Correspondence to: Laurie Erb, Department of Biochemistry, M121 Medical Science Building, University of Missouri-Columbia, Columbia, MO 65212. Tel:(573) 882-1708 Fax:(573) 884-4597 E-mail:erbl{at}missouri.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G proteincoupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against Vß3/ß5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that
V integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signalregulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-
V integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go.
Key Words: purinergic receptors, cell surface receptors, integrins, GTP-binding proteins, signal transduction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular signaling by G proteincoupled receptors (GPCRs)1 not only involves direct coupling to heterotrimeric G proteins, but also involves the formation of larger protein complexes that assist in transmitting an extracellular signal to an intracellular response. For example, GPCRs activated by gamma-aminobutyric acid (GABA) can form a heteromeric complex with other gamma-aminobutyric acid receptors that is essential for activation of inwardly rectifying potassium channels (
In this paper, we have explored protein complex formation between integrins and the P2Y2 nucleotide receptor (P2Y2R), a Go- and Gq-coupled receptor that is stimulated by ATP or UTP and mediates activation of PLC-ß and MAP kinases (Vß3,
Vß5,
Vß11,
5ß1, and
IIbß3 (
Vß3 and
Vß5, and with CD47, a receptor for thrombospondin belonging to the IgG superfamily that is known to couple directly to ß1, ß2, and ß3 integrins and to hetero-trimeric G proteins in the Gi/o family (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies and Peptides
Antihuman 5 and
Vß5 mAbs, antihuman
5ß1 polyclonal antibody (pAb), FITC-labeled goat antimouse F(ab)2 antibody, and human fibronectin were purchased from GIBCO BRL. Antihuman
V mAb was purchased from Zymed Laboratories. Antihuman leukocyte antigen class II mAb was purchased from Immunotech. The production of 7G2 (mouse antihuman ß3 mAb) and B6H12 (mouse antihuman CD47 mAb) has been described previously (
3 mAb, and antimouse IgG conjugated to HRP were purchased from Santa Cruz Biotechnology, Inc. Antimouse IgG conjugated to Cy5 and antirabbit IgG conjugated to Oregon green 488 were purchased from Molecular Probes, and antifocal adhesion kinase (FAK) mAb was purchased from Upstate Biotechnology. Human vitronectin was purchased from Calbiochem. RGDs and RGEs were purchased from Sigma-Aldrich. The peptide corresponding to amino acids 93110 of the human P2Y2R, YARGDHWPFSTVLCKLVR (P2Y293110), and the peptide YARGEHWPFSTVLCKLVR in which the aspartic acid was replaced with glutamic acid (P2Y293110[E97]) were synthesized at the Eppley Core Laboratory (University of Nebraska Medical Center, Omaha, NE). P2Y293110 and P2Y293110(E97) were synthesized on a peptide synthesizer (431A; Applied Biosystems) using Fmoc chemistry and purified on a Waters Delta Prep 4000 system by reverse-phase HPLC with 0.1% trifluoroacetic acid in water versus 0.1% trifluoroacetic acid in acetonitrile using two Delta-Pak C18 40x 100 mm cartridges with 15 µm particle size and 300 Å pore size. Matrix-assisted laser desorption ionization (time of flight) mass spectrometry was used to verify the amino acid composition of P2Y293110 and P2Y293110(E97).
Cell Culture and Transfection
Human K562 erythroleukemia cells (CCL 243; American Type Culture Collection) were cultured in suspension in RPMI 1640 medium (GIBCO BRL) containing 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Human 1321N1 astrocytoma cells (
Immunofluorescent Staining and Visualization
Fluorescence-activated Cell Sorting.
