyl-tail alone (11) or of both the
carboxyl-tail and third cytoplasmic loop (12) of the
AR by the corresponding region of the
AR have been shown to confer agonist-promoted
desensitization to the
AR. However, neither of the
chimeric receptors studied had desensitization profiles comparable with
that of the wild-type
AR. This suggests that other
regions of the receptor are required for a complete
AR-like desensitization.
To identify additional
putative determinants involved in rapid desensitization of the
AR and to assess the respective contribution of
AR intracytoplasmic domains to the sequestration
process, we have constructed a series of chimeric receptors in which
various combinations of the
AR intracellular loops
were replaced by the corresponding domains of the
AR.
Figure 1:
A, topological model of the
human AR. Conserved amino acid residues between the
AR and
AR sequences are represented
by filled circles. Arrows indicate the connecting
sites between the
AR core sequence and the substituted
AR cytoplasmic domains in the chimeric receptors. B, sequence comparison of the cytoplasmic domains of
AR and
AR. Hyphens indicate
identity with the
AR sequence. Triangles indicate protein kinase A phosphorylation sites. Stars indicate potential phosphorylation sites for
ARK. Motifs
identified as potential determinants of sequestration are overlined.
Chimeric receptor constructs were subcloned into the
eucaryotic expression vector pcDNA3/RSV. This vector was generated by
insertion of the BglII-HindIII restriction fragment
from the pRc/RSV vector (Invitrogen) into pcDNA3 (Invitrogen) so as to
replace the cytomegalovirus promoter. Constructs were then stably
transfected in murine L-cells as described previously (12) .
Geneticin-resistant cells were selected in DMEM supplemented with 10%
(v/v) fetal bovine serum, 4.5 g/liter glucose, 100 units/ml penicillin,
100 mg/ml streptomycin, 1 mM glutamine, and Geneticin at a
concentration of 400 µg/ml. Individual clones were screened for
AR expression by radioligand binding assay, using
[
I]CYP as ligand.
Chimeras were named
starting with their receptor subtype followed by four numbers
indicating the origin of the 1st, 2nd, 3rd cytoplasmic loops and of the
carboxyl-tail, respectively. For example -3322
represents a
AR with the first and second cytoplasmic
loop of the
AR and the third cytoplasmic loop and
carboxyl-terminal tail of the
AR.
Adenylyl cyclase activity was measured on these membrane
preparations according to the method of Salomon et
al.(16) . Briefly, the reaction mixture contained: 20
µl of membrane preparation (2-6 µg of protein), 45 mM Tris (pH 7.4), 3 mM MgCl, 1.2 mM EDTA, 0.12 mM ATP, 0.053 mM GTP, 0.1 mM cAMP, 0.1 mM isobutylmethylxanthine, 1 µCi of
[
P]ATP, 2.8 mM phosphoenolpyruvate, 0.2
unit of pyruvate kinase and 1 unit of myokinase in a final volume of 50
µl. Enzymatic activity was determined in the presence of 0-1
mM isoproterenol for 30 min at 37 °C. The reactions were
terminated by the addition of 1 ml of ice-cold stop solution containing
0.4 mM ATP, 0.3 mM cAMP and 25,000 cpm
[
H]cAMP. The cAMP was then isolated by sequential
chromatography on Dowex cation exchange resin and aluminum oxide. Data
are expressed as picomoles of cAMP produced per min per mg of protein.
The results of three to eight experiments were fitted simultaneously
(using the nonlinear least squares regression program SigmaPlot) so as
to give an averaged best fit value.
Figure 2:
Desensitization of
/
-chimeric receptors. Adenylyl
cyclase activity was measured before (open circles) and after
2 (filled circles) and 15 min (open triangle)
treatment with 10 µM isoproterenol as described under
``Experimental Procedures.'' Data represent fits obtained
from the simultaneous analysis of three to eight individual experiments
carried out in duplicate using the computer program SigmaPlot. The
nonlinear least squares regression analysis was performed using the
following equation: f(x)= [a - d]/ (1 + (x/c)b) + d; where a is the maximal activity, b is the slope of the curve, c represent the EC
, and d the basal
activity.
