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J Biol Chem, Vol. 273, Issue 50, 33548-33555, December 11, 1998
From the In the search for the
structural elements participating in signal transduction,
internalization, and resensitization of the bradykinin B2 receptor, we
identified two critical motifs, one in the second intracellular loop
(IC2), the other in the proximal C terminus. We previously described
the contribution of tyrosines within each of the two motifs
(Tyr131 and Tyr322) to signal
transduction and receptor internalization (Prado, G. N., Taylor,
L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638-14642). Here, we investigate the effect of exchanging both
tyrosine residues simultaneously for alanine, phenylalanine, or serine,
termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S)
receptors, respectively. All of these mutants bound bradykinin (BK)
normally, with a Kd of approximately 1.1 nM. However, although phosphoinositide (PI) turnover in
response to BK by Y131A and Y131S proved negligible, the YAYA mutant
returned BK-activated PI turnover to wild type (WT). In contrast, PI
turnover with YSYS remained unresponsive to BK. Importantly, the
pattern of BK-activated arachidonate release differed markedly in the
mutant receptors. For example, whereas Y131S ablated BK-activated
arachidonic acid release, conversion of this mutant to YSYS returned
the BK-activated receptor function to a level above that of WT.
However, YAYA showed only a partial recovery from the poor BK response
of Y131A. These and additional results suggest that Tyr131
and Tyr322 interact cooperatively in conjunction with at
least two separate signaling functions. Given these results, a
molecular model of the receptor was generated with the IC2 and the
proximal C terminus in close spatial proximity. Conformations were
identified to provide structural explanation for these observations.
The conserved Thr137 in the IC2 was next substituted with
proline (T137P) to prevent phosphorylation at this position or with
aspartate (T137D) to emulate phosphorylation. The T137P mutant
demonstrated no change from WT with respect to either BK-activated PI
turnover or arachidonic acid release. However, the mutant exhibited a
markedly reduced capacity to internalize. It also resensitized poorly.
The T137D mutant lacked both BK responsive activities. However, it
internalized and resensitized normally, as did WT. These final results
suggest that Thr137 is functioning as a switch in
termination of signal transduction and the initiation of internalization.
The bradykinin B2 receptor
(BKB2R)1 cDNA has been
successfully transfected into such cell types as hamster lung
fibroblasts, Rat-1 fibroblasts and CHO cells with the expected binding
properties (1-3). The BKB2R is a typical G protein-coupled receptor
(GPCR) that has been reported to be associated with Gq (4-7), Gi, and G12 (8-10). Binding of a GPCR to more than one G The cellular response of GPCRs to agonists is regulated under a tightly
controlled process of desensitization and resensitization (15, 16).
Desensitization prevents cells from uncontrolled stimulation, and
resensitization allows cells to recover and maintain responsiveness
(17). Both processes appear to be regulated at the receptor level.
Recent studies by Haasemann et al. (18) showed that
stimulation of BKB2R results in redistribution of the receptor and its
internalization in caveolae (18). On/off regulatory elements have been
identified in other receptors (19-22). However, to date, no clear
consensus motifs have been identified to determine either
internalization or resensitization. The
DRYXXI/VXXP motif located in the second
intracellular loop (IC2) of the human muscarinic cholinergic receptor
has been proposed as important for receptor internalization (23).
Another important motif is the tyrosine-containing motif located in the
proximal BKB2R C terminus. The motifs in this region have been
implicated in receptor internalization in other GPCRs, such as
neurokinin 1 receptor (17), parathyroid hormone receptor (24), and AT1
receptor (20). Studies have also shown that clathrin-associated protein complexes interact with tyrosine-based motifs (25).
Mutations exchanging single as well as multiple amino acids have been
applied to resolve the actions of GPCRs. The importance of specific
residues within the IC face of GPCRs was demonstrated recently by a
number of investigators, using receptors such as BKB2 receptors (1),
AT1 receptors (26), interleukin-8 receptors (27), and muscarinic
acetylcholine receptors (28). Our previous results showed that
Tyr131 and Tyr322 are crucial for signal
transduction (1). Truncation of the distal C terminus of BKB2R
exhibited normal BK-activated ARA release, PI turnover, and
[Ca2+]i flux. Furthermore, our previous results
showed that at least some of the structural elements involved in the
internalization process are found in the IC2 and the proximal as well
as distal C terminus (1).