K562 cells (106 cells/ml) were centrifuged (100 g at 4°C) and the cell pellet was resuspended in 100 µl of HBSS (GIBCO BRL) with or without antibodies to 5,
Vß5, or ß3 (0.2 mg/ml). After a 30-min incubation at 4°C, the cells were washed twice with 1 ml of PBS (GIBCO BRL) and incubated for 30 min at 4°C in 100 µl of HBSS containing 1:100 dilution of FITC-conjugated antimurine IgG. The cells were washed three times with 1 ml of PBS, fixed with 300 µl of 1% (wt/vol) paraformaldehyde in PBS containing 50 µM CaCl2, and stored at 4°C protected from light. The cells were then transferred to 12 x 75-mm tubes and fluorescence intensity was determined using a flow cytometer (EPICS 753; Beckman Coulter).
Dual Immunofluorescence Labeling.
1321N1 cells expressing HA-tagged P2Y2R constructs were plated on glass coverslips and grown to 40% confluence. The cells were incubated for 1 h at 22°C with 1:100 dilution of rabbit anti-HA pAb in DME containing 3% BSA, washed in PBS, and incubated for 1 h at 22°C with 1:100 dilution of antirabbit IgG conjugated to Cy5 in DME containing 3% BSA. The cells were then washed in PBS, fixed in 1% formalin in PBS for 10 min, lysed with 0.5% Triton X-100 in H2O for 1 min, and washed again in PBS. The fixed cells were incubated for 1 h at 22°C in PBS containing 3% BSA and 1:100 dilution of mouse anti-
V mAb, washed in PBS, and incubated for 1 h at 22°C in PBS containing 3% BSA and 1:100 dilution of antimouse IgG conjugated to Oregon green. The dual-labeled cells were washed in PBS, rinsed in H2O, and mounted on glass slides in Prolong Antifade reagent (Molecular Probes). Digital images of the dual-labeled cells were taken on a confocal microscope (MRC600; Bio-Rad Laboratories), processed with CoMOS software, and visualized with Adobe Photoshop® v5.5 in which red was assigned to Cy5 fluorescence and green was assigned to Oregon green fluorescence. Yellow pixels, representing colocalization of P2Y2Rs and
V integrins, were quantified in single cells from a Photoshop® histogram. In brief, single cell images (1.64 µm/pixel magnification) were selected from a flattened, 24-bit RGB mode document and copied to a new document in CYMK mode where curves for cyan, magenta, and black, representing image noise, were reduced to 0. Pixels within counts 150 of the resulting yellow histogram were recorded as the total number of yellow pixels per cell.
Ligand-coated Bead-binding Assay
Preparation of ligand-coated beads and analysis of their binding to cells were performed as described (
Receptor Constructs
Incorporation of the HA Epitope into the P2Y2R.
Wild-type human P2Y2R cDNA was subcloned into the retrovirus expression vector pLXSN at the EcoRI/BamHI sites of the multiple cloning site. The open reading frame of the wild-type P2Y2R cDNA was modified to incorporate at the NH2 terminus of the expressed protein, the HA epitope (YPYDVPDYA) from influenza virus, by using the PCR. The forward and reverse HA primers were, respectively: 5'-GATCGTGAATTCTGATGTATCCATATGATGTTCCAGATTATGCTGCAGCAGACCTGGAACCCTGG-3' and 5'-GATCGTGGATCCCCTGACTGAGGTGCTATAGCCG-3'. The PCR solution contained primers (0.7 µM each), 10 µl of 10x Vent polymerase buffer (New England Biolabs, Inc.), 100 ng of template DNA, 1 U Vent (exo+) polymerase, and 20 µl of dNTP mixture (0.2 mM dATP, dCTP, dTTP, and dGTP) in a final volume of 100 µl. The PCR parameters were: 96°C for 1 min, 62°C for 1 min, and 72°C for 2.5 min for 25 cycles. After verification of the PCR amplification products by agarose gel electrophoresis, the products were purified using a Promega PCR Wizard kit. The purified PCR products and pLXSN DNA were digested overnight with EcoRI and BamHI and ligated together, followed by transformation of competent Escherichia coli and identification of positive clones. All mutant DNAs were sequenced on both strands to ensure mutagenesis had occurred as predicted using an ABI Prism automated sequencer (PerkinElmer) and fluorescence dideoxy-nucleotide technology.