Substitution of the first cytoplasmic
loop (i1) of the AR with the corresponding region from
AR did not confer any desensitization phenotype as
prestimulation of cells expressing this mutant receptor, for 2 or 15
min, did not affect either the dose-response curves or the maximal
stimulation (Fig. 2, panel B). As expected from
previous studies, substitution of either i3 (Fig. 2, panel
C) or CT (Fig. 2, panel D) conferred
desensitization profiles that are characterized mainly by rightward
shifts of the dose-response curves that became clearly evident
following 15 min of prestimulation. Moreover, as can be seen in panel F, the contribution of these two domains to the
desensitization pattern appear to be additive. Indeed, the
desensitization of the
-3322 for 2 and 15 min lead to
larger shifts of the dose-response curves and provoked a sizable
reduction in the maximal stimulation observed. However, the extent of
desensitization did not reach that observed for the
AR, suggesting that other domains could be required to
obtain a full
AR desensitization phenotype.
Interestingly, the single substitution of the second intracellular loop
(i2) of the
AR by that of the
AR
(
-3233) was sufficient to promote desensitization. In
fact, pretreatment of cells expressing this chimera with isoproterenol
induced reductions in agonist-stimulated adenylyl cyclase activity that
are at least of the same magnitude as those observed for
-3332 and
-3323 (compare panel E with panels C and D). The contribution of i2 to
desensitization is also supported by the observation that
agonist-promoted desensitization observed with the double substitution
of i2 and CT into the
AR was faster and larger than
that conferred by single substitution of CT alone (compare panels G and D). Interestingly, the reduction in
agonist-stimulated adenylyl cyclase activity in cells expressing
-3232 was even faster than that observed in cells
expressing
-3322. Indeed, a 25% reduction in the
maximal stimulation was already evident following a 2-min preincubation
period for the
-3232, whereas no change in the maximal
stimulation was observed at this time for the
-3322.
An additive effect of i2 on desensitization is also evident when
comparing
-3222 with
-3322 (compare panels F and H).
The apparently additive
contribution of the AR i2, i3, and CT to the
desensitization profiles of the chimeric receptors can be easily
appreciated by looking at Fig. 2. Indeed, it can be seen that
co-substitution of these three domains leads to a progressive increase
in the overall extent of desensitization. However, a quantitative
assessment of the additivity is rendered difficult by the fact that
desensitization is reflected by changes in two parameters, i.e. a reduction in the maximal stimulation and a rightward shift of
the dose-response curves. The assessment of the dose-response shifts is
further complicated by the fact that not all chimeras have the same
efficacy to stimulate the adenylyl cyclase activity under basal
conditions and that in several cases, the desensitized adenylyl cyclase
activity did not reach a plateau at the highest isoproterenol
concentration used (1 mM), thus making mathematical analysis
more difficult. Therefore, the reduction in adenylyl cyclase activity
measured for a stimulating concentration of isoproterenol equal to its
EC
for a given chimera was used as an index of the
dose-response rightward shift. Fig. 3illustrates the amplitude
of the changes in stimulation at maximal concentration and at the
EC
for all the chimeric receptors following a
desensitization of 15 min. The effects of the single substitutions of
i2, i3, or CT on the desensitization are reflected mainly by a
reduction of the response at the EC
with only marginal
effects on the maximal stimulation. Double or triple substitution of
these domains conferred agonist-dependent reduction of both the maximal
stimulation and of the stimulation at the EC
.
Interestingly, the substitution of i1, which has no effect on the
desensitization pattern by itself, conferred a slight negative effect
on the desensitization of the maximal response when co-substituted
along with other cytoplasmic domain of
origin
(compare
-3232 and
-3222 with
-2232 and
-2222). This might result
from an unfavorable conformational effect, since the affinity for
[
I]CYP was also significantly reduced by the
co-substitution of i1 (Table 1). Since
-2222 and
-2232 also have slightly reduced affinity for
isoproterenol, as indicated by their higher K
when compared with
AR (Table 1), it might
be suggested that the lower extent of the maximal response
desensitization of these chimeras is an underestimation resulting from
the potentially nonsaturating conditions used. However, this is highly
unlikely, since
-3232 and
-3222 have
similar elevated K
, and yet they display the
greatest desensitization of the maximal response approaching the level
observed for the
AR.
