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RESEARCH COMMUNICATION
1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines
1 Department of Medicine 2 Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
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Abstract |
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integrin cytoplasmic tail to phosphotyrosine-binding (PTB) domain-containing proteins, an interaction proposed to be dynamically regulated by tyrosine phosphorylation. Here we show that replacement of both 1 integrin cytoplasmic tyrosines with alanines, resulting in the loss of all PTB domain interaction, causes complete loss of 1 integrin function in vivo. In contrast, replacement of 1 integrin cytoplasmic tyrosines with phenylalanines, a mutation that prevents tyrosine phosphorylation, conserves in vivo integrin function. These results have important implications for the molecular mechanism and regulation of integrin function.
[Keywords: Integrin; tyrosine phosphorylation; phospho-tyrosine-binding domain; inside-out signaling; outside-in signaling; conditional knock-in]
Received January 9, 2006; revised version accepted February 17, 2006.
). Since the cytoplasmic tails of integrin receptors are relatively short and lack intrinsic enzymatic activity, integrin signals are believed to be mediated by protein–protein interactions. Biochemical studies have identified a large number of intracellular cytoskeletal and signaling proteins that are capable of binding integrin cytoplasmic tails, but a clear picture of how these many potential protein–protein interactions result in bidirectional integrin signals has not yet emerged.
A highly recognizable motif in the integrin cytoplasmic tail is NPxY, two of which are found in the cytoplasmic tails of integrin subunits. Biochemical and structural studies have demonstrated that NPxY motifs bind phosphotyrosine-binding (PTB) domains and that the specificity of NPxY–PTB domain interactions is conferred by both the NPxY motif and the PTB domain (for review, see Uhlik et al. 2005). Some PTB domains (e.g., the Shc [Src homology 2 domain-containing] transforming protein 1 PTB domain) require phosphotyrosines for high-affinity interaction with NPxY motifs, while others (e.g., the talin PTB domain) bind through hydrophobic interaction with nonphosphorylated tyrosine or phenylalanine residues. The specificity and diversity of NPxY–PTB domain interactions have recently been proposed as a molecular mechanism by which integrin cytoplasmic tails transmit a variety of bidirectional signals (Calderwood et al. 2003). Biochemical studies performed in vitro suggest that distinct integrin cytoplasmic tails bind distinct PTB domain-containing proteins, and that tyrosine phosphorylation can alter these interactions through addition of a charged phosphate group to the hydrophobic tyrosine aromatic ring (Calderwood et al. 2003; Garcia-Alvarez et al. 2003). Dynamic regulation of NPxY–PTB domain binding by tyrosine phosphorylation is therefore a potentially elegant means of explaining how short integrin cytoplasmic tails transmit diverse bidirectional signals.
Despite the wealth of biochemical and structural data available, the in vivo role of integrin NPxY motifs is not yet defined. 3 integrins in platelets are critical for hemostasis, and substitution of the two 3 NPxY motifs with NPxF to block phosphorylation-dependent integrin signaling results in a hemostatic defect (Law et al. 1999). Whether this phenotype reflects a broad role for integrin tyrosine phosphorylation or a more restricted role for 3 integrins in platelets is unknown. Furthermore, a Y-F mutation is conservative and predicted to not disrupt many NPxY–PTB domain interactions. Thus the absolute requirement for integrin cytoplasmic tyrosines in vivo is untested. In the present study we have used in vivo mutagenesis and conditional in vivo mutagenesis to address the role of NPxY motifs in 1 integrins. Since 1 integrins are the most utilized integrin subunits in mammals and are highly evolutionarily conserved, mediating adhesion to similar matrix proteins in organisms as diverse as nematodes, flies, and mammals, they provide a means of stringently testing the role of cytoplasmic tyrosines for integrin function in vivo.