Site-directed Mutagenesis of P2Y Receptor cDNA.
A codon for glutamic acid (E) was substituted for D116 and D121 of the turkey P2Y1R and D97 of the human P2Y2R cDNAs by the PCR method described above, except that the forward and reverse P2Y1R-E116 primers were, respectively: 5'-GCCTCCACCAAGTGCTCCCTG-3' and 5'-CCTCTGCAGTTTGCACATGACATCCCCGAAGATCCATTCGGTTTT-3'. The forward and reverse P2Y1E121R primers were, respectively: 5'-GCCTCCACCAAGTGCTCCCTG-3' and 5'-CCTCTGCAGTTTGCACATGACTTCCCCGAA-3'. The 260-bp PCR products were excised from a 0.7% agarose gel, cut with the restriction enzymes PstI and DraIII, and substituted for the wild-type PstI/DraIII cDNA fragment in a P2Y1R-p bluescript construct. The P2Y1R-E116 and P2Y1R-E121 cDNAs were subcloned into the BamHI polylinker site of pLXSN for mammalian cell expression. This protocol was also used to make the F119 to R119 mutation in the turkey P2Y1R cDNA, in which the forward and reverse P2Y1R-R119 primers were, respectively: 5'-GCCTCCACCAAGTGCTCCCTG-3' and 5'-CCTCTGCAGTTTGCACATGACATCCCCGCGGATCCAGT-3'. The forward and reverse P2Y2R-E97 primers were, respectively: 5'-CGCCCGCGGCGAACACTGGCCC-3' and 5'-AGACACCGGTGCACG-3'. The 110-bp PCR product was excised from a 0.7% agarose gel, cut with the restriction enzymes SacII and AgeI, and substituted for the wild-type SacII/AgeI cDNA fragment in the HA-tagged P2Y2R-pLXSN construct. The mutant cDNA sequences were verified by using fluorescence dideoxy-nucleotide sequencing, as described above.
Measurement of Cell Signaling Pathways
Calcium Assay.
The intracellular free calcium concentration ([Ca2+]i) was measured by dual excitation spectrofluorometric analysis of cell suspensions loaded with fura-2 (Sigma-Aldrich) as described previously (
IP Assay.
To measure accumulation of IPs, 1321N1 cells expressing P2Y2R were plated in 12-well dishes and incubated in the presence or absence of specified antibodies for 24 h in 0.7 ml serum-free and inositol-free DME containing 2 µCi of [3H]inositol (20 Ci/mmol). The cells were washed three times and incubated for 30 min at 37°C in 1 ml of LiCl solution (15 mM glucose, 25 mM Hepes, 110 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.9 mM MgCl2, and 30 mM LiCl) followed by stimulation with 0.1 µM UTP for 10 min. The reactions were stopped by aspiration and cell extracts were obtained by addition of 0.3 ml of ice-cold 10% perchloric acid for 15 min. The extracts were neutralized with 0.6 ml of 2 M NaOH and 225 mM Hepes. The IPs in the neutralized extracts were separated from inositol and glycerophosphoinositol on Dowex columns as described previously (
FAK and ERK1/2 Phosphorylation.
