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(Received for publication, December 5,
1995; and in revised form, January 22, 1996) From the
As the
Cellular responses to Rapid desensitization of the 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 Previous
studies have also shown that the To identify additional
putative determinants involved in rapid desensitization of the
Figure 1:
A, topological model of the
human
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
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
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
Figure 2:
Desensitization of
Substitution of the first cytoplasmic
loop (i1) of the The apparently additive
contribution of the
Figure 3:
Quantitative assessment of the
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 To further characterize chimeric receptors showing positive
sequestration, this process was studied for longer periods of time. For
the three single substitutions (
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 The chimeric receptors constructed
in the present study conserved pharmacological properties
characteristic of the As reported previously(12) ,
substitution of the cytoplasmic domains of the All
molecular determinants of Previous studies have suggested the existence of several motifs
located in various cytoplasmic domains of the Our data support the notion that interaction between multiple
intracellular domains of the In conclusion, we have shown that in addition
to the carboxyl tail and the third cytoplasmic loop, the second
cytoplasmic loop of the
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9355-9362
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Adrenergic
Receptor Desensitization and Sequestration
DELINEATION USING CHIMERIC
/
-ADRENERGIC RECEPTORS (*)
-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.
-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).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.AR (8, 9, 10) , but a coherent functional
connection between these molecular determinants is lacking.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.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.
Materials
[
P]ATP and
[H]cAMP were from DuPont NEN and
[
I]CYP was from Amersham Corp. Isoproterenol,
norepinephrine,(-)-alprenolol, DL-propranolol, ATP, GTP,
cAMP, bovine serum albumin, phosphoenolpyruvate, myokinase,
isobutylmethylxanthine, dibutyryl cAMP, forskolin, leupeptin, soybean
trypsin inhibitor, and benzamidine were from Sigma. Pyruvate kinase was
from Calbiochem. DMEM, fetal bovine serum, PBS, trypsin-EDTA, Geneticin
(G418), penicillin, and streptomycin were from Life Technologies, Inc.
CGP12177A and bupranolol were generous gifts from Ciba Geigy and
Schwarz Pharma, respectively. BRL37344 was a gift from SmithKline
Beecham Pharmaceutical, and ICI118551 and ICI201651 were gifts from
Imperial Chemical Industries Zeneca.Construction of Chimeric Receptors and Cell
Culture
Chimeric /AR coding
regions illustrated in Fig. 1were constructed using the
following approaches: (i) for the exchange of the third cytoplasmic
loop and of the carboxyl terminus, silent restriction sites were
generated by site-directed mutagenesis (third cytoplasmic loop: AccI at Val-222 of AR and at Val-218 of
AR; AvrII at Leu-294 of AR
and at Leu-275 of AR and carboxyl terminus: PstI at Arg-348 of AR and at Arg-328 of
AR; ScaI four bases following the stop codon
of the AR. Entire domains of the AR
delimited by these sites were then replaced by the corresponding region
of the AR). (ii) For the exchange of the second
cytoplasmic loop, a AgeI restriction site was created at
Arg-135 of the AR. The AgeI-BstEII
fragment of this AR was excised and replaced by a
double-stranded synthetic oligonucleotide encoding the corresponding
region of the AR. The remaining two amino acids from
the AR sequence (Arg-152 and Cys-153) were mutated to
Asn and Lys by site-directed mutagenesis to complete the sequence of
the AR. (iii) For the first cytoplasmic loop,
site-directed mutagenesis of Trp-64, Thr-65, Pro-66, and Met-71 to Lys,
Phe, Glu, and Val, respectively, was performed to generate a
AR sequence. All mutations were confirmed by dideoxy
sequencing.
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.
AR expression by radioligand binding assay, using
[I]CYP as ligand.-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.Radioligand Binding Assay
Nearly confluent cells,
grown as monolayers, were washed with PBS, incubated for 5 min with 2%
trypsin/EDTA at 37 °C and resuspended in DMEM supplemented with 10%
(v/v) fetal bovine serum. The cells were then centrifuged at 450
g for 5 min at 4 °C and washed twice with ice-cold
PBS. Binding assays were carried out using 100 µl of cell
suspension in a final volume of 250 µl containing 50 mg/ml bovine
serum albumin and 1 µM desipramine.
