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Vol. 55, Issue 2, 339-347, February 1999
Institut de Pharmacologie et de Toxicologie, Faculté de Médecine, 1005 Lausanne, Switzerland (S.M-K., A.S., L.A., D.D., S.C.); and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina (L.S.B., M.G.C.)
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Summary |
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 Top
 Summary
 Introduction
Experimental procedures
Results
Discussion
References
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We compared the phosphorylation and internalization properties of
constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at
A293 in the distal part of the third intracellular loop and at
D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of
agonist-independent phosphorylation were 150% and 50% higher than
those of the wild-type alpha-1b AR, respectively. On the
other hand, for the constitutively active D142A and D142T mutants, the
basal levels of phosphorylation were similar to those of the wild-type
alpha-1b AR and did not appear to be further stimulated
by epinephrine. Overexpression of the guanyl nucleotide binding
regulatory protein-coupled receptor kinase GRK2 further increases the
basal phosphorylation of the A293E mutant, but not that of D142A
mutant. Both the wild-type alpha-1b AR and the A293E
mutant could undergo 
-arrestin-mediated internalization. The
epinephrine-induced internalization of the constitutively active A293E
mutant was significantly higher than that of the wild-type
alpha-1b AR. In contrast, the D142A mutant was impaired
in its ability to interact with -arrestin and to undergo
agonist-induced internalization. Interestingly, a double mutant
A293E/D142A retained very high constitutive activity and regulatory
properties of both the A293E and D142A receptors. These findings
demonstrate that two constitutively activating mutations occurring in
distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.
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Introduction |
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Top
Summary
Introduction
Experimental procedures
Results
Discussion
References
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The
alpha-1b adrenergic receptor (AR) belongs to the superfamily
of guanyl nucleotide binding regulatory protein (G protein)-coupled receptors (GPCR). The seven transmembrane domains (TMD) common to all
GPCRs contribute to the formation of the ligand binding pocket, whereas
amino acid sequences of the intracellular domains appear to mediate
receptor-G protein coupling (Savarese and Fraser, 1992
). Agonist
binding to a GPCR is believed to induce a conformational change of the
receptor that results in its productive coupling to heterotrimeric G
proteins, thus leading to intracellular signaling events.
After receptor activation, exposure to the agonist can induce a series
of biochemical events, resulting in desensitization of various GPCRs. A
prominent role in agonist-induced desensitization of several GPCRs is
played by GPCR kinases (GRK) whose activation does not require the
production of second messengers (Premont et al., 1995). Once the
receptor is occupied by the agonist, it is recognized by the kinase and
becomes phosphorylated. The subsequent uncoupling of the receptor and G
protein is then mediated by arrestin proteins that specifically bind to
the phosphorylated receptor (Wilden et al., 1986; Benovic et al.,
1987). GRKs have the unique ability to recognize and phosphorylate
their GPCR substrates only in their active (i.e., agonist-occupied) conformation.
Exposure to the agonist can also induce receptor internalization that
results in the decrease of cell surface receptors and their
translocation toward an intracellular compartment (Fonseca et al.,
1995; Koenig and Edwardson, 1997). Several lines of evidence indicate
that internalization does not mediate receptor desensitization because
it occurs when receptors are already uncoupled from the G protein. On
the other hand, internalization seems to play an important role in the
resensitization of the receptor as has been shown for the
beta-2 AR (Pippig et al., 1995; Krueger et al., 1997).
Recently, elegant studies provided evidence that binding of
-arrestin to the phosphorylated receptor represents an early step in
the agonist-induced internalization of beta-2 AR (Ferguson et al., 1996; Goodman et al., 1996).
We recently described that mutations of Asp-142 belonging to the highly
conserved DRY sequence lying at the end of TMDIII and of Ala-293 in the
distal part of third intracellular (i3) loop could
constitutively activate the alpha-1b AR (Kjelsberg et al.,
1992; Scheer et al., 1996). A combined approach with experimental and
computer-simulated mutagenesis of the receptor suggested that these two
amino acids play a key role in the activation process of the
alpha-1b AR, i.e., its transition from the inactive to active state (Scheer et al., 1996, 1997).
