(Received for publication, February 27, 1997, and in revised form, March 28, 1997)
From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
Presently, little is known of the amino acid motif(s) participating in bradykinin B2 receptor-mediated signal transduction processes. In this report we investigate the potential role of the two existing tyrosine (Tyr) residues in the intracellular regions and the carboxyl terminus in the regulatory function of this receptor. Rat-1 cells, which do not contain detectable bradykinin B2 receptor, were transfected with wild type and mutant receptor cDNAs. Tyr-131 and Tyr-321 were each mutated to corresponding alanine-, serine-, and phenylalanine-containing sequences. The last 34 amino acid residues of the carboxyl terminus were truncated. Rat-1 cells transfected with the mutant forms of the receptor cDNA including the truncated COOH-terminal cDNA all bound [3H]bradykinin with essentially the same Kd of approximately 2.2 nM as cells transfected with the wild type bradykinin B2 receptor. However, mutating Tyr-131 resulted in important changes in bradykinin-stimulated phosphoinositide turnover and arachidonate release. For example, exchanging Tyr-131 for alanine led to an 80% decreased arachidonate release (p < 0.005), 90% decrease in inositol phosphate (IP) accumulation (p < 0.001), with receptor uptake at 15 min remaining essentially unchanged. Mutating the same Tyr to phenylalanine resulted in unchanged bradykinin-stimulated IP accumulation, only a slightly lowered arachidonate release, and unchanged receptor uptake at 15 min. Mutating Tyr-321 to alanine resulted in a very different pattern. There was a small but significant reduction in arachidonate release (p < 0.03) and IP accumulation (p < 0.008) with a large, 30%, increase in receptor uptake at 15 min (p < 0.010). Truncation of a portion of the carboxyl tail also proved meaningful, with a 60% decrease in arachidonate release and an 80% decrease in IP accumulation. The truncation also resulted in a large, 130%, decrease in receptor uptake at 15 min (p < 0.023). Taken together, these results point to Tyr-131 as an important element in determining bradykinin-stimulated arachidonate release and IP accumulation. Tyrosine phosphorylation at this site apparently does not play a major role. Tyr-131, Tyr-321, and the carboxyl tail appear to be important in determining receptor uptake.
Bradykinin is a nine-amino acid peptide hormone exerting diverse biological action ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, generation of pain, and renal homeostasis (1, 2). Bradykinin expresses its physiological effect through activation of a B2 type receptor. The bradykinin B2 receptor cDNA has been isolated from several species such as mouse, rat, and human (3-5). The receptor is G-protein-coupled with a structure characterized by seven transmembrane regions (4). Upon binding, the receptor activates a number of enzyme systems involved in signal transduction including phospholipase C and cytosolic phospholipase A2 (6). When stably transfected into such cells as hamster CCL39 the receptor retains its signal paths (7).
Investigation of the regulatory regions or domains of the bradykinin B2 receptor which participate in the activation of second messenger responses has been hampered by the unavailability of immunoprecipitating antibodies against the receptor. Herein we target single amino acid and amino acid sequence moieties that could prove crucial to the function of this receptor in signal transduction. These include the two existing tyrosines at positions 131 and 321 and the carboxyl terminus consisting of 34 amino acid residues including four serines and two threonines. We analyze the ability of normal and mutant bradykinin B2 receptors to stimulate inositol phosphate (IP)1 production and release of arachidonate. Our results show that tyrosine (Tyr) 131 and 321 and the carboxyl terminus participate variably in function of both paths as well as in receptor internalization.
Materials[3H]Bradykinin (78 Ci/mmol)
was obtained from Amersham Corp.
myo-[1,2-3H]Inositol (45-80 Ci/mmol) and
[3H]arachidonate (60-100 Ci/mmol) were obtained from
DuPont NEN. Analytical grade Dowex X8 (AG 1-X8, 100-200 mesh) was
obtained from Bio-Rad. Restriction endonucleases were purchased from
DuPont NEN. Taq polymerase was obtained from Promega Corp.
(Madison, WI). Oligonucleotides were synthesized from an in-house
Applied Biosystems DNA synthesizer. All other reagents were from Sigma unless stated otherwise.