1321N1 cells expressing P2Y2R constructs were grown to 80% confluence in six-well plates and incubated at 37°C in medium without serum for 24 h before the experiment. After stimulation, the cells were washed with ice-cold PBS and lysed with 250 µl of lysis buffer (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 10 mM Na4(P2O7), 25 mM ß-glycerophosphate, 25 mM p-nitrophenylphosphate, 0.5 mM EGTA, 0.5% Triton X-100, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 nM okadaic acid). The extracts were centrifuged (8,200 g for 10 min at 4°C) to remove insoluble material, solubilized in 100 µl of 2x Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 2% SDS, 10% sucrose, 1 mM EDTA, 50 mM dithiothreitol, 0.003% bromophenol blue), heated for 3 min at 96°C, subjected to 7.5% SDS-PAGE, and transferred to nitrocellulose membranes for protein immunoblotting. Immunoblotting of tyrosine phosphorylated FAK was performed by using 1:1,500 dilution of rabbit antiphospho FAK Tyr397 IgG (Upstate Biotechnology) as the primary antibody and 1:2,000 dilution of HRP-conjugated antirabbit IgG as the secondary antibody. Immunoblotting of phosphorylated ERK1/2 was performed by using 1:1,000 dilution of mouse antiphospho p42/p44 MAP kinase IgG (Cell Signaling) as the primary antibody and 1:2,000 dilution of HRP-conjugated antimouse IgG as the secondary antibody. Chemiluminescence in the blots was visualized on autoradiographic film with the LumiGlo chemiluminescence system (New England BioLabs, Inc.) and was quantitated by using a GS-525 molecular imager and MultiAnalyst software (Bio-Rad Laboratories). For normalization of the signal, the membranes were stripped of antibodies by a 30 min incubation at 60°C in stripping buffer (62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS), washed with TTBS, and reprobed with 1:1,000 dilution of anti-FAK or anti-p42/p44 MAP kinase as the primary antibodies, which bind to FAK or ERK1/2 independent of their phosphorylation state.
Alternatively, tyrosine phosphorylation of FAK was analyzed by immunoprecipitation of FAK as described by the antibody manufacturer (Upstate Biotechnology) followed by immunoblotting of the precipitated protein with 1:1,500 dilution of mouse antiphosphotyrosine monoclonal IgG as the primary antibody and 1:1,500 dilution of HRP-conjugated antimouse IgG as the secondary antibody.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
P2Y293110 Interacts Specifically with Vß3/ß5 Integrins and CD47
To investigate whether the RGD domain of the P2Y2R facilitates binding to a specific integrin, we coupled a peptide derived from the human P2Y2R, YARGDHWPFSTVLCKLVR (P2Y293110) to fluorescent beads and assessed the ability of the peptide-coated beads to adhere to human K562 erythroleukemia cells. Integrin receptor expression in K562 cells has been reported to be limited to the fibronectin-binding integrin 5ß1 (
2,
3,
V, and ß3) can be induced by treatment of the cells with the protein kinase activator phorbol dibutyrate (PDBu;
5,
Vß5, or ß3 and subjected to fluorescence-activated cell sorting. We found that 88, 63, and 74% of the K562 cells were positively stained by antibodies to
5,
Vß5, and ß3, respectively, and that an overnight treatment of K562 cells with 100 nM PDBu did not increase the fluorescence intensity or percentage of cells stained by these antiintegrin antibodies (not shown). This indicates that the K562 cell line used in this study expresses vitronectin-binding integrins (
Vß3 and
Vß5) and that cell surface expression of these integrins does not require PDBu-mediated stimulation of protein kinase C. Results of the peptide-binding studies indicated that P2Y293110-coated beads bound to K562 cells with an AI of 580 ± 71 (mean ± SEM, n = 6), whereas fibronectin- and vitronectin-coated beads exhibited AI values of 318 ± 14 (n = 3) and 300 ± 28 (n = 3), respectively (Fig 1 A). The AI for nonspecific binding, determined with human serum albumincoated beads, was 46 ± 10 (n = 3), and the AI for beads coated with an RGE mutant P2Y2 peptide, YARGEHWPFSTVLCKLVR (P2Y293110[E97]), was 70 ± 18 (n = 3). Previous studies indicated that the RGE sequence has a low affinity for integrins (