Because constitutively active receptor mutants are thought to mimic, at
least in part, the agonist-occupied form of wild-type GPCRs, it has
been postulated that they can be substrates for GRK-mediated
phosphorylation in the absence of the agonist. This hypothesis is
supported by previous findings that indicated that constitutively
active beta-2 AR and alpha-2 AR carrying
mutations in the distal portion of i3 loop can be phosphorylated by
GRK2 in the absence of the agonist (Ren et al., 1993; Pei et al.,
1994). These results suggested that activating mutations in the
C-terminal portion of the i3 loop of the ARs induce receptor
conformations with docking complementarity with both the G protein
heterotrimers and regulatory proteins, including GRKs. However,
activating mutations have been recently described in different
structural domains of GPCRs (Scheer and Cotecchia, 1997). Thus, an
important question is whether constitutively active receptors carrying
mutations in different receptor domains share similar regulatory properties.
To investigate the relationship between constitutive activity and the regulatory properties of receptor mutants, in this study we compared the phosphorylation and internalization properties of constitutively active alpha-1b ARs carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of i3 loop and at D142 lying at the end of TMDIII. Our results indicate that the constitutively active alpha-1b AR mutated at either one of the two positions are strikingly different with respect to their regulatory properties. This study provides information that might have several implications for the elucidation of the mechanisms underlying the activation and regulation of the alpha-1b AR both at a biochemical and structural level.
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Experimental Procedures |
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Top
Summary
Introduction
Experimental procedures
Results
Discussion
References
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COS-7 Cell Culture and Transfections.
COS-7 cells were grown
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and gentamicin (100 µg/ml) and transfected
by the diethylaminoethyl-dextran method. The cDNA encoding the hamster
alpha-1b AR (Cotecchia et al., 1988) and its mutants
were subcloned in pRK5 (Didsbury et al., 1991), that of GRK2 and its
K220R mutant (Kong et al., 1994) in pCMV5 (Andersson et al., 1989) and
that of the -arrestin-1 mutant V53D (Ferguson et al., 1996) in
pCDNA1. For phosphorylation assays, cells were plated in 55-mm dishes
(1 × 106 cells) for most experiments and in 100-mm
dishes (3 × 106 cells) for experiments involving the
D142A and D142T mutants. The transfected DNA encoding the receptors was
2.5 µg per 1 × 106 cells. For inositol phosphate
(IP) determination, COS-7 cells (0.15 × 106) were
plated in 12-well plates and the transfected DNA encoding the receptors
ranged from 0.2-3 µg per 1 × 106 cells as
indicated in the figures. For internalization experiments, cells
(0.3 × 106) were grown in six-well plates and the
transfected DNA was 0.2-3 µg per 1 × 106 cells for
the receptors. The amount of transfected DNA encoding GRK2, its K220R
mutant and the -arrestin-1 mutant V53D was 3-fold higher than that
of the receptors. Assays were performed 48 h after transfection.
Mutagenesis of the alpha-1b AR. The cDNA of the hamster alpha-1b AR was mutated by polymerase chain reaction-mediated mutagenesis with Taq DNA polymerase. The mutated DNA fragments were sequenced by automated DNA sequencing (Microsynth, Balgach, Switzerland) and subcloned in the pRK5 expression vector containing the wild-type alpha-1b AR.
IP Measurement.
Transfected cells were labeled for 12 h
with myo-[3H]inositol at 4 µCi/ml in inositol-free DMEM
supplemented with 1% FBS. Cells were then preincubated for 10 min in
phosphate-buffered saline containing 20 mM LiCl, and then stimulated
for 45 min with epinephrine. Total IPs were extracted and separated as
described previously (Cotecchia et al., 1988).
32P-Labeling and Immunoprecipitation of the
Receptors.
Transfected COS-7 cells were equilibrated in
phosphate-free DMEM for 2 h and then incubated in the same buffer
containing 32Pi (0.2 mCi/ml) for 2 h at
37°C. The incubation was then continued in the absence or presence of
epinephrine as indicated. A separate set of dishes was incubated under
similar conditions, but in the absence of 32Pi
to measure receptor binding. Immunoprecipitation of the phosphorylated receptors was performed as described previously (Lattion et al., 1994).