Reports of the use of models of BKB2R to explore ligand binding have
appeared in the literature (29-31). For example, Kyle et
al. (29) proposed that ligand binding to the receptor is in a
twisted S shape with the C-terminal Herein, we further investigate the relationship between
Tyr131 and Tyr322 in terms of signaling,
uptake, and resensitization. In conjunction with our results, we
generated a molecular model of BKB2R with these two regions in close
spatial proximity. We also investigate the role of an apparently
critical threonine at position 137 in IC2. Alignment of GPCRs of the
peptide subfamily shows that proline is generally conserved at this
position. However, the position occupied by this proline is a
tryptophan in endothelin receptors and a serine in galanin receptors.
Our results suggest that threonine 137 participates in the regulation
of BKB2R internalization and resensitization, whereas BKB2R
desensitization does not involve this residue.
Materials--
[3H]BK (78 Ci/mmol) was obtained
from Amersham Pharmacia Biotech,
myo-[1,2-3H]inositol (45-80 Ci/mmol) was
obtained from NEN Life Science Products. Analytical grade Dowex-X8
(AG-1-X8, 100-200 mesh) was obtained from Bio-Rad. Restriction
endonucleases were purchased from New England Biolabs. QuikChange
mutagenesis kit was obtained from Strategene Corp. (La Jolla, CA).
Oligonucleotides were either synthesized from an in-house Applied
Biosystems DNA synthesizer or purchased from Life Technologies, Inc.
All other reagents were from Sigma unless stated otherwise.
Site-directed Mutagenesis--
The QuikChange Site-Directed
Mutagenesis method (Stratagene) was used to generate mutant BKB2
receptors. Briefly, two primers complementary to each other were
designed to contain the desired mutation. The mutation-containing DNA
was synthesized using Pfu DNA polymerase. The parental
strand, which is dam-methylated, was removed by digestion
with Dpn 1. The mutant strand was transformed into XL1-Blue
supercompetent cells and then DNA isolated by the miniprep method. The
synthesized mutant DNA was sequenced by an in-house facility using an
automatic DNA sequencer (Applied Biosystem Inc., model 370A). Using the
BKB2R as template, T137P, T137D, and Y322F were constructed using
oligonucleotides T137P (sense strand, 5'-ctggtgaagcccatgtccatg-3';
antisense strand, 5'-catggacatggacttc accag-3'), T137D (sense strand,
5'-ctggtgaaggacatgtccatg-3'; antisense strand,
5'-catggacatgtccttcaccag-3'), and Y322F (sense strand, 5'-agaggtttcccaggcaata-3'; antisense strand, 5'
tattgcctgggaaacctct-3'), respectively. To create double mutants YAYA,
YFYF, and YSYS, the following parental strands and oligonucleotides
combinations were used: For YAYA, Y131A was used as a parental strand
and oligonucleotides (sense strand, 5'gaggtggcccaggcaata-3'; antisense
strand, 5'tattgcctgggccacctct-3') were used to mutate Y322A. For YSYS,
Y321S was used as a parental strand, and oligonucleotides (sense
strand, 5'-atcgaccgatccctggcgctg-3'; antisense strand,
5'-cagcgccagggatcggtcgat-3') were used to mutate Y131S. For YFYF, Y322F
was used as a parental strand, and oligonucleotides (sense strand,
5'-atcgaccgattcctggcgctg-3'; antisense strand, 5'-cagcgccaggaatcggtcgat-3') were used to mutate Y131F. The
oligonucleotides used to mutate Y322A and Y131S were previously
described (1).
Cell Culture and Transfection--
Rat-1 cells were obtained
from Dr. Robert Weinberg (Whitehead Institute, Massachusetts Institute
of Technology) as a generous gift. Rat-1 cells were seeded at 100,000 cells/well in a six-well plate in Dulbecco's modified Eagle's medium,
containing 5% fetal bovine serum supplemented with 50 units/ml
penicillin and 50 µg/ml streptomycin. Cells were grown at 37 °C in
a humidified atmosphere with 5% CO2. For transfection, two
solutions were prepared. One solution contained 2 µg of DNA in 20 µl of Opti-MEM (Life Technologies, Inc.), and the other solution
contained 6 µl of LipofectAMINE (Life Technologies, Inc.) and 14 µl
of Opti-MEM. Both solutions were combined and incubated at room
temperature for 15 min. The DNA-liposome complex was added to 1 ml of
Opti-MEM and added to each well. Sixteen hours later, transfection
medium was replaced with complete medium. Transfected Rat-1 cells were
then trypsinized and detached the next day and seeded in a
60-cm2 dish containing complete medium and 500 µg/ml of
Geneticin (G418, Life Technologies, Inc.). Neomycin-resistant clones
were isolated for 2 weeks and independently expanded in a 24-well
plate. Positive clones were identified by the amount of specific
binding to [3H]BK.