Rat-1 cells were obtained from Dr. Robert Weinberg (Whitehead Institute, MIT) as a generous gift. Cells were grown at 37 °C in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum supplemented with 50 units/ml penicillin and 50 µg/ml streptomycin. All tissue culture dishes were obtained from Fisher Scientific.
Site-directed MutagenesisThe overlap extension polymerase
chain reaction method was used to generate mutants of the bradykinin B2
receptor utilizing as template the rat bradykinin B2 cDNA clone in
the vector pRC/CMV (Invitrogen). For each mutant receptor construct,
four oligonucleotides (two flanking and two internal primers) were
utilized. The two flanking primers, an upstream primer located 33 base
pairs upstream from the start site with an internal HindIII
site and a downstream primer designed to contain a stop codon followed
by an endonuclease restriction site XbaI, were designed to
facilitate subcloning into the HindIII/XbaI site
of the plasmid pRC/CMV. The two internal primers were complementary to
each other and contained the desired mutant sequences with melting
temperatures between 55 and 65 °C. The Tyr at position 131 was
altered to either Ala, Ser, or Phe. The Tyr-131 Ala (Y131A) mutant
was constructed using a sense oligonucleotide
5
-tatcgaccgagccctggcgct-3
and an antisense oligonucleotide
5
-agcgccagggctcggtcgata-3
(bold letters indicate the
nucleotides that were altered). The other bradykinin B2 receptor
mutants were constructed using the following: Y131S (sense strand,
5
-atcgaccgatccctggcgctg-3
and antisense strand,
5
-cagcgccagggatcggtcgat-3
); Y131F (sense strand, 5
-atcgaccgattcctggcgctg-3
and antisense strand,
5
-cagcgccaggaatcggtcgat-3
); Y321A (sense strand,
5
-agaggtggcccaggcaata-3
and antisense strand,
5
-tattgcctgggccacctct-3
). The bradykinin B2
receptor-deleted carboxyl tail (DC34) was constructed using a
downstream primer containing nucleotide sequences encoding amino acids
327-331, a stop codon, and a XbaI restriction site. Two
overlapping fragments from two polymerase chain reactions with paired
external and internal primers were mixed for a second polymerase chain
reaction as described (8). The polymerase chain reaction product of the
each receptor construct was sequenced to confirm the identity of the
receptor mutants.
Rat-1 cells were incubated with 1 µCi/ml
myo-[3H] inositol in 1 ml of growth medium
for 16-24 h. Ten min prior to ligand stimulation, cells were exposed
to Dulbecco's modified Eagle's medium containing 20 mM
LiCl2 and 20 mM Hepes, pH 7.4. Cells were then
exposed with 10 nM bradykinin for 30 min at 37 °C, and
incubations were terminated by the addition of 0.5 ml of 10 mM ice-cold formic acid. Cells were scraped off and the
formic acid soluble material isolated by centrifugation and neutralized
by adding 10 ml of 5 mM sodium tetraborate. Total
[3H]IPs were extracted using a Dowex AG 1-X8 formate
resin in an anion exchange column and eluted with 2 M
ammonium formate, pH 5.0, as described (9). Following the addition of 4 ml of Ecolite scintillation fluid (ICN Biomedical, Inc., Aurora, OH),
samples were counted for radioactivity in a Pharmacia Biotech Inc.
liquid scintillation counter.
Rat-1 cells were prelabeled with
[3H] arachidonate (0.2 µCi/well) for 16 h as
described previously (10). Briefly, cells were washed and incubated
with 500 µl of Dulbecco's medium containing 2 mg/ml bovine serum
albumin (radioimmunoassay grade, Sigma). Cells were then incubated with
100 nM bradykinin for 20 min at 37 °C. Medium was
removed and centrifuged at 800 × g. Radioactivity was
determined in a scintillation counter after addition of 2 ml of
Ecolite scintillation fluid.