For most experiments, antibodies raised against the last 24 amino acids
(residues 492-515) of the alpha-1b AR were used at 1:50
dilution. Similar results were obtained with antibodies raised against
the first 22 amino acids of the receptor (data not shown). After
autoradiography, the amount of radioactivity associated with the
phosphorylated receptor was quantified by liquid scintillation
spectroscopy. To assess receptor phosphorylation, the receptors were
expressed at similar levels and the 32P content (counts per
minute) of the immunoprecipitated receptors was directly compared for
statistical analysis.
Western Blot Analysis of GRK2 and -Arrestin.
Overexpression of GRK2, of its mutant K220R and of -arrestin-1
mutant V53D was assessed on the cytosolic fractions of transfected cells as described previously (Diviani et al., 1996). For
immunodetection of GRK2 and its mutants K220R, the antiserum (used at
1:1000 dilution) was raised against a glutathione
S-transferase fusion protein encoding residues 467 to
688 of rat GRK3, as described previously (Diviani et al., 1996). For
immunodetection of the -arrestin-1 mutant V53D, the antiserum (used
at 1:1000 dilution) was raised against a glutathione
S-transferase fusion protein encoding residues 172-268
of bovine -arrestin.
Ligand Binding.
Membrane preparations derived from cells
expressing the alpha-1b AR or its mutants and ligand
binding assays with
{-(4-hydroxy-[125I]iodophenyl)ethylaminomethyl}tetralone
([125I]HEAT) were performed as described previously
(Cotecchia et al., 1992). Prazosin (10-6 M) was
used to determine nonspecific binding. [125I]HEAT
concentration was 250 pM for measuring receptor expression at a single
saturating concentration and 80 pM for competition binding analysis of
epinephrine. Intact cell receptor binding assays were performed as
described (Lattion et al., 1994) by incubating cell monolayers grown in
35-mm dishes with [3H]prazosin (2 nM) in 2.5 ml of DMEM
at 4°C for 7-10 h. After binding, cells were washed three times with
ice-cold phosphate-buffered saline containing 0.1% bovine serum
albumin, scraped in water, and counted. Phentolamine (10-4
M) was used to determine nonspecific binding that was 50%
of total binding. Data were analyzed by nonlinear least-square
regression analysis (DeLean et al., 1982).
-Arrestin-Green Fluorescent Protein (GFP) Distribution.
The construction of the plasmid encoding a -arrestin-GFP conjugate,
transfection of HEK-293 cells and fluorescence microscopy were as
described previously (Barak et al., 1997). The transfected DNA per
106 cells was 0.025-1.0 µg for the
alpha-1b AR and A293E mutant, 3 µg for the D142A
receptor, 0.5 µg for the -arrestin-GFP, and 1 µg for GRK2.
Statistical Analyses. Results are expressed as mean ± S.E. Statistical significance was assessed by paired Student's t test.
Materials. COS-7 cells were purchased from American Tissue Culture Collection (Manassas, VA); DMEM, gentamicin, FBS, and restriction enzymes were purchased from Life Technologies, Inc. (Gaithersburg, MD); Taq polymerase was purchased from Boehringer Mannheim (Indianapolis, IN); [125I]HEAT and [3H]inositol were purchased from DuPont/NEN (Wilmington, DE); Dowex AG1-X8 from Bio-Rad (Richmond, CA); epinephrine was purchased from Sigma Chemical Co. (St. Louis, MO); and prazosin was purchased from Research Biochemical International, Inc. (Natick, MA).
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Results |
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Top
Summary
Introduction
Experimental procedures
Results
Discussion
References
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Phosphorylation of Constitutively Active Mutants Carrying Mutations
at Position A293 and D142.
We previously reported two series of
constitutively active mutants resulting from all-amino acid scanning
mutagenesis at two positions, i.e., at A293 in the distal part of the
i3 loop and at D142 of the DRY motif lying at the end of TMDIII
(Kjelsberg et al., 1992; Scheer et al., 1996). To compare the
phosphorylation properties of the constitutively active receptors
mutated at A293 or D142, we have chosen a mutant with high (D142T and
A293E) and a mutant with intermediate (D142A and A293I) levels of
constitutive activity for each series of receptors. We previously
reported that the maximal expression levels of the D142A and D142T
mutants in COS-7 cells was 3- to 4-fold lower than that of the
receptors mutated at position 293. Thus, to directly compare their
properties the receptor mutants were expressed at similar expression
levels of 0.3-0.4 pmol/mg of protein.