Phosphoinositide Turnover--
Rat-1 cells were incubated with 1 µCi/ml of myo-[3H]inositol in 1 ml of growth
medium for 16-24 h, and the levels of inositol phosphates were
determined 1 day later as described previously (1).
Arachidonic Acid Release--
Rat-1 cells were labeled with
[3H]arachidonate (0.2 uCi/well) for 16 h as
described previously (1). Briefly, cells were washed with DMEM
containing 2 mg/ml bovine serum albumin and incubated with 100 nM BK for 20 min at 37 °C. Medium was removed and
centrifuged at 800 × g. Radioactivity in the
supernatant was determined in a Receptor Binding Assay and Internalization--
Receptor binding
studies and internalization of the BKB2 receptors in intact Rat-1 cells
were carried out as described previously (1). Briefly, 80-100%
confluent cell monolayers in 24-well plates (Costar, Cambridge MA) were
incubated in binding buffer containing various concentrations of
[3H]BK in the absence (total binding) or presence
(nonspecific binding) of 10 Calcium Mobilization Assays--
Stably transfected Rat-1 cells
were detached and washed once in DMEM containing 5% fetal bovine
serum. Cells were then washed a second time in physiological buffer
solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 0.9 mM CaCl2, 15 mM HEPES, 0.1% bovine
serum albumin). Cells were resuspended at 1 × 107
cells/ml and incubated with Fura-2/AM for 30 min (2 µM
final concentration). After 30 min, the cell suspension was diluted 10× with physiological buffer solution and incubated for another 30 min. Cells were pelleted and resuspended at 1 × 106
cells/ml. Ca2+ mobilization experiments were performed
using a Perkin-Elmer LS-3B fluorescence spectrophotometer.
Intracellular calcium increase in the presence or absence of BK was
measured as described (5). Data were analyzed using the FURA program.
To examine desensitization, cells were allowed to equilibrate at
37 °C for 5 min and then stimulated sequentially with 10 nM BK at 2-min intervals, without an intervening wash.
Resensitization was examined by exposing cells to a desensitizing dose
of 10 nM BK for 2 min at 37 °C. The cells were then
washed with physiological buffer and resuspended at 1 × 106 cells/ml. Calcium mobilization assays as described
above were carried out by stimulating cells with a second dose of 10 nM BK at various times after the first desensitizing dose
to 10 nM BK.
Molecular Modeling--
The molecular model of the BKB2R was
built up using the topological arrangement of the transmembrane helices
of rhodopsin (32, 33) following standard procedures using the program
WHAT IF (34). The loops connecting the transmembrane helices were then
added to complete the model. In an attempt to develop the conformational preferences of IC2 and the C terminus of the receptor, the corresponding sequences were submitted to a BLAST search (35). The
homologous regions of each of these protein structures were then
analyzed for conformational features. The secondary structural features
were then incorporated into the molecular model (36).
To refine the molecular model, molecular dynamics (MD) simulations and
energy minimizations were carried out with the GROMACS program (37).
The membrane environment was mimicked by a 40-Å layer of decane
molecules, with approximately 40-Å layers of water above and below
(38). The simulation cell was 132 × 81 × 71 Å and
consisted of 12,906 waters and 518 decanes. The seven helical bundle of
the receptor was placed in the decane layer with the extra- and
intracellular regions within the water phases. Twenty different MD
simulations, with alternative starting structures, were carried out for
200 ps on an SGI Origin 2000 computer.
Mutation of the Intracellular Tyrosines of the BKB2
Receptor--
Previous experiments pointed to Tyr131
within the DRY motif, which is located in IC2, as important for both PI
turnover and ARA release. These experiments also pointed to a
Tyr131/Tyr322 interaction. The location of
Tyr131 and Tyr322 in the BKB2 receptor is shown
in Fig. 1. To investigate this interaction further, we generated critical double mutants at the Tyr131 and Tyr322 positions. Both tyrosine
residues were converted to a small hydrophobic (alanine), a large
hydrophobic (phenylalanine), and a small hydrophilic (serine)
hydroxyl-bearing residue. All the mutants generated showed little
change in binding affinity compared with the WT receptor, as
illustrated in Table I. Levels of
expression obtained in Rat-1 cells stably transfected with the double
mutants are also shown in Table I.