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Results and Discussion |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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integrin subunit cytoplasmic tyrosines in vivo mice were generated in which the cytoplasmic tyrosines (Y783 and Y795) encoded by the Itgb1 gene were replaced with either alanines or phenylalanines (Fig. 1A). The phenylalanine (YF) mutation blocks interactions requiring tyrosine phosphorylation but retains those requiring the hydrophobic aromatic ring of the amino acid, while the alanine (YA) mutation ablates both. Itgb1YA/+ and Itgb1YF/+ mice were healthy and fertile (data not shown) and were intercrossed to generate 1YA (Itgb1YA/YA) and 1YF (Itgb1YF/YF) animals. Analysis of the progeny of heterozygous matings revealed expected numbers of Itgb1YA/+ mice but failed to reveal live-born 1YA animals (Supplementary Table 1), indicating that 1YA animals die during embryonic life. Timed Itgb1YA/+ matings identified resorptions that could be genotyped as Itgb1YA/YA animals but no live 1YA embryos as early as embryonic day 6.5 (E6.5) (Fig. 1B,C; Supplementary Table 1). Histologic examination of uteri collected at E6.5 revealed that presumptive 1YA embryos were resorbed with only a residual population of trophoblast cells visible, a phenotype similar to that reported for 1 integrin-deficient embryos (Fig. 1C; Fassler and Meyer 1995; Stephens et al. 1995). Mouse embryos lacking 1 integrins form normal blastocysts but fail to develop an inner cell mass, a critical early step in mammalian embryogenesis (Fassler and Meyer 1995; Stephens et al. 1995). Direct histologic comparison with E6.5 embryos from Itgb1+/– intercrosses revealed identical findings in presumptive Itgb1–/– embryos (Fig. 1C), demonstrating that 1 cytoplasmic tyrosines are required for integrin function in early mouse embryonic development.
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1YA animals, Itgb1YF/+ intercrosses generated normal numbers of live-born 1YF animals that were healthy and fertile (Supplementary Table 1; data not shown). Consistent with these genetic findings, 1 integrin tyrosine phosphorylation could not be detected in phosphotyrosine immunoblots of 1 integrin immunoprecipitated from wild-type E12.5 or E16.5 embryos (data not shown). Thus the requisite role for the 1 integrin subunit in the development and function of tissues as varied as the skin, blood, neural crest, and cartilage is independent of 1 integrin cytoplasmic tyrosine phosphorylation.
Essential roles for 1 integrins in the development and function of specialized tissues that develop after the blastocyst stage of embryogenesis have been demonstrated through analysis of chimeric animals generated by injecting 1-deficient embryonic stem (ES) cells into wild-type blastocysts. These studies utilized lineage tracing to demonstrate that 1 integrins are cell autonomously required to form liver and blood cells but not skin or skeletal muscle cells (Fassler and Meyer 1995; Hirsch et al. 1996). To determine if the requirement for 1 cytoplasmic tyrosines in vivo was restricted to early embryogenesis or if these residues were more broadly required for 1 integrin function during development we injected 1YA (Itgb1YA/YA) and 1YF (Itgb1YF/YF) ES cells that were tagged with GFP using a lentivirus that is ubiquitously expressed in vivo into wild-type blastocyts (Fig. 2A,B). Chimeric animals with high levels of ES cell contribution to the dermis of the skin (>90% based on analysis of agouti coat color, which is derived exclusively from the SV/129 ES cells following injection into C57Bl/6 blastocysts) were obtained using both 1YA ES cells and control 1YF ES cells (Fig. 2C; data not shown). 1YA and 1YF contribution to hematopoietic cells was determined using flow cytometry to detect GFP+ bone marrow cells and contribution to other tissues measured by immunodetection of GFP protein in tissue lysates. As expected, 1YF cells contributed robustly to all tissues, but 1YA cells failed to contribute to hematopoietic cells in the bone marrow or spleen (Fig. 2D,E) or to liver despite contribution to skeletal muscle (where fusion with wild-type cells can rescue mutant cell function), brain, and dermis (Fig. 2C,E; Supplementary Table 2). Thus 1YA embryos and 1YA chimeras phenocopy those lacking 1 entirely, indicating that 1 cytoplasmic tyrosines are universally required for integrin function during development.