Figure 3:
Quantitative assessment of the
/
-chimeric receptor desensitization.
Isoproterenol-stimulated adenylyl cyclase activity was measured in
membrane preparations derived from cells pretreated or not with
isoproterenol (10 µM) for 15 min. Data are expressed as
percent reduction of the stimulated activity (A) at the
maximal stimulatory concentration (1 mM of isoproterenol) (B) at the EC
values for each chimera determined
in the absence of pretreatment. Data shown are mean ± S.E. for
three to eight experiments carried out in duplicate. For
-3222, the EC
was calculated by taking
the stimulation attained at 1 mM as the maximum
stimulation.
Figure 4: Sequestration of chimeric receptors following stimulation with 10 µM isoproterenol for 5 and 15 min. Receptor sequestration was determined by differential centrifugation of plasma membrane and light vesicular fractions as described under ``Experimental Procedures.'' The level of sequestration is expressed as percentage of total receptor number. Data are means ± S.E. for three independent experiments carried out in triplicate.
Sequestration of the AR in
Ltk
cells reached a maximum of 34% after 15 min of
isoproterenol stimulation. Thus, we first screened L cells expressing
the chimeric receptors described above by measuring sequestration
following incubation with 10 µM isoproterenol for 5 and 15
min (Fig. 4). The single substitution of either CT, i2 or i1 of
the
AR with the corresponding regions of the
AR partially restored agonist-promoted sequestration
(
-3332: 12%;
-3233: 8%;
-2333: 7%, following a stimulation of 15 min). In
contrast, substitution of i3 had no apparent effect on the
sequestration pattern. Positive effects on sequestration of the CT, i2,
and i1 appeared partially additive as the level of sequestration
observed for
-3232 and
-2232 tended
to be greater than those observed when each of these domains were
substituted alone. However, it should be noted that substitution of i3
had a dominant negative effect over all other substitutions. Indeed, no
agonist-promoted sequestration was observed in any of the
AR chimeric receptors containing an i3 of
AR origin. Based on these data, one could postulate
the existence of a specific sequence located in i3 of the
AR that inhibits sequestration. If such a negative
sequence exists, substitution of this
AR domain by the
corresponding region of the
AR, would be expected to
lead to a ``super-sequestration'' profile. We studied the
sequestration profile of such a chimeric
-2232
receptor: the sequestration pattern was indistinguishable from that of
the
AR wild type (data not shown) arguing against the
existence of specific sequences within the i3 loop that inhibit
sequestration. Alternatively, one could argue that the lack of
sequestration of
-2222 results from its reduced
affinity for isoproterenol, which is reflected by a 14-fold increase in
the K
when compared with the
AR (Table 1). This is highly unlikely since
-3232
has an even higher K
, but undergoes
sequestration which reached levels comparable with that attained for
the
AR. Also, increasing the concentration of
isoproterenol used to promote sequestration to up to 1 mM did
not induce any sequestration of
-2222 (data not
shown).
To further characterize chimeric receptors showing positive
sequestration, this process was studied for longer periods of time. For
the three single substitutions (-2333,
-3233,
-3332), agonist-promoted
sequestration reached its maximum between 15 and 30 min of stimulation
(10%-15%) and remained at that level for up to 60 min (Fig. 5).
Although sequestration was observed for these three chimeras, the level
of sequestration never reached that observed for the
AR (30%). In contrast, sequestration of
-3232 and
-2232 attained levels
observed for the
AR albeit with somewhat slower
kinetics. Indeed, sequestration levels were equivalent to those of the
AR after 60 min of stimulation. These results suggest
that three
AR cytoplasmic domains, i1, i2, and CT,
provide positive sequestration signals that may be somewhat additive
but are not sufficient to restore an entirely normal
AR sequestration profile.
Figure 5: Time course of agonist-promoted sequestration. Cells were treated with 10 µM isoproterenol at 37 °C for the indicated times and percentage of sequestered receptors determined as described in Fig. 4. Data are means of three independent experiments carried out in triplicate.