; Scheer et al., 1996), the IP response mediated
by the constitutively active mutants could be stimulated by epinephrine with the exception of the A293E mutant for which the agonist-induced response was observed in previous studies only at its maximal expression levels.
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). This cluster of
serines was mutated in the wild-type alpha-1b AR as well as
in the A293E and D142A mutants. The cluster of serines containing the
main sites of phosphorylation for GRK (Ser-404, Ser-408, and Ser-410) and PKC (Ser-394 and Ser-400) was mutated in the wild-type, A293E, and
D142A, so as to construct receptors the S394 to 415A, A293E/S394 to
415A, and D142A/S394 to 415A, respectively. The
Ki of epinephrine for the A293E/S394
to 415A and D142A/S394 to 415A was not significantly different than
that of the A293E and D142A, respectively (data not shown).
In agreement with previous findings (Diviani et al., 1997), all the
receptors carrying mutations of the phosphorylation sites displayed low
levels of basal as well as agonist-induced receptor phosphorylation
(Fig. 3). These results strongly suggest
that the increased basal phosphorylation of the A293E mutant mainly occurs at the same sites involved in GRK- and PKC-mediated
phosphorylation of the wild-type alpha-1b AR.
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; Perez et al.,
1996).
Effects of GRK2 on Phosphorylation of A293E and D142A Mutants.
Previous findings indicated that constitutively active
beta-2 AR and alpha-2 AR mutants were
substrates for GRK2-mediated phosphorylation in the absence of agonist
(Ren et al., 1993; Pei et al., 1994). Thus, we wanted to investigate
the effect of GRK2 on the phosphorylation of the constitutively active
receptors mutated at position 293 or 142. For these studies, the A293E
and D142A mutants were chosen because they displayed similar
constitutive activity when expressed at similar levels (Fig. 1A).
) were
transiently coexpressed with either the wild-type or mutated receptors
in COS-7 cells (Fig. 4). Western blot
analysis indicated that for both GRK2 and its dominant negative mutant
the expression level was 5- to 10-fold higher than that of the
endogenous kinase, as reported previously (Diviani et al., 1996), In
agreement with previous findings (Diviani et al., 1996), in cells
overexpressing GRK2 epinephrine-induced phosphorylation of the
alpha-1b AR was significantly increased above that of the
receptor expressed alone. Overexpression of GRK2 resulted also in a
significant increase of the basal phosphorylation of the A293E that was
31% above that of the receptor expressed alone. This result strongly
suggests that the constitutively active A293E mutant is a substrate for GRK2-mediated phosphorylation in the absence of the agonist. This hypothesis is further supported by the observation that coexpression of
the dominant negative GRK2 mutant resulted in ~50% decrease in the
agonist-independent phosphorylation of the A293E mutant (data not
shown). Overexpression of the dominant negative GRK2 mutant partially
impaired epinephrine-induced phosphorylation of the wild-type
alpha-1b AR in agreement with previous findings (Diviani et
al., 1996), but did not have any effect on that of the D142A mutant.
|
Effects of Combined Mutations at A293 and D142 Positions. Our findings indicate that mutations at position A293 and D142 of the alpha-1b AR share similar effects in triggering receptor activation, but induce different effects on receptor phosphorylation. To further investigate the relative role of the structural domains of the alpha-1b AR including A293 and D142 we constructed a receptor mutant A293E/D142A carrying the double mutation.
As shown in Fig. 1A, the D142A/A293E mutant displayed a constitutive activity that was 71% and 93% higher as compared with that of the A293E or D142A receptors, respectively. On the other hand, the levels of agonist-independent phosphorylation of the A293E/D142A mutant was significantly decreased by 40% as compared with that of the A293E receptor (Fig. 1B). The Ki of epinephrine for the A293E/D142A mutant was not significantly different than that of the D142A or A293E receptors. (data not shown). These findings have two main implications. First, they provide additional evidence that the conservation of D142 is important for receptor phosphorylation. Second, they suggest that the effects of mutations at A293 and D142 are additive on the constitutive activity whereas they have opposite effects on the phosphorylation of the alpha-1b AR.Agonist-Induced Internalization of A293E and D142A Mutants.