[I]CYP at 200 pM (for
AR) or at 600 pM (for AR)
was used as the radioligand. Specific binding was defined as binding
displaced by 10 µMD/L-propranolol
(AR) or 50 µM bupranolol
(AR). Assays were carried out for 90 min at 25 °C
and terminated by rapid filtration through Whatman GF/C glass fiber
filters previously soaked in PBS containing 0.3% polyethyleneimine (to
reduce nonspecific binding). Protein concentrations were determined on
broken cell preparations by the method of Bradford (14) using
the Bio-Rad protein assay system with bovine serum albumin as standard. Sequestration Assays
Cells grown in 75-cm flasks were incubated in the presence of 10 µM isoproterenol in DMEM containing 10 µM ascorbic acid
or the vehicle alone for the indicated periods of time. The flasks were
then placed on ice, washed twice with ice-cold PBS, and the cells
detached mechanically in a buffer containing 5 mM Tris, 2
mM EDTA, pH 7.4, 5 mg/liter soybean trypsin inhibitor, 5
mg/liter leupeptin, and 10 mg/liter benzamidine (buffer A). Cell
suspensions were homogenized with a Polytron homogenizer (Janke &
Undel Ultra-Turrax T25) for 5 s at maximal setting. The lysate was
centrifuged at 450 g for 5 min at 4 °C. The
supernatant was layered on top of a 35% sucrose cushion and centrifuged
at 150,000 g for 90 min. As reported
previously(15) , the light membrane vesicular fraction was
found at the 0-35% interface, whereas the plasma membrane
fraction sedimented at the bottom of the sucrose cushion. Each fraction
was collected, diluted in buffer A and centrifuged at 200,000 g for 60 min. The pelleted membranes were resuspended in 50
mM Tris, 5 mM MgCl, pH 7.4, and used
immediately for radioligand binding assays. Binding assays were
conducted as described above but using membrane preparations instead of
cell suspensions. Sequestration levels measured for the wild type
AR using this technique were identical to those
measured in whole cell binding assays in which sequestration is defined
by the number of [I]CYP binding sites
inaccessible to the hydrophilic ligand CGP12177A (data not shown).Adenylyl Cyclase Assay
Cells grown in 75-cm flasks were incubated in DMEM containing 10 µM ascorbic acid with or without 10 µM isoproterenol for
the indicated periods of time. Incubations were stopped by washing the
cells twice with ice-cold PBS. Cell were then detached mechanically and
homogenized in ice-cold buffer A using a polytron homogenizer (Janke
& Undel Ultra-Turrax T25). Lysates were centrifuged at 450 g for 5 min at 4 °C. Supernatants were centrifuged at
43,000 g for 20 min at 4 °C and the pellets washed
twice in buffer A. The washed membranes were then resuspended in a
buffer containing 75 mM Tris, pH 7.4, 5 mM MgCl, 2 mM EDTA, and protease inhibitors. , 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.Determination of Intracellular Cyclic AMP
Levels
Cells were washed once in PBS and incubated in the
absence or presence of 10 µM of isoproterenol or CGP12177A
for 15 min at 37 °C in PBS 0.5 mM isobutylmethylxanthine,
0.5 mM ascorbic acid. The incubation buffer was discarded and
cells lysed in 1 M NaOH for 30 min at 37 °C. The lysate
was neutralized with 1 M acetic acid and centrifuged in a
microcentrifuge at maximum speed for 5 min. The supernatant was used
for cAMP determination using [H]cAMP
radioimmunoassay system (Amersham Corp.).