Binding studies
of bradykinin to intact Rat-1 cells was carried out as described
previously (10). Briefly, 80-100% confluent cell monolayers in
24-well plates (Costar, Cambridge MA) were incubated in binding buffer
containing various concentrations of [3H]bradykinin in
the absence (total binding) or presence of 100-fold excess of
bradykinin (nonspecific binding) for 2 h at 4 °C. Unbound radioactivity was removed by washing three times with binding buffer
containing 0.15% bovine serum albumin. Cells were solubilized with 0.5 ml of 0.2% sodium dodecyl sulfate. Cell-bound radioactivity was
determined in a counter after the addition of 2 ml of Ecolite scintillation fluid. For internalization studies, cells were incubated with 100 nM bradykinin at different time point at 37 °C.
Cells were washed with ice-cold buffer to remove unbound bradykinin. Surface bound ligand was stripped by a 5-min incubation on ice 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]bradykinin-specific binding remaining after acid
washing.
All values are represented as means ± S.E. Student's t test was used for statistical analysis of data.
A diagrammatic representation of wild type bradykinin B2 receptor
construct is illustrated in Fig. 1. Tyr-131 was mutated to the corresponding alanine, serine, and phenylalanine, designated Y131A, Y131S, and Y131F, respectively. Tyr-321 was mutated to alanine,
designated Y321A. Carboxyl-terminal truncation mutant missing the
terminal 34 amino acids is designated DC34.
Although previous binding studies have shown some endogenous bradykinin
B2 receptors in Rat-1 cells (11), our control Rat-1 cells did not
express detectable levels of [3H]bradykinin binding or
bradykinin-stimulated phosphoinositide turnover and arachidonate
release. Rat-1 cells were then stably transfected using an expression
vector pRC/CMV, containing cDNA inserts encoding the full-length or
the mutated rat bradykinin B2 receptors. Tritium-labeled ligand binding
analysis was performed in intact Rat-1 cells, untransfected or stably
transfected with wild type or mutant bradykinin B2 receptors to assess
receptor expression. Fig. 2 illustrates saturation
binding of untransfected cells, which did not bind bradykinin and wild
type bradykinin B2 receptor-transfected cells, which bound bradykinin
with a Kd of 2.24 nM. The binding
properties of mutant receptors to bradykinin when expressed in Rat-1
cells are summarized in Table I. As seen in Table I, all
mutant receptors bound with a dissociation constant similar to that for
the wild type receptor. The Y131F mutant receptor showed a slight
increase in binding affinity, whereas the Y321A receptor showed a
binding affinity identical to that of the wild type receptor. For the
purposes of this study we chose cell clones with a
Bmax ranging from 4.8 × 105 to
13 × 105 receptors/cell.
|
To determine the consequence of the bradykinin B2 receptor mutation on
receptor-phospholipase C interaction we measured total IP production in
response to bradykinin by the variously transfected Rat-1 cells. We
showed previously that bradykinin-stimulated IP production was not
receptor number-dependent in cells possessing between
25,000 and 140,000 bradykinin B2
receptors/cell.2 Rat-1 cells transfected
with Y321A bradykinin B2 receptor showed a 30% reduction in
bradykinin-stimulated phosphoinositide turnover compared with wild type
transfected cells (Fig. 3). Truncation of the
carboxyl-terminal 34 amino acids reduced IP production by 70%.
Mutation of Tyr-131 to alanine or serine resulted in the most dramatic
alterations in bradykinin-stimulated IP production. The response to
bradykinin in the Y131A- and Y131S-transfected cells was reduced by 91 and 93%, respectively. However, bradykinin-caused IP accumulation in
Y131F-transfected cells remained unchanged. Based on these results, it
appears that Tyr-131 residue has an important role in the action of
bradykinin on phosphoinositide turnover.
We next determined the ability of the mutant receptors to regulate the
release of free arachidonate from Rat-1 cells transfected with the
various mutant cDNAs. In cells containing between 28 and 120 × 105 receptors bradykinin-caused release was not receptor
number-dependent (data not shown). As shown in Fig.
4, mutation of Tyr-131 proved important to this process.
In cells transfected with Y131A, bradykinin lost 80% of its
stimulating activity. The Y131S-transfected cells lost all response to
bradykinin, and the Y131F-transfectants displayed a 50% loss in
bradykinin response compared with the wild type. Bradykinin-stimulated
release of arachidonate in Y321A receptor mutant and DC34-truncated
receptor cells was also reduced by approximately 50%.
