Article |
Address correspondence to Eric J. Brown, Program in HostPathogen Interactions, University of California, San Francisco, Campus Box 2140, 600 16th St., San Francisco, CA 94143-2140. Tel.: (415) 514-0167. Fax: (415) 514-0169. email: ebrown{at}medicine.ucsf.edu
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Abstract |
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Key Words: cell migration; CD47; chemokines; migration; signal transduction
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Introduction |
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To migrate in response to a chemotactic signal, cells need to modulate their adhesive properties in a regulated manner. This involves integrin activation, which can in turn be modulated by association with membrane partners such as tetraspannins, growth factor receptors, or CD47 (Brown, 2002). CD47 is a ubiquitous integral membrane glycoprotein, which is physically and functionally associated with integrins vß3,
2ß1,
IIbß3, and
4ß1. CD47 ligation has been shown to activate PTX-sensitive G proteins, suggesting a mechanism through which CD47 might regulate migration (Brown and Frazier, 2001).
PLIC-1 and PLIC-2 are two closely related proteins originally identified through their interaction with the cytoplasmic tail of CD47. Sequence analysis reveals ubiquitin-like (Ubq) domains in the amino termini of both proteins, and a ubiquitin-associated (Uba) domain in each carboxy terminus. The region between the Ubq and the Uba domains contains several Sti1 motifs (Kaye et al., 2000) of unknown function. It is this internal region that contains CD47 binding sites. Despite high homology between the two proteins, PLIC-1 binds more tightly to CD47 than PLIC-2, perhaps because it has two internal repeats that interact with CD47 (Wu et al., 1999).
A connection between PLICs and both actin cytoskeleton and intermediate filaments suggested that PLICs may participate in CD47 regulation of adhesion and migration (Wu et al., 1999). Here, we have investigated the role of the PLICs in cell migration and have found that PLIC-1, but not PLIC-2, inhibits cell migration. Surprisingly, this regulation occurs through effects on Gi signaling rather than directly on integrin function. Thus, PLIC-1 is involved in communication between integrins and GPCRs and likely is a molecular component of the mechanism through which CD47 regulates cell motility and Gi signal transduction.
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Results |
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PLIC-1 blocks SDF-1induced PLCß activation
To test whether PLIC-1 directly affected Gi signaling, we assessed the ability of PLIC-1 to alter [Ca2+]i responses. As shown in Fig. 2 A, whereas SDF-1 induced an increase in [Ca2+]i in both JC and JPLIC-2, JPLIC-1 was unable to mount any increase in [Ca2+]i in response to this agonist. This difference between the effects of PLIC-1 and PLIC-2 on SDF-1
induced calcium also was reproduced in independently derived clones. PTX completely blocked SDF-1
induced [Ca2+]i in JC, confirming its dependence on Gi signaling (unpublished data). In contrast, PLIC-1 had no significant effect on the [Ca2+]i increase induced by cross-linking the T cell antigen receptor (Fig. 2 C), which is dependent on tyrosine kinase rather than heterotrimeric G protein signaling (Mustelin and Tasken, 2003).
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To determine whether PLCß-mediated increase in [Ca2+]i was required for migration of Jurkat or A431, we examined migration in cells treated with the intracellular Ca2+ chelator BAPTA or with the PLC inhibitor U73122. Both BAPTA and U73122 prevented chemotaxis, suggesting a role for PLC in migration of these cells (Fig. 2 E). Thus, PLIC-1 inhibition of SDF-1induced PLCß activation likely contributes to its inhibition of migration.
PLIC-1 inhibits Gi- and Gq-, but not Gs-coupled signaling
To determine whether PLIC-1 affected migration mediated through Gi-coupled receptors other than CXCR4, JPLIC-1, JPLIC-2, and JC were transiently transfected with the Gi-coupled receptor activated solely by a synthetic ligand (RASSL) Ro2, which is similar to -opioid receptors but binds spiradoline rather than an opioid ligand (Coward et al., 1998). JC and JPLIC-2 transfected with this receptor migrated in response to spiradoline, but JPLIC-1 cells did not (Fig. 3 A), despite equal expression of the transfected RASSL (not depicted). Thus, PLIC-1 inhibits migration through two different Gi-coupled receptors in Jurkat cells.