PI Turnover of Tyr131 and Tyr322
Mutants--
The ability of the single mutants at Tyr131
and Tyr322 and double mutants YSYS, YAYA, and YFYF to
signal PI turnover is illustrated in Fig.
2. Results show that in Rat-1 cells
expressing YAYA, the double mutant containing the small hydrophobic
alanine, the BK-activated receptor signals a PI turnover equal to that
of WT. This is in stark contrast to the actions observed with the Y131A
mutant (Fig. 2). However, the action of YFYF, which contains the large
hydrophobic phenylalanine, unlike the single mutant Y131F, was markedly
reduced by 90% compared with WT. PI turnover by YSYS, containing the
small hydrophilic serine, was reduced by 68% as compared with WT. This marked reduction in response is approximately the same as that of Y131S
and Y322S (Fig. 2).
ARA Release of Tyr131 and Tyr322
Mutants--
To analyze another signaling pathway, the release of ARA
in response to BK was measured in stably transfected Rat-1 cells. The
ability of the double mutant receptors to stimulate ARA release proved
considerably different from that observed with PI turnover (Fig.
3). In this case, Rat-1 cells expressing
the double hydrophilic mutant, YAYA or YFYF, showed significant
reduction in response to BK. This result is similar to single point
exchanges of tyrosine for alanine or phenylalanine (1). On the other
hand, the double mutant YSYS showed an ARA response above WT, which is
in contrast to the single substitution of tyrosine for serine at either
position 131 or 322, resulting in a poorly responsive receptor.
Role of Thr137 in Signal Transduction and
Internalization--
The IC2 of BKB2R contains a threonine at position
137, as shown in Fig. 1. This residue is the exact counterpart of
proline in the DRYXXI/VXXP motif, an
internalization motif found in other GPCRs (18). The proline residue is
conserved in a number of GPCR families, including the AT1 and BKB1
receptors. We tested the effect of changing threonine to proline at
this position, T137P. The mutant receptor functioned normally, as did
WT, with respect to both ARA release and PI turnover, as illustrated in Fig. 4, A and B,
respectively. However, as illustrated in Fig. 5, the function of T137P changes markedly
with regard to internalization. T137P is internalized only minimally,
at 5 and 15% after 30 min and 45 min, respectively.
We investigated the role of the putative phosphorylation site,
threonine 137 in IC2. To mimic the effect of phosphorylation, we
introduced an aspartate residue, a negatively charged amino acid. The
exchange of aspartate or glutamate for serine or threonine allows for
the assessment of a single phosphorylated residue on the structure and
function of a protein (39, 40). Our results show that substitution of
threonine 137 with aspartate (T137D) caused a drastic reduction in both
PI turnover and ARA release compared with both WT and the T137P mutant
receptor (Fig. 4, A and B). The T137D mutant
receptor also exhibited a very different internalization profile in
comparison to T137P. At 5 min, 38 and 46% of the receptors are
internalized in T137D and WT receptors, respectively. This is in
contrast to a negligible amount of T137P internalized. At 30 min, 63, 47, and 5% of the receptors were internalized for WT, T137D, and
T137P, respectively (Fig. 5). These results suggest that the aspartate
substitution at position 137 introduced a conformational change similar
to that of a phosphorylated threonine, allowing for receptor internalization.
Internalization of WT and Tyrosine Mutant Receptors--
The
Tyr131/Tyr322 mutants were next compared with
the WT receptor for their capacity to internalize following BK receptor
activation. At 15 min, only YSYS internalized to approximately the same
level as WT, at about 42% uptake. The YFYF and YAYA mutants displayed slow receptor uptake, at 19 and 30% uptake, respectively (Fig. 6). Interestingly, YAYA acted like WT
with respect to PI turnover, whereas it displayed the
slowest receptor uptake. On the other hand, YSYS, which showed poor
linkage to PI turnover, displayed a very similar rate of uptake to WT.
The action by these two mutant receptors, YAYA and YSYS, indicate that
receptor signaling does not correlate with ligand-mediated
internalization of BK.