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1 integrin function in 1YA embryos and 1YA chimeras could result from the disruption of interactions with intracellular proteins required for integrin function or could reflect loss of stable surface expression of the mutant receptor. The latter possibility is suggested by biochemical studies of integrin cytoplasmic tails demonstrating that the Y-A mutation disrupts interaction with the cytoskeletal protein talin (Calderwood et al. 2002) and the observation that talin-deficient ES cells lose surface expression of 1 integrins (Priddle et al. 1998). To address this question, mutant 1 integrin expression and function were next studied in homozygous 1YA and 1YF ES cells. 1YA ES cells expressed levels of total and cell surface 1 integrin that were indistinguishable from those of 1YF and wild-type ES cells (Fig. 3A,B). 1YA ES cells appeared more loosely adherent than wild-type ES cells when grown on gelatin or embryonic fibroblasts (Fig. 3C; data not shown), however, and were unable to bind laminin, a 1-specific ligand, or fibronectin, a ligand for 1 and 3 integrins, when 3 integrin function was blocked by RGD peptide (Fig. 3D). Finally, in contrast to wild-type or 1YF ES cells, 1 integrins on the surface of 1YA ES cells were not recognized by the activation-specific anti-1 antibody 9EG7 (Fig. 3B). Thus 1YA integrins are expressed normally on the cell surface, but are in an inactive conformation and unable to bind matrix protein ligands.
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IIb3, a fibrinogen receptor, is maintained in a strictly inactive conformation until activated by inside-out signals generated by other receptors on the platelet surface (O’Toole et al. 1991). Following inside-out activation and fibrinogen binding, the cytoplasmic tail of the 3 subunit is tyrosine phosphorylated, an event that initiates a secondary wave of integrin signaling referred to as outside-in. In vivo replacement of the 3 cytoplasmic tyrosines with phenylalanine does not interrupt inside-out activation of platelet integrins, but results in defective hemostasis due to the loss of outside-in integrin signals (Law et al. 1999). Mouse and human platelets express the 21 integrin, which functions as a receptor for vessel wall collagen. The 21 integrin is essential for the adhesion of flowing platelets to exposed collagen under arterial levels of hemodynamic shear (Sarratt et al. 2005) and is believed to require inside-out activation similar to that described for IIb3 (Jung and Moroi 2000). To address the role of 1 cytoplasmic tyrosines in the context of defined inside-out and outside-in integrin signaling, we therefore studied platelet collagen responses mediated by 21.
1YA platelets were generated using conditional 1YA knock-in (1cYA) animals that grew to adulthood without phenotypic abnormalities (Fig. 4; data not shown). Cre-mediated excision of the endogenous Itgb1 terminal exon in the 1cYA allele resulted in splicing to the mutant terminal exon and expression of the 1YA mRNA (Fig. 4B). Cre expression was induced in the hematopoietic cells of 1cYA animals carrying the MX1-Cre transgene. Cre-induced 1cYA animals had circulating platelets with normal surface levels of 1 integrin (Fig. 4C). Following ADP stimulation, 1 integrins on the surface of wild-type but not 1YA platelets became 9EG7 positive (Fig. 4D), however, consistent with an inability to activate the 1YA integrin. Inside-out activation of 21 was directly measured using the 21 ligand soluble collagen. In contrast to wild-type and 1YF platelets, 1YA platelets failed to bind soluble collagen after ADP stimulation, although binding of the IIb3 ligand fibrinogen was preserved in 1YA platelets (Fig. 4E; Supplementary Fig. 1). Thus cytoplasmic tyrosines are required for inside-out activation of 1 integrins in platelets.