Recent evidences suggest that receptor phosphorylation is required to
facilitate the internalization of the beta-2 AR and muscarinic receptors (Tsuga et al., 1994; Ferguson et al., 1995). In
particular, it has been proposed that binding of -arrestin to the
phosphorylated receptor represents an early step in the agonist-induced
internalization of beta-2 AR (Ferguson et al., 1996;
Goodman et al., 1996). This was supported by the finding that a
dominant negative mutant of -arrestin-1 (V53D) could partially inhibit receptor internalization (Ferguson et al., 1996). Thus, the
internalization of the A293E and D142A mutants was compared with that
of the wild-type alpha-1b AR in the absence or presence of a dominant negative mutant of -arrestin-1 (V53D).
-arrestin-1 mutant reduced receptor
internalization to 18%. Western blot analysis indicated that the
expression of the -arrestin-1 mutant was a ~5-fold higher than
that of the endogenous arrestin (data not shown). A more profound
inhibition of receptor internalization could not be achieved in our
experiments probably because the amount of -arrestin-1 mutant
expressed was not maximal despite the large amount of transfected DNA.
Alternatively, -arrestin might be only partially involved in the
internalization process of the alpha-1b AR.
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-arrestin-1 mutant (Fig. 5).
On the other hand, the D142A receptor mutant was impaired in its
ability to undergo epinephrine-induced internalization that was never
>6%.
As previously reported for the beta-2 AR (Ferguson et al.,
1996), coexpression of the dominant negative -arrestin-1 mutant resulted in a significant 52% increase of epinephrine-induced phosphorylation of the wild-type alpha-1b AR as compared
with that of the receptor expressed alone (data not shown). However, overexpression of the V53D -arrestin-1 mutant did not have any significant effect on the phosphorylation of the D142A receptor (data
not shown).
It could be expected that the A293E mutant displaying increased basal
phosphorylation could also be constitutively internalized in the
absence of the agonist. Overexpression of the V53D -arrestin-1 mutant did not significantly increase the number of cell surface receptors for any of the receptors tested as compared with receptors expressed alone (data not shown). However, these observations cannot
exclude a tonic internalization of the A293E receptor mutant that would
be hardly measurable in our experimental approach measuring ligand
binding to intact cells.
Interaction of Receptors with -Arrestin-GFP Conjugate.
Previous studies demonstrated that -arrestin-GFP conjugate was a
valuable tool to investigate the role of -arrestin in the internalization process of the beta-2 AR (Barak et al.,
1997). Thus, to better characterize the interaction between
-arrestin and the constitutively active mutants, we examined how a
-arrestin-GFP responded to agonist-induced activation of the
receptor by fluorescence microscopy. For these studies, HEK cells were
used because, as compared with COS-7 cells, their smaller size results
in a better contrast produced by the redistribution of
-arrestin-GFP. The use of HEK cells is supported by previous
evidences indicating that several regulatory properties of GPCRs are
comparable in HEK and COS-7 cells. 1) The relationship between receptor
phosphorylation and arrestin translocation was demonstrated both in HEK
and COS-7 cells (Barak et al., 1997; Menard et al., 1997). 2) Our
previous findings indicated that the regulatory properties of the
alpha-1b AR coexpressed with various GRKs and arrestins
were virtually identical in COS-7 and HEK cells (Diviani et al., 1996).
-arrestin-GFP and GRK2 results in a clear translocation of
-arrestin toward the cell membrane. This is demonstrated by an
enhancement of the cell membrane fluorescence and a concomitant loss of
cytosolic fluorescence in Fig. 6B as compared with Fig. 6A.
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-arrestin-GFP to the cell membrane resulting in a decrease of
cytosolic fluorescence (Fig. 6F). These findings strongly suggest that
in cells expressing the constitutively active A293E receptor mutant
-arrestin are, at least in part, constitutively translocated to the
cell membrane. The translocation of -arrestin-GFP in cells
expressing either the alpha-1b AR or the A293E mutant was
smaller when GRK2 was not overexpressed (data not shown).
On the other hand, agonist-induced stimulation of the constitutively
active D142A receptor mutant did not induce any apparent translocation
of the -arrestin-GFP that remained diffused throughout the cytosol
both in the absence and presence of epinephrine (Fig. 6, C and D).