Receptor Desensitization and
Resensitization--
Bradykinin-induced receptor desensitization was
examined in cells stably transfected with WT and with mutants that show
a slow rate of receptor uptake, namely T137P, YAYA, and YFYF. With BK
stimulated ARA release, we observed differential desensitization. As
illustrated in Fig. 7, WT and YFYF
desensitized at 100%, YAYA desensitized to 80%, and T137P
desensitized to only 50%. Interestingly, T137P displayed the slowest
rate of internalization, whereas internalization of YFYF at 30 min
equaled that of WT (Figs. 5 and 6). With regard to BK activated
Ca2+ flux, all four receptors desensitized completely
within seconds of exposure to BK. This was evidenced by total inability
of BK to induce a second burst of Ca2+ flux after an
initial exposure to BK (Fig.
8A).
Receptor resensitization was evaluated in WT, T137P, YAYA, and YFYF, as
shown in Fig. 8B. Cells were treated with a saturating concentration of BK for 2 min. Excess BK was removed by washing. Five
minutes after washing, there was no observed
[Ca2+]i response to a subsequent dose of
saturating BK in WT or the mutant receptors (data not shown). However,
complete resensitization to BK occurred within 15 min for WT (Fig.
8B). We then determined whether the different mutant
receptors resensitized at 15 min. The resensitization process proved
related to the rate of receptor internalization. Although the initial
[Ca2+]i response was low for YFYF, it recovered
normally, as did WT, within 15 min, reaching its original
[Ca2+]i. On the other hand, YAYA showed only
partial recovery after 15 min. No recovery was observed for T137P at 15 min and even up to 60 min (data not shown).
Molecular Modeling--
The results from the BLAST search of the
C-terminal domain indicated a high probability of an We have generated a number of mutants within the IC2 and proximal
C terminus of the BKB2R. All constructs retained the binding kinetics
of the WT receptor. However, the mutations showed marked and varied
effects on BKB2R signal transduction, receptor internalization, and
resensitization. These results suggest that no one motif within the IC
face of the BKB2R is solely responsible for a given receptor function.
Instead, cooperative interactions appear to be taking place among the
various IC domains.
For example, replacement of Tyr322, located at the proximal
C terminus, with the small, uncharged alanine had little effect on either BK-stimulated ARA release or PI turnover. However, replacement with the small, hydroxyl-containing serine reduced both events markedly. Replacement of Tyr131 with serine also markedly
reduced both events. However, when both tyrosine residues,
Tyr131 and Tyr322, were mutated to serine
(YSYS), the consequent reduction of a bulky residue only impaired the
activation of phospholipase C. The double mutation did not affect
receptor-stimulated ARA release. In contrast, double mutation of
Tyr131 and Tyr322 residues to alanine (YAYA)
impaired ARA release but not PI turnover. In this case, a hydrophobic
residue at both locations promotes phospholipase C action but hinders
the release of ARA. However, a double substitution with phenylalanine,
a bulky hydrophobic residue, hinders both signaling pathways. These
results demonstrate that discrete sequences within the DRY motif of IC2
and the tyrosine-based motif in the proximal C terminus interact with
each other and cause differential effects on BK-activated signal
pathways. A possible explanation for this differential signaling is
that Tyr322 of the BKB2R is phosphorylated and acts like
the YIPP motif, similarly found in the proximal C tail of the AT1
receptor (41). However, our results and results reported by Blaukat
et al. (42) indicate that this tyrosine residue is not
phosphorylated. A more solid explanation is that the two IC regions are
cooperatively involved in coupling to more than a single subset of G
protein (Gq, Gi, or Gi-like proteins). Interaction between the IC loops and the proximal C tail has been proposed in other GPCRs (43). In the
To further understand the role of IC2 in receptor uptake, we
investigated the DRYXXI/VXXP Our results further suggest that internalization and resensitization
are linked, whereas desensitization is a more complicated process
related to specific metabolic events regulated by BK. When
resensitization was tested by measuring [Ca2+]i,
T137P, which was only minimally taken up even after 60 min, showed no
resensitization. On the other hand, YFYF, with a moderate rate of
internalization, resensitized similarly to WT. YAYA, which was also
taken up poorly, but not to the extreme of T137P, resensitized
somewhere between YFYF and T137P. It remains to be resolved which
receptor motif(s) determine internalization, which determine
desensitization, and what correlation exists between these two actions.