To assess the contribution of Tyr-131, Tyr-321, and the carboxyl
terminus to the regulation of receptor internalization, we analyzed the
ability of bradykinin to induce loss of further bradykinin surface
binding. Rat-1 cells expressing wild type or the mutant receptors were
treated with excess ligand, 100 nM bradykinin, for 1, 5, 15, 30, 45, and 60 min at 37 °C to promote receptor internalization.
The receptor-associated bradykinin was then removed by an acid wash,
and the number of remaining binding sites was determined by incubating
the cells with a saturating concentration of
[3H]bradykinin at 4 °C. As shown in Fig.
5, all receptors exhibited a time-dependent
decrease in [3H]bradykinin binding. In 15 min
approximately 45% of wild type binding sites were removed. Truncation
of amino acid residues between positions 332 and 366 led to a decrease
in uptake compared with wild type, to only a 15% receptor uptake at 15 min and 30% at 30 min. On the other hand, mutation of Tyr-131 to Ser
and Tyr-321 to Ala resulted in transfectants with very rapid receptor
uptake of approximately 70% at 15 min. The corresponding Y131F
transfectant showed only a small decrease in receptor uptake of
approximately 35% at 15 min; the Y131A mutant displayed a larger
decrease in uptake of approximately 20% at 15 min.
To begin to identify the molecular determinants of the rat bradykinin B2 receptor-mediated intracellular signaling, we generated a truncated mutant of the receptor in which the last 34 amino acids of the carboxyl tail were deleted. This sequence contains four serine and two threonine residues and two protein kinase C consensus sequences. We also focused on the two intracellular Tyr residues that have been implicated in signal transduction leading to prostaglandin synthesis (12). Tyr-131, located in the second intracellular loop (i2) within the DRY motif, is within a region that is conserved among the family of G-protein coupled receptors (13). Tyr-321, located in the proximal carboxyl tail, is also conserved in all species of the bradykinin B1 and B2 receptor family. We analyzed the functional roles of these conserved residues, Tyr-131 and Tyr-321, in terms of their involvement in bradykinin-induced receptor internalization and signal transduction.
Our findings indicate that both Tyr residues and the carboxyl terminus influence receptor internalization and signal transduction but do so in different ways. The exchange of Tyr-131 or Tyr-321 for alanine, Tyr-131 for serine or phenylalanine, or truncation of the last 34 amino acids of the carboxyl terminus did not impede the insertion of the receptor into the membrane or the binding affinity for the ligand. All mutant receptors, including the wild type, yielded clones with various number of receptors/cell (between 48,000 and 115,000). Within this receptor range, we found bradykinin-stimulated phosphoinositide turnover and arachidonate release to be independent of receptor number.
Tyr-131 appears focal for all three processes examined here, namely IP generation, arachidonate release, and receptor internalization. With regard to receptor interaction with phospholipase C, i.e. IP accumulation, the importance of Tyr-131 is evident with almost total loss of activity in both Y131A and Y131S mutant receptors. These two amino acids are considerably smaller than Tyr and do not contain the aromatic ring. Serine, however, possesses a hydroxyl group and can be phosphorylated and/or form hydrogen bonds (14). Telling in this mechanism is the total retaining of activity in the Y131F-transfected cells. Since phenylalanine does not possess a hydroxyl group, Tyr phosphorylation or the charge contribution of the hydroxyl group does not appear critical. Instead, the hexane ring itself or the amino acid bulk may be a determinant of the specificity of this site. This finding agrees with a previous report that shows that Tyr residues of the bradykinin B2 receptor are not phosphorylated as indicated by phosphoamino acid analysis.(15).
Tyr-131 is also critical for bradykinin-activated arachidonate release. This is indicated by an 80% reduction in response in Y131A and a total loss of response in Y131S. The difference in response between Y131S and Y131A is probably due to the large receptor uptake of the Y131S. In addition, the Y131F mutant does not revert completely to the wild type response. Instead, it displays a small but statistically significant loss in response compared with wild type. It is possible that in this case charge or the aromatic acid bulk plays a cooperative role in this activity.