To rule out that the reduced signal in the -arrestin-GFP
translocation assay in cells expressing the D142A receptor derived from
the lower expression of this mutant, arrestin translocation was
compared in HEK cells expressing similar levels of D142A and wild-type
alpha-1b AR. Fluorescence microscopy was used to visually look for positives in a large cohort of cells (~50,000 cells per dish). As shown in Fig. 7, in cells
expressing the alpha-1b AR agonist-induced increase of
fluorescence at the cell margin was proportional to receptor expression
(compare Fig. 7, D and F). However, when the alpha-1b AR was
expressed at levels similar to those of the D142A agonist-induced
translocation of arrestin was observed in cells expressing the wild
type, but not in those expressing the mutated receptor (Fig. 7, B and
D).
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-arrestin toward the receptor
that could lead to increased agonist-induced internalization of the
receptor mutant. On the other hand, the decreased ability of the D142A
to undergo agonist-induced phosphorylation might result in its apparent
lack of interaction with -arrestin. as well as of
epinephrine-induced receptor internalization.
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Discussion |
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Top
Summary
Introduction
Experimental procedures
Results
Discussion
References
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|---|
The goal of this study was to investigate the relationship between the constitutive activity and the regulatory properties of different constitutively active alpha-1b AR mutants. Our findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR, i.e., at A293 in the distal portion of the i3 loop and at D142 lying at the end of TMDIII, have opposite effects on the regulatory properties of the receptor.
Active Conformation of Alpha-1b AR Induced by
Mutations at A293 is a Substrate for GRK2.
The analysis of the
A293E and A293I mutants suggests a correlation between the constitutive
activity induced by the mutations of Ala-293 and agonist-independent
phosphorylation of the receptor (Fig. 1). Three lines of evidence
suggest that the increased basal phosphorylation of the A293E mutant
is, at least in part, mediated by GRK2. 1) Coexpression with GRK2
significantly increased both the basal and agonist-dependent
phosphorylation of the A293E receptor (Fig. 4). 2) Mutations of the
phosphorylation sites on the receptor, including those for GRK2,
dramatically decreased both basal and agonist-induced phosphorylation
of the A293E mutant (Fig. 3). 3) Overexpression of a kinase-deficient
GRK2 mutant could partially reduce the basal phosphorylation of the
A293E receptor (data not shown). These results are in striking
agreement with previous findings that showed that for the
beta-2 AR and alpha-2 AR, mutations of
the amino acid homologous to A293 in the alpha-1b AR,
resulted in both increased constitutive activity and
agonist-independent phosphorylation mediated by GRK2 (Ren et al., 1993;
Pei et al., 1994).
A293E Mutant Displays Increased -Arrestin-Mediated
Internalization.
In this study we report for the first time, that
agonist-induced internalization of the alpha-1b AR might
be, at least in part, mediated by -arrestin as recently shown for
the beta-2 AR (Ferguson et al., 1996; Goodman et al.,
1996). This was mainly demonstrated by the fact that coexpression of
the alpha-1b AR with the dominant negative
-arrestin-1 mutant V53D inhibited receptor internalization by
~40% (Fig. 5).
). This is also in agreement with a
recent study that showed that the down-regulation of the Gq
subunit
induced by a constitutively active alpha-1b AR mutant carrying a triple mutation in the distal portion of the i3 loop was
greater than that mediated by the wild-type receptor (Lee et al.,
1996).
On the other hand, our findings strongly suggest that the affinity of
the A293E mutant for -arrestin is increased as compared with that of
the wild-type receptor. This was mainly demonstrated by the fact that
in cells expressing the constitutively active A293E mutant the
-arrestin-GFP conjugate was, at least in part, constitutively
translocated to the cell membrane as compared with cells expressing the
wild-type alpha-1b AR (Fig. 6).
The constitutive translocation of -arrestin toward the receptor as
well as the enhanced agonist-induced receptor internalization are
consistent with the increased basal phosphorylation of the constitutively active A293E mutant. Our findings further support the
notion suggested for different GPCRs that GRK-mediated phosphorylation of the receptor can facilitate its interaction with -arrestin that
promotes receptor internalization (Tsuga et al., 1994; Ferguson et al.,
1995).