Our present and previous results, in sum, clearly indicate
cooperativity or interaction between the C-terminal tail and IC2 of the
receptor. We postulate the presence of Concerning Thr137 and the In summary, our results using BKB2R mutant receptors and consequent
molecular modeling involving the distal IC2 and proximal C terminus
point to important interaction between these two regions, not only with
regard to regulation of signal transduction but also in conjunction
with receptor internalization and resensitization. Apparently, the
motifs encompassing Tyr131 and Tyr322, perhaps
in conjunction with elements within the IC3, dictate BKB2R-G protein interaction.
*
This work was supported in part by National Institute of
Health Grants HL25776 from NHLBI, AG00115 from NIA, and GM54082 (to D.F.M.)The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 617-638-4717;
Fax: 617-638-5339.
The abbreviations used are:
BKB2R, bradykinin B2
receptor; BKB2, bradykinin B2; GPCR, G protein-coupled receptor; AT1, angiotensin II type 1; MD, molecular dynamics; WT, wild type; ARA, arachidonic acid; PI, phosphoinositide; DMEM, Dulbecco's modified
Eagle's medium; IC, intracellular loop; TM, transmembrane.
Motif Mutation of Bradykinin B2 Receptor Second Intracellular
Loop and Proximal C Terminus Is Critical for Signal Transduction,
Internalization, and Resensitization*
§,, and
Department of Biochemistry, Boston
University School of Medicine, Boston, Massachusetts 02118 and the
¶ Department of Molecular Pharmacology, Division of Biology & Medicine, and Department of Chemistry, Brown University,
Providence, Rhode Island 02912
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunit is not unprecedented. For example, the angiotensin II type 1 (AT1) receptor couples to both Gi and Gq (11). Whereas Gq has been
linked to phospholipase C activation (6, 7), the release of
arachidonate, via phospholipase A2 activation, has been linked to Gi
and Gs (12-14).
-turn lodged inside the
receptor. Doughty and Reynolds (30) generated a BKB2R model showing an
interaction between TM6 and TM7 through an
Arg281/Asp266 attraction. However, at this
time, the conformational features of the intracellular loops and
termini of the BKB2R have not been reported.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
counter. To examine
desensitization, cells were pretreated with DMEM containing saturating
doses of BK followed by an intervening wash and then restimulated with
BK. The control group was treated identically but pretreated with DMEM
containing no BK. Radioactivity in the supernatant for both groups was
measured as described above.
7 M BK for 2 h at 4 °C. Cells were washed three times with ice cold buffer and
then solubilized with 0.2% sodium dodecyl sulfate. Radioactivity was
determined in a counter. The affinity and the number of binding
sites were then determined. For internalization experiments, cells were
incubated with 100 nM BK at different time points at
37 °C. Cells were washed with ice-cold buffer and acid-stripped with
0.2 M acetic acid, pH 3.0, containing 0.5 M NaCl. The number of binding sites was then determined by performing a
binding assay as above. Data were expressed as the percentage of
[3H]BK-specific binding remaining after an acid wash.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Schematic representation of the rat BKB2
receptor. The schematic illustration shows the amino acid sequence
of IC2 and the carboxyl tail. The sites of substitution
(Tyr131, Thr137, and Tyr322) are
highlighted (shaded circles) and indicated by the
arrows.
Binding parameters of [3H]BK in transfected Rat-1 cells
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Fig. 2.
Bradykinin-induced PI turnover in Rat-1 cells
expressing single and double mutant BK receptors. PI turnover was
measured in myo-[3H]inositol-labeled cells
expressing WT and the indicated mutant receptors. Data represent
triplicate samples of each cell type from three experiments in which
various cell clones were analyzed in parallel. Statistical data
analysis was carried out by Student's t test. p
values < 0.05 were considered to indicate a significant
difference from WT.
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Fig. 3.
Bradykinin-induced ARA release in Rat-1 cells
expressing single and double mutant BK receptors. Rat-1 cells in
24-well plates were labeled with [3H]arachidonate (0.2 µCi/well) for 16 h, washed, and then stimulated with 100 nM bradykinin for 20 min as described under "Experimental
Procedures." Data represent triplicate wells from three experiments.
Statistical data analysis was carried out by Student's t
test. p values < 0.05 were considered to indicate a
significant difference from WT.
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Fig. 4.