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To determine if other Gß functions were inhibited by PLIC-1, we tested CXCR4 endocytosis after addition of SDF-1
, because release and activation of Gß
from the heterotrimeric G protein is required for GRK-mediated internalization of GPCRs (Penn et al., 2000). As shown in Fig. 4, endocytosis of CXCR4 after SDF-1
addition was decreased in JPLIC-1 compared with JC or JPLIC-2.
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To test the possibility that PLIC-1 could affect Gß functions by interfering with proteasome activity, we examined the effects of the proteasome inhibitor lactacystin on [Ca2+]i increase in response to SDF-1
. Lactacystin, which induced a significant accumulation of ubiquitinated proteins in the cells (inset), did not significantly affect SDF-induced calcium changes (Fig. 7). Therefore, we conclude that PLIC-1's inhibition of Gß
signaling does not require any effect it may have on proteasome activity.
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Discussion |
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We considered several alternative possibilities for the mechanism by which PLIC-1 affected CXCR4 signaling and cell migration. First, we considered that it might affect Ca2+ release from cytoplasmic stores in general. However, release of Ca2+ by ligation of CD3 was normal, demonstrating that activation of PLC and subsequent release of Ca2+ stores was unaffected by PLIC-1. We considered that as a protein potentially involved in proteasome function, PLIC-1 might affect the concentration of one or more of the proteins involved in signaling to PLCß; however, CXCR4 itself, Gß
, and PLCß2 expression all were unaffected by PLIC-1. Furthermore, SDF-1
binding to CXCR4 was unaffected by PLIC-1 expression. Finally, we considered that PLIC-1 inhibition of proteasome activity might lead to the signaling aberrations. This is unlikely for several reasons: first, lactacystin, which clearly inhibited proteasome function, had minimal effect on CXCR4-mediated cytoplasmic Ca2+ rise; second, to the extent that it has been studied, PLIC-2 is equivalent to PLIC-1 for proteasome inhibition (Kleijnen et al., 2000); and, finally, we saw no detectable accumulation of ubiquitinated proteins in the PLIC-1 transfectants. Furthermore, although ubiquitination of CXCR4 leads to its degradation, endocytosis of this receptor is independent of its ubiquitination (Marchese and Benovic, 2001), suggesting the effect of PLIC-1 on its internalization is not ubiquitin dependent. Thus, the data are most consistent with PLIC-1mediated inhibition of Gß
function independent of its ability to interfere with proteasome activity, and likely through its binding to Gß
. PLIC-1 has functional similarities with the Pd family of G protein signaling regulators (Schulz, 2001). Pd and its homologue PhLP both bind Gß
directly with high affinity and inhibit Gß
-dependent functions by sequestration. Pd, by binding to Gß
, inhibits ß2-adrenergic receptor kinase translocation to the plasma membrane and internalization of the receptor; PhLP blocks internalization of the
-opioid receptor (Schulz, 2001). Although Pd is expressed exclusively in the retina, PhLP has a broad distribution, and we have found it in Jurkat cells (unpublished data).
Inhibition of Gß function is sufficient to account for the ability of PLIC-1 to block cell migration, without postulating additional effects on integrin or cytoskeletal function. Previous papers by the Bourne and Charo groups have established that Gi-mediated chemotaxis absolutely requires release of Gß
from G
i, with subsequent activation of Gß
effectors (Arai et al., 1997; Neptune and Bourne, 1997). Although deletion of the PLCß2 gene in mice caused primary PMN and lymphocytes to migrate faster (Jiang et al., 1997), in our system, both Ca2+ clamping and a PLC inhibitor dramatically decreased cell migration, to about the same extent as PTX. Although reasons for the difference between our results and the knockout are unknown, the fact that there is PTX-sensitive migration in PLCß2-/- cells suggests that there may be other Gß
-dependent effector mechanisms important in migration that also are affected by PLIC-1.