D142 Plays a Role in Interaction of Alpha-1b AR with Regulatory Proteins. A striking finding of our work is that mutations of D142 can impair receptor phosphorylation and internalization (Figs. 1 and 5). Our results, however, indicate that mutation of D142 into alanine did not entirely abolish receptor phosphorylation. In fact, overexpression of GRK2 could partially rescue the phosphorylation of the D142A mutant resulting in a significant increase of epinephrine-induced phosphorylation of the receptor (Fig. 4). In addition, the basal phosphorylation of the double mutant D142A/A293E was still significantly higher than that of the wild-type alpha-1b AR (Fig. 1B). Altogether, these findings suggest that the mutation of D142 into alanine reduces the affinity of the receptor for GRK2.
The expression of the D142A mutant was 3- to 5-fold lower than that of the wild-type alpha-1b AR. This is consistent with what has been reported for other GPCRs in which mutations of the homologous aspartate could dramatically reduce receptor expression (Lu et al., 1997). One might speculate that constitutive internalization might be
responsible for the apparent lack of agonist-induced internalization of
the constitutively active D142A mutant. This hypothesis seems to be
ruled out by several observations. 1) Overexpression of the dominant
negative mutant of -arrestin-1 did not increase the expression of
the D142A mutant at the cell surface or agonist-induced phosphorylation
of the receptor (data not shown). 2) in HEK cells that express high
amounts of -arrestin agonist-induced stimulation of the D142A
receptor did not induce any apparent translocation of the
-arrestin-GFP conjugate to the cell membrane (Figs. 6 and 7). We
conclude that the mutation of D142 into alanine might reduce the
affinity of the alpha-1b AR for GRK2 and/or -arrestin or
for both.
These findings suggest that the aspartate of the DRY sequence plays a
role in the interaction of the alpha-1b AR with regulatory proteins, including GRK2 and -arrestin. This observation is in agreement with recent findings that suggest that sequences from both
the i2 and i3 loops are involved in the regulatory mechanisms of other
GPCRs (Moro et al., 1994; Bourne, 1997). It is now well established
that the interaction of rhodopsin kinase (GRK1) with the intracellular
loops of rhodopsin underlies the activation process of the kinase that
can subsequently phosphorylate residues located in the C-tail of the
receptor (Palczewski et al., 1991; Shi et al., 1995; Thurmond et al.,
1997). However, despite these observations, the structural determinants
of GPCRs that interact with different GRKs and/or arrestin proteins, as
well as the molecular mechanisms underlying the activation of these
regulatory proteins, remain largely unknown. One interpretation of our
findings could be that the negative charge of D142 is directly
interacting with members of the GRK and/or arrestin family.
Alternatively, mutations of D142 in the alpha-1b AR can
perturb the conformation of the i2 or i3 loops that might directly bind
and activate GRK2, leading to the interaction of the phosphorylated
receptor with -arrestin.
An important question is to what extent the apparent constitutive
activity of the D142A mutant reflects enhanced activation versus
reduced desensitization. The definition of receptors carrying mutations
at position D142 as "constitutively active" is supported by their
peculiar pharmacological properties (extremely high affinity for
agonist binding) as well as by their features described by the
molecular dynamics analysis of the theoretical receptor structures (Scheer et al., 1996; Scheer et al., 1997). In addition, several lines
of evidence seem to exclude that reduced desensitization is the main
mechanism underlying the high degree of constitutive activity of the
D142A mutant. 1) Removal of the phosphorylation sites does not
significantly enhance the constitutive activity of the wild-type
alpha-1b AR (as reported in Results section). Thus, the attenuation of desensitization could hardly explain the high
spontaneous activity of the constitutively active receptor mutants. 2)
A marked degree of constitutive activity is detectable for both the
A293E and D142A mutants also in a membrane assay (in which
desensitization should not occur) measuring receptor-mediated stimulation of GTP
35S binding to Gq
(our unpublished results).
However, an intriguing hypothesis is that the output of both the D142A
and A293E receptor mutants results from the balance between two
opposite processes
activation and desensitization. On one hand, the
constitutive activity of the D142A mutant in intact cells might be
enhanced because of its reduced desensitization. On the other hand, the
constitutive activity of the A293E mutant might be underestimated
because of its increased desensitization. This hypothesis might also
apply to the interpretation of the properties of other constitutively
active GPCRs.