|
The specificity of binding between K562 cells and P2Y293110-coated beads was determined by preincubating the cells with antibodies raised against specific integrins or integrin-associated proteins. P2Y293110-coated bead binding to K562 cells was inhibited by mAbs to ß3, Vß5, or CD47, but not by antibodies to the
5ß1 integrin (Fig 1 B). These results suggest that the RGD peptide corresponding to the first extracellular loop of the P2Y2 receptor binds specifically to
Vß3/ß5 integrins, but not to the
5ß1 integrin expressed in K562 cells. The finding that anti-CD47 antibodies could also inhibit binding of P2Y293110-coated beads to K562 cells supports the involvement of an
Vß3 integrin, since CD47 was shown previously to associate with ß3 integrins and regulate vitronectin binding to the
Vß3 integrin (
RGD-dependent Colocalization of the P2Y2R with V Integrins
To determine whether the full-length P2Y2R interacts with Vß3/ß5 integrins, we expressed HA-tagged wild-type or RGE mutant P2Y2Rs in human 1321N1 astrocytoma cells and used immunofluorescence to analyze the distribution of these receptors relative to the
V integrin subunit. Confocal microscopy of dual-labeled 1321N1 cell transfectants showed that endogenously expressed
V exhibited
10-fold higher levels of colocalization with the wild-type P2Y2R than with the RGE mutant P2Y2R (Fig 2). Furthermore, colocalization of
V and the wild-type P2Y2R occurred whether or not the cells were treated with 100 µM UTP (Fig 2), indicating that association of these proteins is not dependent on P2Y2R activation.
|
The RGD Domain of the P2Y2R Regulates Go-mediated Ca2+ Mobilization and Other Signaling Events
Functional studies indicated that the RGE mutant P2Y2R required 1,000-fold higher concentrations of the agonists ATP or UTP than the wild-type P2Y2R to stimulate PLC-dependent mobilization of intracellular Ca2+ in human 1321N1 cell transfectants (Fig 3 A). The difference in agonist potencies between the wild-type and RGE mutant P2Y2Rs in the cell transfectants was not due to differences in the level of cell surface receptor expression, since fluorescence-activated cell sorting of intact cells labeled with anti-HA antibodies confirmed that both HA-tagged wild-type and RGE mutant P2Y2Rs had similar expression levels (not shown). It is also unlikely that the RGE mutation in the first extracellular loop of the P2Y2R affects ligand binding since receptor modeling and mutagenesis studies have indicated that the ligand-binding determinants for both the P2Y1 and P2Y2 receptor subtypes are located within the third, sixth, and seventh transmembrane domains (
|
|
The Gi/o inhibitor pertussis toxin caused a 30% inhibition of Ca2+ mobilization mediated by the wild-type P2Y2R expressed in 1321N1 cells, whereas Ca2+ mobilization mediated by the RGE mutant receptor was insensitive to pertussis toxin treatment (Fig 3 B). This supports other studies indicating that the P2Y2R activates both Go- and Gq-mediated calcium signaling (
Previous studies indicated that the P2Y2R mediates tyrosine phosphorylation of FAK (1,000-fold higher concentrations of the agonist UTP, as compared with the wild-type P2Y2R. FAK phosphorylation mediated by the wild-type and RGE mutant P2Y2Rs was dependent on intracellular Ca2+ mobilization, since preincubation with the calcium chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibited UTP-induced FAK phosphorylation in 1321N1 cell transfectants (Fig 4 B). These results are consistent with the conclusion that the RGD sequence of the P2Y2R promotes efficient activation of the calcium-dependent signaling pathway.