PI turnover and ARA release by
Thr137 mutant receptors. Effects of T137P and T137D
receptors on the BK-stimulated PI turnover (A) and ARA
release (B) relative to WT. The method of measurement
is described under "Experimental Procedures."
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Fig. 5.
Internalization profile of Thr137
mutant receptors. Rat-1 cells expressing WT ( 
), T137P (
),
and T137D (
) were incubated with 100 nM BK for the times
indicated. After acid stripping and three washes with ice-cold buffer,
[3H]BK binding to Rat-1 cells was measured as described
under "Experimental Procedures." Results are the mean of duplicate
wells from three separate experiments.
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Fig. 6.
Internalization profile of double mutant BK
receptors. Rat-1 cells expressing WT ( ), YSYS (), YAYA
(
), and YFYF () were incubated with 100 nM BK for the
times indicated. After acid stripping and extensive washing with
buffer, [3H]BK binding to cells was measured as described
under "Experimental Procedures." Results are the mean of duplicate
wells from two separate experiments.
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Fig. 7.
Receptor desensitization with ARA release as
parameter. Rat-1 cells expressing WT, YAYA, YFYF, and T137P
receptors were labeled with [3H]arachidonate for 16 h as described under "Experimental Procedures." The experimental
group was pretreated with DMEM containing saturating doses of BK
followed by an intervening wash and then restimulated with BK. The
control group was treated identically but pretreated with DMEM
containing no BK. Radioactivity in the supernatant for both groups was
determined. Percentage of desensitization was calculated by dividing
the second BK stimulated ARA release in the experimental group by the
BK-stimulated ARA release in the control group and subtracting from
100. Statistical data analysis was carried out by Student's
t test. p values < 0.05 were considered to
indicate a significant difference between ARA release in the
experimental group versus the control group.
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Fig. 8.
Receptor desensitization and resensitization
with Ca2+ mobilization as parameter.
A, for desensitization, Rat-1 cells expressing WT ( 
),
T137P (), YAYA (), and YFYF (
) receptors loaded with Fura-2
were stimulated with 10 nM BK and followed by a second dose
of 10 nM BK. Arrows indicate time of BK
administration. B, for resensitization, Rat-1 cells were
stimulated with 10 nM BK for 2 min. Cells were washed and
allowed to recover at 37 °C for 15 min before a second dose of 10 nM BK. Data is a representative of at least three
independent measurements.
-helix in the
region just after transmembrane 7 (TM7), encompassing residues
310-329, and a second, less well defined helix, encompassing residues
335-350. The first helix is amphipathic in nature, with a well defined
hydrophobic and hydrophilic face. Given the amphipathicity of the
helix, we assumed that it is lying on the surface of the membrane, with the hydrophilic amino acids projecting into the aqueous phase. During
the MD simulations, this helix always adopted such an orientation with
respect to the water/decane interface, even when starting completely in
the water or decane phase. The results for IC2 indicate the presence of
a -turn centered around Thr137 (i + 1 position of the turn). Both of these conformational features were
incorporated into the molecular model of the receptor. Alternative starting structures were generated by moving the -helical domain of
residues 310-329 to different positions with respect to the helical
bundle and IC2 (i.e. projecting toward TM1-TM2, on the other
side adjacent to TM6-TM5, or directly underneath the helical bundle).
During the simulations, a weak harmonic force with a target distance of
12 Å was applied to residues Tyr131 and
Tyr322. In this manner, the conformations of the
cytoplasmic domains can adjust to adapt to this constraint. The results
from the simulation with the helix near TM6-TM5 are shown in Fig.
9.
View larger version (55K):
[in a new window]
Fig. 9.
The BKB2R conformation generated by MD
simulations in a decane/water two-phase simulation cell. The MD
simulations of the BKB2 are shown in side (A) and bottom
(B) views. The transmembrane helices are shown as
cylinders, with TM3 displayed in cyan. IC2 and
IC3 are shown as light blue and dark blue
ribbons, respectively. Amino acid residues Tyr131,
Thr137, and Tyr322 are shown in
orange in ball-and-stick format.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2-adrenergic receptor, the C-terminal portion of IC3 and the proximal C tail are important for Gs activation (44). In the BKB2R,
a small hydrophobic residue such as alanine at Tyr131 and
Tyr322 favors interaction with a G protein responsible for
PI turnover (Gq). In all, residue size, hydrophobicity, and charge at
these positions play important roles in signal transduction. Another example to illustrate the effect of charge is the substitution of the
hydrophilic threonine residue at location 137 with either proline or
aspartate. Exchange of threonine for proline had no effect on either
ARA release or PI turnover. However, the T137D mutant receptor, which
displays a negative charge, showed impaired PI turnover and ARA
release. Our previous studies demonstrated that point mutations at
Tyr131 to serine, alanine, or phenylalanine also affected
the rate of receptor internalization as compared with that of WT (1).