Conformations of Alpha-1b AR Underlying Its Activation and Regulatory Processes. A challenging task for most GPCRs is the identification of the structural motifs and conformations of the receptor that can selectively interact with a large number of signaling proteins that regulate the balance between receptor activation and desensitization.
In the rhodopsin system, it has been shown that, first, the receptor domains interacting with transducin and rhodopsin kinase are in part overlapping, and second, the light-activated meta II state can both activate transducin and be phosphorylated by rhodopsin kinase (Palczewski et al., 1991; Shi et al., 1995; Thurmond et al., 1997).
Altogether, these findings suggest that the receptor conformations
underlying the activation and desensitization processes of rhodopsin
are similar. This hypothesis is also supported by studies that show
that constitutively active rhodopsin mutants carrying mutations in
different transmembrane domains, can display light-independent
phosphorylation (Rim and Oprian, 1995).
Our findings indicate that the constitutively active
alpha-1b AR mutants carrying mutations of A293 might display
receptor conformations with docking complementarity for both the G
proteins, on one hand, and GRK2 and -arrestin, on the other. Thus,
by analogy with rhodopsin, it can be suggested that also in the
alpha-1b AR the structural domains involved in the
interaction of the receptor with the G proteins and the regulatory
proteins GRKs and arrestins can overlap.
On the other hand, the finding that the D142A mutant was constitutively
active, but impaired in the processes of receptor phosphorylation and
internalization suggests that, among the multiple conformations
underlying receptor activation, not all favor the interaction of the
receptor with GRK2 and/or -arrestin.
Conclusions.
Our study demonstrates that the
agonist-independent activity of different alpha-1b AR
constitutively active mutants does not necessarily correlate with their
ability to undergo enhanced phosphorylation and internalization. The
results of this study have also other implications. 1) The DRY sequence
is identified as an important structural determinant for the
phosphorylation and internalization of the alpha-1b AR.
2) Additional evidence is provided about the role of -arrestin in
the regulation of the alpha-1b AR. 3) The divergent
regulatory properties of the constitutively active mutants might help
to interpret the structural differences observed in the molecular
dynamics analysis of their putative structures (Scheer et al., 1996,
1997). This might improve our theoretical model describing the receptor
conformers associated with the active and inactive states of the
alpha-1b AR.
| |
Acknowledgments |
|---|
We are sincerely grateful to Dr. R. J. Lefkowitz for the
antiserum against GRK2; Dr. J. F. Benovic for the K220R mutant of GRK2; Dr. M. G. Caron for the dominant negative mutant of
-arrestin1 (V53D); and Dr. F. Mayor, Jr., for the antiserum against
-arrestin. We acknowledge the excellent technical help of M. Munoz
and M. Nenniger in cell culture. We thank Dr. Peter Greasley for
critical revision of the manuscript.
| |
Footnotes |
|---|
Received May 29, 1998; Accepted October 30, 1998
This work was supported by Fonds National Suisse de la Recherche Scientifique Grant 31-51043.97 and by European Community Grant BMH4-CT97-2152.
Send reprint requests to: Dr. Susanna Cotecchia, Institut de Pharmacologie et de Toxicologie. 27, Rue du Bugnon, Faculté de Médecine, 1005 Lausanne, Switzerland. E-mail: susanna.cotecchia{at}ipharm.unil.ch
| |
Abbreviations |
|---|
AR, adrenergic receptor(s);
G protein, guanyl
nucleotide-binding regulatory protein;
GRK, G protein-coupled receptor
kinase;
GPCR, G protein-coupled receptors;
i2 and i3, second and third
intracellular (loops), respectively;
IP, inositol phosphate;
DMEM, Dulbecco's modified Eagle's medium;
TMD, transmembrane domain;
FBS, fetal bovine serum;
PKC, protein kinase C;
[125I]HEAT, {-(4-hydroxy-[125I]iodophenyl)ethylaminomethyl}tetralone;
GFP, green fluorescent protein.
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References |
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Top
Summary
Introduction
Experimental procedures
Results
Discussion
References
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