Despite extensive investigation during the past years,
molecular mechanisms involved in short term AR regulation have not
been completely elucidated. We took advantage of the high degree of
homology existing between the
AR and the
AR (69% within putative membrane spanning domains and
corresponding junctions with intracellular loops) and of their distinct
profile of regulation to identify novel molecular determinants of
receptor desensitization and sequestration. Current hypothetical models
suggest that molecular determinants of
AR regulation
are located in intracellular domains(18) . Sequence homology
between
AR and
AR facilitated the
exchange of unmodified intracellular domains and the construction of
functional chimeric receptors. We previously showed that the chimeric
receptor strategy is particularly adapted to study molecular basis of
receptor function(19) . This approach, complementary to
site-directed mutagenesis studies, allows assessment of the
contribution of entire structural domains without preconceived notions
of the precise residues involved.
The chimeric receptors constructed
in the present study conserved pharmacological properties
characteristic of the AR. In particular,
-2222 which contains the largest proportion of
AR derived sequence maintained all the pharmacological
trademarks of the
AR including the agonistic
properties of the
AR antagonist CGP12177A. Previous
studies, based on molecular modelling and pharmacological
characterization of the
- and
AR
suggested that
-antagonists with
-agonist properties, such as CGP12177A, may adopt a
stacked conformation in the
AR binding pocket, leading
to antagonistic effects while they would adopt an extended conformation
in the less encumbered
-binding site. This last
conformation may allow interactions with specific residues implicated
in signal transduction(20) . The
-like
pharmacological properties maintained in
-2222 suggest
that the intracellular domains do not affect the overall organization
of the binding pocket determined by the positioning and the orientation
of the transmembrane domains.
As reported previously(12) ,
substitution of the cytoplasmic domains of the AR with
those of
AR containing all known specific consensus
sequences for receptor phosphorylation by
ARK and protein kinase A
(i3, CT) (4, 5, 15, 21, 22) failed
to confer a
AR-like desensitization profile. The
present report clearly shows that additional molecular determinants
involved in receptor desensitization are also present in the
AR i2. The presence of this domain alone is sufficient
to confer a desensitization level at least equivalent to that provided
by CT and i3. Furthermore, when substituted in combination with the
other domains, additive effects on the level of agonist-promoted
desensitization were found. Consistent with the contribution of i2 to
receptor desensitization is the observation that Phe-139 located in i2
of the
AR is apparently involved in G-protein coupling (8) . The recent finding that phosphorylation of Tyr-141 within
the
AR i2 favors its coupling with G
(23) also suggest that this domain plays an important
role in the regulation of receptor-G
interaction. The
contribution of i2 to the desensitization process could result from its
interaction with previously identified proteins, which regulate
receptor function, such as
ARK or
-arrestin thus stabilizing
their interactions with domains already characterized. Alternatively,
i2 may contain new unidentified sites which promote receptor
uncoupling. The observation that substitution of i2 alone is sufficient
to confer receptor desensitization would support the latter.
All
molecular determinants of AR uncoupling identified so
far correspond to phosphorylation targets for protein kinases. In a
previous report mutation of all putative
ARK, protein kinase A and
protein kinase C phosphorylation sites significantly reduced
agonist-promoted phosphorylation and desensitization but did not
completely abolish them(5) . This is consistent with the idea
that additional phosphorylation sites may exist and be involved in
receptor desensitization. Two serine residues Ser-137 and Ser-143
present in i2 of
AR are absent from the
AR. These residues might be the target of another
kinase. One serine (Ser-137) is contained in the potential
phosphorylation consensus site S/T P X K/R, which has been
shown to be a preferred substrate for cdc2 kinase(24) .
Additional experiments are required to assess whether this region
contains phosphorylation sites involved in receptor uncoupling and to
identify the putative kinase participating in such regulation.