|
P2Y2R activation in 1321N1 cells also leads to phosphorylation of the MAP kinases, ERK1/2. Similar to results from experiments on calcium mobilization and calcium-dependent FAK phosphorylation (Fig 3 and Fig 4), ERK1/2 phosphorylation by the RGE mutant P2Y2R requires 1,000-fold higher concentrations of UTP, as compared with the wild-type P2Y2R (Fig 5 A). Depending on the cell type, ERK1/2 phosphorylation by the P2Y2R has been found to occur by the calcium-independent activation of protein kinase C
and phospholipase D (
|
P2Y2R-mediated Signaling Is Inhibited by Anti-V Antibodies and by RGDS
We investigated whether an antibody raised against the V integrin subunit could interfere with UTP-stimulated P2Y2R signal transduction. To analyze the affect of this antibody on the P2Y2R calcium signaling pathway, we measured IP formation since inositol 1,4,5-trisphosphate (IP3) is the second messenger for intracellular calcium mobilization (
V antibody partially inhibited IP formation induced by UTP (Fig 6 A), consistent with the decreased calcium response seen for RGE mutant receptors as compared with wild-type P2Y2R (Fig 3). Similarly, phosphorylation of FAK, a calcium-dependent response (Fig 4 B), was partially inhibited by anti-
V antibody (Fig 6 B). ERK1/2 phosphorylation was inhibited by anti-
V antibody to a lesser extent than FAK phosphorylation or IP formation (Fig 6 C), consistent with the presence of both calcium-dependent and calcium-independent pathways for ERK1/2 phosphorylation (Fig 5 B). Anti-
3 integrin antibody had no effect on the ability of the P2Y2R to stimulate any of these signaling events (Fig 6, AC). These findings suggest that interaction of
V integrin with the P2Y2R is important for optimum P2Y2R activation.
|
Previous studies have indicated that overnight treatment with the tetrapeptide RGDS, which inhibits integrin dimerization and disrupts the formation of focal adhesions, can also inhibit GPCR-mediated ERK1/2 activation and FAK phosphorylation in certain cell types (V antibody.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study suggest that an integrin-binding domain (RGD) in the P2Y2R allows this receptor to associate with Vß3/ß5 integrins and the integrin-associated thrombospondin receptor, CD47. Furthermore, mutation of the RGD sequence to RGE in the P2Y2R or addition of an anti-
V integrin antibody was found to inhibit P2Y2R signal transduction (i.e., IP formation and phosphorylation of FAK and ERK1/2). This suggests that interaction between P2Y2Rs and integrins either shifts the P2Y2R into a high affinity state and/or allows efficient coupling of this receptor to its intracellular signaling proteins. Currently, there is no reliable assay for measuring ligand binding to the P2Y2R, making it difficult to distinguish between these two possibilities. However, there is evidence that the latter is affected. We found that the Gi/o inhibitor, pertussis toxin, partially inhibited Ca2+ signaling by the wild-type P2Y2R, but not by the RGE mutant receptor. Since CD47 has been found to form a complex with integrins and proteins in the Gi/o family (
Activation of CD47 has been shown to shift several integrins into a high affinity state. For example, the CD47 agonist 4N1K stimulates platelet spreading on a fibrinogen-coated surface and causes platelet aggregation by activating the integrin IIbß3 (
Vß3 integrin-dependent cell spreading on a vitronectin-coated surface (
2ß1 integrin-dependent chemotactic migration toward soluble collagen (
Vß3 integrin with peptides derived from type IV collagen causes Ca2+ mobilization and chemotaxis, both of which require CD47 (
Vß3/ß5 integrins and CD47 and support the idea that functional diversity among members of the GPCR superfamily is provided by differential linkage to scaffolding proteins involved in the formation of a protein-signaling complex (
The presence of an RGD sequence in an extracellular domain of a GPCR is rare, occurring only in the first extracellular loop of several species homologues of the P2Y2Rs and P2Y6Rs and the third extracellular loop of the H2 histamine receptor (GPCR database: http://www.gpcr.org). In contrast to the human and murine P2Y2Rs, the rat homologue contains a QGD instead of an RGD sequence (
Although the potential for a GPCR to physically interact with an integrin has not been explored previously, there is precedence for interactions between integrins and other types of receptors containing an RGD sequence. For example, the urokinase-type plasminogen-activated receptor has been found to coimmunoprecipitate with the Mß2 integrin (
Vß3 integrins (
Vß3 and the PDGF receptor increase endothelial cell migration (
V integrin thus far have been unsuccessful. Studies that have successfully coimmunoprecipitated GPCRs and other proteins have found that (a) covalent crosslinking of the proteins is necessary because of the harsh conditions required to solubilize GPCRs and (b) overexpression of both proteins is necessary for Western blot detection (
V integrin colocalize in the plasma membrane of 1321N1 astrocytoma cells and that the extent of colocalization between these proteins is
10-fold greater than the level of colocalization between
V and the RGE mutant receptor (Fig 2). Although these data are suggestive of a direct interaction between an integrin and the RGD sequence of the P2Y2R, it is possible that the interaction observed by microscopy reflects a transient, low-affinity interaction or an interaction with the detergent insoluble actin cytoskeleton. If this is the case, it will require more sophisticated techniques to demonstrate this interaction by coimmunoprecipitation. Whatever the nature of this RGD-dependent interaction, our data strongly suggest that it is directly relevant to the regulation of P2Y2R-mediated signal transduction.