The role of Tyr-131 in receptor internalization is more difficult to
assess. Replacement of Tyr with either phenylalanine or alanine limits
uptake of the bradykinin B2 receptor as indicated by the reduced
receptor uptake in Y131A and Y131F. In this regard, the bulk of the
amino acid at this position appears to have a stabilizing effect with
the smaller alanine moiety being more restrictive to uptake than the
large phenylalanine and underscores the role for a hydrophilic amino
acid. Indeed, our results show that replacement of a hydrophilic Tyr by
a smaller hydrophilic amino acid such as Ser promotes rapid receptor
internalization. Serine residues have been linked to receptor
internalization from mutagenesis studies of other G-protein-coupled
receptors such as muscarinic receptors, monocyte chemoattractant
protein-1 receptor, and 2-adrenergic receptor (16-20).
In addition to the likely conformational changes, it is possible that
insertion of serine at position 131 within the conserved DRY motif of
the receptor, permits phosphorylation of this site by a Ser/Thr kinase,
which then facilitates receptor uptake. As shown in a reported
phosphoamino acid analysis of the bradykinin B2 receptor, serine
residues are the prime targets for bradykinin-induced phosphorylation,
whereas tyrosine moieties are not (15). Interestingly, the highly
conserved DRY region has been linked to the internalization of the
gonadotropin-releasing hormone receptor (21). The Tyr in the DRY region
of the gonadotropin-releasing hormone receptor is normally replaced by
Ser. When this Ser is mutated to Tyr, a 60% increase in uptake takes
place (21). Thus, the presence of Tyr-131 within the highly conserved
DRY region may be part of a motif that acts as a determinant of
bradykinin B2 receptor uptake.
Truncation of the carboxyl terminus has been shown repeatedly, in a
number of different receptor cDNAs, to influence receptor internalization (22-24). Our results show that the carboxyl tail of
the bradykinin B2 receptor is critical for regulation of receptor internalization. Truncation of the terminal 34 amino acids leads to a
slower rate of receptor uptake. It is interesting to note that receptor
uptake in the truncated mutant becomes indistinguishable from the wild
type receptor at 60 min. This suggests that other sites in the carboxyl
terminus may also be involved in receptor internalization regulation.
We then focused on a conserved Tyr residue in the proximal region of
the carboxyl terminus of the bradykinin B2 receptor. The exchange of
Tyr-321, at the proximal region of the carboxyl tail, to alanine led to
a dramatic increase in receptor uptake. At 60 min 40% more Tyr-321
receptor is taken up compared to wild type. Thus the presence of Tyr at
position 321 which is part of the carboxyl terminus inhibits receptor
internalization. The same mutation leads to only a small inhibition of
bradykinin-activated IP accumulation or arachidonate release. This
inhibition is probably a manifestation of the dramatic uptake of this
receptor during the initial 10 min following ligand-receptor
interaction. These observations on the effect of Y321A mutation on
receptor internalization emphasize the importance of aromatic amino
acids in maintaining efficient endocytosis. It is possible that Tyr-321
may be part of a recognition feature involving a limited number of
residues. Endocytotic motifs containing tyrosines in the carboxyl tail
have been identified in many single transmembrane domain proteins (24). Internalization motifs have been proposed to contain six residues involving a type I- turn with an exposed aromatic (Tyr) residue (23). In addition to such motifs, the overall topological and conformational structure of the carboxyl terminus appears to play an
important role in receptor internalization.
In conclusion, we analyzed the functional significance of two residues, a conserved Tyr-131 and a unique Tyr-321, and the carboxyl terminus of the rat bradykinin B2 receptor. The conserved Tyr-131 appears to play a significant role in receptor G-protein coupling (i.e. stimulation of IP production and arachidonate release) and influences internalization of the receptor. In contrast, Tyr-321 is not required for G-protein coupling but is involved in agonist-induced internalization of the receptor. The carboxyl terminus containing the putative phosphorylation sites Ser/Thr residues also appears to play a significant role in receptor internalization.