Previous studies have suggested the existence of several motifs
located in various cytoplasmic domains of the AR
involved in sequestration. However, no clear connection could be
established between these motifs that leads to an unequivocal
identification of the molecular determinants triggering the
sequestration process. In their studies, Hausdorff et al.(25) showed that site-directed mutagenesis of a subset of
serine residues, believed to be
ARK phosphorylation sites, blocked
agonist-promoted sequestration. In particular, substitution of Ser-356
and Ser-364 by glycine residues completely blocked sequestration.
However, mutations of additional serines and threonines in this region
restored a normal sequestration phenotype(4) . The authors
concluded that Ser-356 and Ser-364 are not required for sequestration
but that their mutation leads to conformational changes interfering
with the sequestration process. Also suggesting that receptor
conformation may influence sequestration is the recent report by Green
and Liggett(9) , indicating that a proline-rich sequence
located in the third cytoplasmic loop of the
AR
prevents the efficient sequestration of this receptor subtype.
Recently, a tyrosine residue (Tyr-326) located at the interface between
the seventh transmembrane domain and the carboxyl tail has been
proposed as a specific determinant for
AR
sequestration(10) . Although this residue may be required, it
is certainly not sufficient to confer an agonist-promoted sequestration
phenotype. Indeed, a tyrosine residue within a NPXXY motif
identical to that of the
AR is also present in a
similar position in the
AR. However, the
AR subtype is not sequestered upon agonist stimulation (11, 12) . In addition, mutation of the tyrosine
residue located in the NPXXY motif of the gastrin-releasing
peptide receptor or of the Type 1 angiotensin II receptor did not
affect their agonist promoted sequestration arguing against a general
role for this sequence (26, 27) . More recently,
Ferguson et al.(28) proposed that the reduction of
sequestration caused by the mutation of Tyr-326 in the
AR resulted from the inability of this mutant receptor
to act as a substrate for
ARK. They proposed that
ARK-mediated phosphorylation facilitates
AR
sequestration. Although that may be the case, it is clear from previous
studies that phosphorylation by
ARK is not an absolute requirement
nor is it the signal initiating the sequestration process. Indeed, it
has been shown that
AR lacking all putative
ARK
phosphorylation sites can readily be sequestered upon agonist
stimulation(3, 7, 10, 28) . The
presence of an hydrophobic residue in the
DRYXXI(V)XXPZ sequence (where Z is the hydrophobic
residue) within the second cytoplasmic loop of the
AR
has also been proposed as being important for receptor
sequestration(8) . Such a hydrophobic residue is conserved in
identical position in the
AR (DRYLAVTNPL), suggesting that the presence of this
residue is not sufficient to facilitate agonist-promoted sequestration.
Our data support the notion that interaction between multiple
intracellular domains of the AR contribute to
sequestration phenotypes. Clearly, none of the cytoplasmic domains
(which contain the various sequestration signals previously proposed),
when substituted alone, could confer a
AR-like
sequestration pattern. In fact, i1, i2 and CT allowed very modest
agonist-promoted sequestration, while the association of the second
intracytoplasmic loop with the carboxyl terminus of the
AR in the chimeric
-3232 and
-2232 receptor restored sequestration levels similar
to that of the
AR albeit with slower kinetics. These
results suggest that CT and i2 of the
AR play major
roles in the sequestration process. The contribution of CT is
consistent with the recently proposed facilitator role of CT
ARK
phosphorylation sites in the sequestration(28) . However, the
mere presence of these motifs is not sufficient to assure a
sequestration phenotype. Indeed, no sequestration was detected in any
of the chimeric receptor harboring the third cytoplasmic loop from
AR origin. This negative effect is clearly not
attributable to the presence of a specific signal preventing
sequestration, since it is compatible with normal sequestration of the
wild type
AR. These data therefore suggest that,
together with the concerted participation of multiple cytoplasmic
domains, the adoption of an appropriate conformation resulting from
specific interactions among intra-cytoplasmic domains is required for
proper sequestration.
In conclusion, we have shown that in addition
to the carboxyl tail and the third cytoplasmic loop, the second
cytoplasmic loop of the AR is involved in the process
of agonist-promoted desensitization. Also, sequestration does not
merely depend on the presence of specific domains (i.e. i2 and
CT) but largely relies on the proper arrangement of all the cytoplasmic
domains.