Effects of Intracellular Tyrosine Residue Mutation and Carboxyl Terminus Truncation on Signal Transduction and Internalization of the Rat Bradykinin B2 Receptor*

(Received for publication, February 27, 1997, and in revised form, March 28, 1997)

Gregory N. Prado Dagger , Linda Taylor and Peter Polgar

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

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.

Cell Culture

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 Mutagenesis

The 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 right-arrow 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.

IP Formation

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 beta  counter.

Release of Arachidonate

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 beta  counter after addition of 2 ml of Ecolite scintillation fluid.

Receptor Binding Assay and Internalization

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 beta  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.

Data Analysis

All values are represented as means ± S.E. Student's t test was used for statistical analysis of data.


RESULTS

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.


Fig. 1. Schematic representation of the structure of the rat B2 bradykinin receptor. Wild type (WT) rat bradykinin B2 receptor is shown with three putative glycosylation sites and two putative intracellular Tyr residues that were mutated. The second intracellular loop Tyr at position 131 was mutated to either alanine, serine, or phenylalanine, and the proximal carboxyl terminus at position 321 was mutated to alanine. The lower left panel and right panel show the wild type sequence aligned in relation to the mutated region of interest. The single letter amino code denotes the original and the substituted amino acid, and the number in between corresponds to the amino acid location. Deletion of 34 amino acids from residue 332 to 366 of the distal carboxyl terminus is also shown and is designated as DC34.
[View Larger Version of this Image (31K GIF file)]

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.


Fig. 2. Saturation binding analysis of [3H]bradykinin (BK) to untransfected Rat-1 cells and bradykinin B2 receptor expressed in Rat-1 cells. The specific [3H]bradykinin binding to wild type receptors (bullet ) and untransfected Rat-1 cells (black-square) is shown and described under "Experimental Procedures." Inset, Scatchard transformation of the data. Data are representative of three experiments.
[View Larger Version of this Image (15K GIF file)]

Table I. Binding parameters of [3H]bradykinin in transfected Rat-1 cells

Data represent the means ± S.E. obtained from three experiments with each point performed in duplicate.

Receptor Kd Bmax

nM sites/cell
Wild type 2.24 50,160  ± 4,917
Y131A 2.173 101,240  ± 9,640
Y131S 1.84 131,357  ± 5,613
Y131F 1.17 103,056  ± 6,314
Y321A 2.32 113,866  ± 9,108
DC34 1.89 47,772  ± 1,590

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.


Fig. 3. Bradykinin-stimulated IP formation in Rat-1 cells expressing mutant bradykinin receptors. IPs were measured in myo-[3H]inositol-labeled cells expressing wild type (WT) and the indicated mutant receptors. Data represent triplicate dishes 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.
[View Larger Version of this Image (38K GIF file)]

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%.


Fig. 4. Bradykinin-stimulated release of arachidonic acid in Rat-1 cells expressing mutant bradykinin receptors. Confluent cultures of Rat-1 cells in 24-well plates were labeled with [3H]arachidonate for 18 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. WT, wild type.
[View Larger Version of this Image (37K GIF file)]

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.


Fig. 5. Internalization of bradykinin receptors. Rat-1 cells expressing (panel A) wild type (WT, bullet ), Y321A (triangle ), and DC34 (open circle ) and (panel B) Y131A (open circle ), Y131F (bullet ), and Y131S (black-square) were treated with 100 nM bradykinin for various periods of time. After acid stripping and extensive washing with buffer, [3H]bradykinin binding to Rat-1 cells was measured as described under "Experimental Procedures." Data are mean of duplicate wells from two experiments.
[View Larger Version of this Image (10K GIF file)]


DISCUSSION

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 beta 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-beta 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.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants HL25776 from NHLBI and A600115 from NIA.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.
Dagger    To whom correspondence should be addressed: Dept of Biochemistry, Boston University School of Medicine, Boston, MA 02118. Tel.: 617-638-4717; Fax: 617-638-5339.
1   The abbreviation used is: IP, inositol phosphate.
2   D. A. Ricupero, P. Polgar, L. Taylor, M. O. Sowell, G. Yunling, G. Bradwin, and R. M. Mortensen, submitted for publication.

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