The Vß3 integrin is expressed in vascular endothelial cells and has been shown to play a vital role in angiogenesis, the de novo growth of blood vessels, a process essential to the growth of solid tumors that has become an important target in cancer therapy (
v integrin expression that is inhibited by the protein kinase C inhibitor GF109238 (Erb, L., C. Clamp, and G.A. Weisman. 1998. International Society for Heart ResearchXVI World Congress. J. Mol. Cell. Cardiol. 30:499). In endothelial cell cultures, P2Y2R expression is readily apparent (
Vß3, may occur under conditions that promote angiogenesis or atherosclerosis. Considering the wide-spread distribution of P2Y2Rs, it seems likely that interactions between P2Y2Rs and integrins, such as
Vß3, could have a range of physiological consequences yet to be delineated.
Here we have presented data suggesting that P2Y2Rs, Vß3/ß5 integrins, and CD47 interact within the same cell. Is it also possible for these receptors to interact intercellularly and affect cellcell adhesion? Several studies have indicated that extracellular ATP and UTP can stimulate cellcell adhesion, although the molecular/biochemical basis for this interaction has not been identified. For example, ATP and UTP have been shown to stimulate cell adhesion in a monocyte/macrophage lineage (
Vß3/ß5 integrins was not explored. The
Vß3 integrin on monocytes mediates their adherence to endothelial and epithelial cells (
Vß3 and CD47 play a role in transendothelial monocyte and neutrophil migration (
Vß3 or CD47 has not been assessed. Studies by our group have found that activated polymorphonuclear cells bind more readily to K562 cells transfected with human P2Y2R cDNA than to untransfected or vector-transfected K562 cells (H. Gresham, unpublished observation). Therefore, cellcell interactions between P2Y2Rs,
Vß3/ß5 integrins, and CD47 are likely to have physiological consequences and should be further investigated. Since peptides modeled after the first extracellular loop of the P2Y2R bind selectively to
Vß3/ß5 integrins and CD47 (Fig 1 B), the potential for P2Y2R-derived peptides to inhibit intercellular interactions between these receptors also should be investigated. Such inhibitors of
Vß3 integrin, CD47, and P2Y2R interactions could prove useful as pharmacotherapeutic agents in the treatment of atherosclerosis, diabetes, cancer and inflammation.
![]() |
Footnotes |
---|
1 Abbreviations used in this paper: AI, adhesion index; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; ERK1/2, extracellular regulated kinase; FAK, focal adhesion kinase; GPCR, G proteincoupled receptor; HSA, human serum albumin; IP, inositol phosphate; HA, hemagglutinin; MAP, mitogen-activated protein; pAb, polyclonal Ab; P2YR, P2Y nucleotide receptor; RGD, arginine-glycine-aspartic acid.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Simon Robson (Harvard University, Cambridge, MA) for helpful discussions and advice.
This work was supported by grants from the American Diabetes Association, American Heart Association, Cancer Research Center of Missouri, Monsanto, National Institutes of Health, North American Cystic Fibrosis Foundation, and the University of Missouri-Columbia F21C Program.
Submitted: 31 October 2000
Revised: 21 March 2001
Accepted: 26 March 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|