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21, which mediates firm attachment of the growing thrombus to exposed collagen, an interaction that is likely to require stabilizing outside-in signals analogous to those required for IIb3 to stabilize platelet–fibrinogen binding within the thrombus (Law et al. 1999). To further test the role of 1 integrin tyrosines, we next tested the ability of 1YA and 1YF platelets to mediate collagen adhesion under flow. Consistent with a complete loss of integrin function, 1YA platelets exhibited severe defects in collagen adhesion under flow similar to those of platelets lacking 21 entirely (Fig. 4F; Supplementary Fig. 1). Despite normal inside-out responses, however, 1YF platelets also exhibited mildly reduced adhesion to collagen under flow (Fig. 4E), a result most consistent with an outside-in signaling defect.
These studies provide an in vivo test of a model of integrin function that has arisen from extensive biochemical and in vitro studies. In the present model integrin and chain interaction is believed to hold the receptor in an inactive conformation that requires allosteric release by intracellular molecular events (Takagi et al. 2002; Vinogradova et al. 2002). Protein binding to conserved cytoplasmic tyrosine (NPxY) motifs in the chain has been proposed as a key step and point of regulation in this process (Calderwood et al. 2003; Tadokoro et al. 2003). Our studies reveal that cytoplasmic tyrosines play an essential role in the regulation of 1 integrin function in virtually all cells, regardless of whether they are known to tightly regulate integrin adhesion or not. What is the critical function of these tyrosines? Genetic studies in lower organisms suggest that one critical function may be to mediate interaction with the cytoskeletal protein talin, as the FERM domain of talin requires the aromatic ring of tyrosine or phenylalanine to bind integrin cytoplasmic tails (Garcia-Alvarez et al. 2003), and loss of talin in both flies and nematodes confers phenotypes that closely mimic integrin subunit loss (Brown et al. 2002; Cram et al. 2003). Given the large number of other proteins capable of binding these motifs, however, more specific loss-of-function mutations will be required to determine which play critical roles for integrin function in vivo.
In light of the complete loss of 1 integrin function observed in 1YA animals and cells and the many essential roles played by 1 integrins in vivo, the normal phenotype of 1YF mice is a particularly striking and unexpected finding. Although less utilized integrin subunits such as 2 contain NPxF motifs, 1 integrin tyrosine residues are evolutionarily conserved from nematodes to man and 1 tyrosine phosphorylation has been demonstrated to drive cell migration and transformation in vitro (Sakai et al. 2001). The ability of cells to regulate integrin adhesion and the large number of PTB domain-containing proteins that can potentially interact with integrins have led to the hypothesis that tyrosine phosphorylation might provide a mechanism by which cells dynamically regulate integrin function. Binding of the cytoskeletal protein talin to integrin NPxY motifs is thought to be necessary for inside-out activation of integrins (Tadokoro et al. 2003) and is predicted to be disrupted by tyrosine phosphorylation (Garcia-Alvarez et al. 2003). As a result, tyrosine phosphorylation has been proposed as a molecular switch by which the integrin–talin interaction, and thereby integrin adhesion, may be regulated (Calderwood et al. 2003). The viability of 1YF animals demonstrates that tyrosine phosphorylation is not an essential mechanism by which integrin function is regulated in vivo. Whether phosphorylation of 1 tyrosine residues plays a critical role in more specialized functions that require the transmission of outside-in signals from 1 integrins will require further investigation. This finding supports a model of integrin function in which NPxY–PTB domain interactions are critical for integrin function but are either not dynamically regulated or are regulated through other mechanisms; e.g., through structural changes driven by events at neighboring regions of the integrin cytoplasmic domain, or more indirectly through modulation of critical intermediary proteins such as talin itself. Further studies to determine how integrin interaction with intracellular proteins is dynamically regulated will provide fresh insight into the biological roles of these receptors as well as new therapeutic possibilities.