Y131A displayed the slowest rate of uptake, whereas Y131S displayed a
rate considerably faster than WT. Our present results further demonstrate that the double mutant YAYA also displayed a very slow rate
of uptake, notably slower than WT or the other double mutants. These
results confirm the importance of the DRY region in receptor
internalization. Apparently, mutant receptors that display either the
slowest or the most rapid rates of receptor internalization still show
a degree of PI turnover similar to that of WT receptor. This suggests
that signal transduction in relation to the
Tyr131/Tyr322 interaction is not involved
directly with receptor uptake. A similar lack of correlation between
internalization and signal transduction was observed in muscarinic
(18), angiotensin/AT1 (45), and 2-adrenergic receptors
(46).
motif as proposed
in muscarinic cholinergic receptor (23). We aligned this motif with 184 GPCRs belonging to the peptide subfamily. We found that proline is
conserved in 71% of the receptors. Conversion of threonine in the
BKB2R to proline resulted in a complete loss of receptor uptake.
However, substitution of proline at this site did not affect signal
transduction. Substitution with a negatively charged aspartate to
generate T137D reversed these effects. T137D did not stimulate ARA
release or PI turnover but returned receptor uptake to that of WT. This
result points to the importance of a negative charge at this site and suggests that phosphorylation at Thr137 participates in
termination of receptor function and initiation of receptor uptake.
Blaukat et al. (42) demonstrated threonine/serine phosphorylation to be taking place in conjunction with BKB2R
internalization and resensitization.
-helices (for the C terminus)
and a -turn (residues 137-138 of IC2) based on homology analysis
with structurally defined proteins, following a procedure recently
reported for the 
-opiate receptor (36). However, the relative
orientation of the loops and termini, containing the -helices or
-turns, needs to be established. The aim of our modeling effort is
to investigate the conformations with these two regions (IC2 and C
terminus) in close proximity. Given the constraints (i.e.
presence of the -helices and -turn), we looked for conformations
that are energetically feasible. There are three possible orientations
that would place the C-terminal helix (residues 310-329) in close
proximity to Tyr131: it could lie 1) on the membrane
surface near TM6-TM5, 2) on the membrane surface near TM1-TM2, or (3)
across the cytoplasmic ends of the helix bundle. All attempts at
placing the C-terminal helix toward TM1-TM2 failed. The topological
orientation of the helical bundle does not allow for Tyr131
and Tyr322 to come close without disrupting the C-terminal
helix or the TM bundle. In contrast, during the simulations starting
with the other two orientations, low energy conformations were adapted early and maintained for the remainder of the 200 ps of MD simulation. Of these two, we prefer the first, in which the C-terminal helix lies
on the membrane surface, mimicked by a layer of decane molecules in the
simulation, near TM6-TM5. In the resulting structure, the hydrophobic
face of the amphipathic C-terminal helix projects into the membrane
surface. This conformation also places Cys326, postulated
to be a site of palmitoylation, adjacent to the membrane surface. One
role of the palmitoylation of this residue would be to securely anchor
this helix to the membrane surface. Based on this, we exclude the low
energy conformations that place the -helix of the C terminus across
the helix bundle, even though from an energetic perspective based on
the MD simulations, it is feasible. In Fig. 9, two views of the
resulting conformation from the MD simulation, placing
Tyr131 and Tyr322 8.1 Å distal from each
other, are shown. The resulting conformation places IC3 juxtaposed to
IC2 and the C terminus. This is important given the well documented
role of IC3 in coupling to the G proteins.
-turn, the resulting
conformations have this turn well exposed, projecting down into the
solvent, readily available for phosphorylation. Mutations at this
position indicate that following receptor activation, leading to signal transduction, phosphorylation at this position results in termination of the activated receptor structure. This termination is then followed
by internalization and potential resensitization of the receptor. A
model of the T137P mutation suggests that it stabilizes the -turn
structure, while removing the possibility of phosphorylation.
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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