Pharmacological Characterization of the
In order to
assess the contribution of the various cytoplasmic domains of the
/AR ChimerasAR to agonist-promoted uncoupling and sequestration,
chimeric receptors were constructed which comprised extracellular and
transmembrane regions of AR origin and various
combinations of intracellular domains from AR and
AR. Conserved amino acid sequences between the
- and the AR within the putative
transmembrane regions and the corresponding junctions with
intracellular loops facilitated the exchange of intracellular domains (Fig. 1). For the chimeric receptor identified as
-2222, all the cytoplasmic domains of the
AR were substituted by those of the
AR. Eight additional
/AR chimeric receptors were
generated. Clones isolated from Ltk
cells stably
expressing between 150 and 960 fmol of receptor/mg of protein were
selected for further study. As shown in Table 1, all the chimeras
constructed retained affinity constants for
[I]CYP comparable with that of the wild type
AR (500-3000 pM), which is
significantly higher than that of the AR (51
pM). Further characterization of -2222
clearly showed that, despite a moderate decrease in the affinity for
several ligands, this receptor which contains the largest contribution
of AR sequences, still retained a general order of
potency for -adrenergic ligands which is characteristic of the
AR (Table 2). Particularly revealing is the
relatively low affinity of the -2222 for
isoproterenol, alprenolol, bupranolol, and ICI118551. Furthermore,
CGP12177A which is an antagonist at the AR but has
partial agonistic properties toward the AR, stimulated
adenylyl cyclase with an intrinsic activity of 0.6 in cells expressing
-2222, thus confirming the AR-like
pharmacology of -2222 (Table 2).
Agonist-promoted Desensitization of
Previous
reports showed that substitution of the third cytoplasmic loop (i3)
and/or the carboxyl tail (CT) in the -Adrenergic-stimulated Adenylyl Cyclase ActivityAR by its 2
equivalents partially restored a -like rapid
desensitization profile(11, 12) . However, neither the
CT alone nor the combination of i3 and the CT restored desensitization
of a similar magnitude to that observed for the wild type
AR. The chimeras described above were thus used to
determine the contribution of each cytoplasmic loop to the
desensitization pattern. Desensitization induced by pretreatment of the
cells with isoproterenol (10 µM) for 2 and 15 min are
shown in Fig. 2. As reported previously, isoproterenol caused a
rapid desensitization in cells expressing the wild type
AR (Fig. 2, panel I). Desensitization
was characterized by a rightward shift of the isoproterenol
dose-response curve and by a robust time-dependent reduction of the
maximal stimulation promoted by the agonist. Also, consistent with
previous observations, sustained stimulation of the
AR-expressing cells (Fig. 2, panel A)
induced only a modest shift in the isoproterenol dose response curve
and a very small decrease in the maximal stimulation following a
desensitization of 15 min. This confirms that the AR
is largely resistant to rapid agonist-induced desensitization compared
with the AR.
/-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.
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).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.
/-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.
Molecular Determinants of Isoproterenol-dependent
Sequestration
As reported previously(17) , agonist
stimulation leads to a rapid and time-dependent translocation of
AR from the plasma membrane to a light vesicular
fraction (Fig. 4). In contrast, no such sequestration is
observed in cells expressing the AR. In fact,
isoproterenol promoted an apparent enrichment of the plasma membrane
fraction in -binding sites (expressed in Fig. 4as negative sequestration). In previous attempts aimed at
identifying molecular determinants of sequestration, the exchange of
intracellular domains of the AR with the corresponding
regions of the AR (11, 12) led to
apparently contradictory observations: the exchange of the CT alone
partially restored agonist-promoted sequestration, but no sequestration
could be observed in a chimeric receptor harboring both the i3 and the
CT of the AR. Therefore, we tested whether any single
or multiple exchanges between intracellular regions of receptors could
restore a sequestration profile similar to that observed for the
AR.
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).-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.
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.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.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.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. 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. 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.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.
)AR, -adrenergic receptor; ARK, -adrenergic receptor
kinase; CYP, iodocyanopindolol; DMEM, Dulbecco's modified
Eagle's medium; PBS, phosphate-buffered saline; CT, carboxyl
tail.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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