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Materials and methods |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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CMVcre, Mx1cre, FLPe transgenic mice, and C57BL6 were purchased from the Jackson Laboratory. 2 knockout mice were generously provided by Drs. Sam Santoro and Mary Zutter (Vanderbilt University, Nashville, TN). Conditional 1 mice were obtained from the Jackson Laboratory and generously provided by Dr. Cord Brakebusch (Max Planck Institute, Martinsreid, Germany). 1YA, 1YF, and conditional 1YA knock-in ES cells and mice were generated using standard protocols. Targeting constructs contain a 4.4-kb 5' arm, a 4-kb 3' arm, and a 1.5-kb target fragment covering exon 15 of the 1 gene, where sequences encoding Tyr 783 and Tyr 795 were mutated to alanines or phenylalanines. Homozygous targeted ES cells were generated by selection in 1.0 mg/mL G418.
ES cell adhesion assays
ES cells were trypsinized and cultured on gelatin plates for 1 h for removal of mouse embryonic fibroblast (MEF) cells. ES cells were then treated with 0.25 mg/mL GRGDS peptide or control peptide GRGES (American Peptide Co.) or nothing for 30 min. ES cells were plated onto 96-well plates coated with 0.5 µg/well of fibronectin, laminin, or collagen at 1 x 106 cells per well. After 1 h incubation at 37°C, the plates were washed three times in PBS. Adherent cells were fixed in 95% ethanol, stained in 0.1% crystal violet, washed in water, and lysed in 0.2% Triton X-100 for 10 min for each step, and OD 595 was recorded.
Chimeric mouse studies
GFP-expressing double knock-in YA, YF, or wild-type ES cells were analyzed by flow cytometry. Bone marrow cells of 1YA-GFP and 1YF-GFP chimeric mice were stained with 1 µg/mL PE-Cy5 anti-CD45 antibody and then analyzed by flow cytometry. GFP signals in chimeric tissues were detected by Western blot following standard protocols.
Detection of 1YA mRNA in conditional Y-A mice
RT–PCR was performed using the PCR primers 5'-GCAAGGAGAAG GACATTGATGA-3' and 5'-CAAGTCTTCTGTCAGTCCCTG-3', encompassing exon 13 to exon 15 (mutation-targeted exon) were used to generate a 521-base-pair (bp) product. The MwoI enzyme was added directly to the PCR reactions and the products analyzed by gel electrophoresis.
Induction of 1YA integrin expression in platelets in conditional YA mice
Cre was induced by six intraperitoneal injections of 0.25 mg polyinocinic acid–polycytidylic acid (Fluka #81354) at 2-d intervals. Treated mice were used in experiments at least 4 wk after induction.
Platelet integrin 1 expression and integrin inside-out activation assays
Platelet-rich plasma was stimulated with 0 or 50 µM ADP and staining reagents (50 µg/mL FITC-collagen or 100 µg/mL Alexa Fluor 488-fibrinogen or 3 µg/mL Alexa Fluor 647-9EG7 antibody or 3 µg/mL Alexa Fluor 647-HM1-1) added. The platelets were fixed with 4% paraformaldehyde for 5 min and analyzed by flow cytometry.
Platelet flow assays
Whole anticoagulated blood was perfused over a collagen-coated glass slide in a tapered plate chamber, and platelet adhesion was quantified using ImagePro software as previously described (Sarratt et al. 2005).
Statistics
Means and standard deviations or standard error of the mean are shown with the number of samples for each group indicated as N. P values shown were calculated using the two-tailed Student’s t-test.
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Acknowledgments |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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1fl/fl mice; and Sam Santoro and Mary Zutter for Itg2–/– mice. |
Footnotes |
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E-MAIL markkahn{at}mail.med.upenn.edu; FAX (215) 573-2094. 
Supplemental material is available at http://www.genesdev.org.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1408306
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