These data are the first to demonstrate a significant biological difference between members of the PLIC family. We suggest that the difference in function results, at least in part, from the difference in subcellular localization of PLIC-1 and PLIC-2. Based on what is known of function so far, it appears that PLIC-1 is most closely associated with plasma membrane, PLIC-2 with cytosolic proteasomes, and A1u (called Ubin in the mouse) is likely predominantly expressed in the nucleus. Its ability to associate with the plasma membrane likely is necessary for PLIC-1 to prolong the half-life of GABA receptors and presenilins. It is intriguing that the three membrane proteins (including CD47) with which PLIC-1 has been associated all span the membrane multiple times. It may be that this architecture is important for PLIC-1 interaction.
Finally, it is tantalizing that CD47 is a plasma membrane binding site for PLIC-1 because CD47 ligation has been associated with a nonclassical mechanism for Gi activation (Brown and Frazier, 2001). It may be that CD47 ligation results in loss of association with PLIC-1, releasing an inhibition of Gi signaling, or it may be that activation of Gi signaling through another mechanism recruits PLIC-1 to CD47 at the plasma membrane to restore homeostasis. In either case, the interaction of PLIC-1 with CD47 may represent a novel mechanism for regulation of G protein signaling, and understanding the mechanisms involved in regulating the interaction of these two proteins is likely to reveal additional potential pathways for control of this major plasma membrane signaling pathway.
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Materials and methods |
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cDNA constructs
cDNA encoding myc or GST-tagged PLIC1 or PLIC-2 were described previously (Wu et al., 1999). GSTsyntaxin 2 cDNA was a gift from K. Mostov (University of California San Francisco, San Francisco, CA [UCSF]). HA-tagged M3 muscarinic receptor and Flag-tagged ß2 adrenergic receptor cDNAs were provided by H. Bourne (UCSF; Neptune and Bourne, 1997). The cDNA encoding the RASSL Ro2 (Coward et al., 1998) was a gift of B. Conklin (UCSF). Pd-like protein construct was provided by B. Willardson (Brigham Young University, Provo, UT).
To generate deletion mutants of PLIC-1 in fusion with GST, PCR products encompassing the coding regions of PLIC-1 (1538), PLIC-1 (534582), and PLIC-1 (100533) were cloned in frame with the coding region of GST in pGEX-KG vector. The different domains were generated by PCR using the following primers: PLIC-1 (534582): 5'-GCGAATTCCGCAGAGTCCAGAAGTCAGATT-3' and 5'-TGCACTCGAGCTATGACGGCTGGGAACCCAGC-3'; PLIC-1 (1538): 5'-TGACGGAATTCTTGCCATGGCCGAGAGCGCAGAGAGCG-3' and 5'-TCGGCCCTCGAGCTATC-AGACTTCTGGACTCTGCAGCTGAGGGTT-3'; PLIC-1 (100533): 5'-CAGGCGGAATTCGACCGCAAGATAATTCAGCTCAGCAAACA-3' and 5'-TCGGCCCTCGAGCTATCAGACTTCTGGACTCTGCAGCTGAGGGTT-3'. The PCR products were digested with EcoRI/XhoI and cloned into pGEX-KG, and subsequently sequenced to verify that no errors had been introduced during PCR or cloning.
mAbs and reagents
SDF-1 was from PeproTech. Purified Gß
, PTX, U-73122, BAPTA-AM, lactacystin, GTP
-S, isoproterenol, and carbachol were purchased from Calbiochem. Spiradoline was purchased from Sigma-Aldrich. Fura-2AM was purchased from Molecular Probes. The anti-CD3 mAb (OKT3) was purchased from American Type Culture Collection. Gß antibodies were purchased from Upstate Biotechnology or BD Transduction Laboratories. Anti-myc antibodies were purchased from Upstate Biotechnology or Invitrogen. Lck, PLCß2, and G
i antibodies were purchased from Santa-Cruz Biotechnology, Inc. IP3 and cAMP assay kits were purchased from Amersham Biosciences and BIOMOL Research Laboratories, Inc., respectively.
Cell migration
Chemotaxis of Jurkat T cells in response to SDF-1 was determined using a 24-well plate with 3-µm-pore inserts (BD Biosciences). After filling the lower chamber with medium alone or medium containing 500 ng/ml SDF-1
, 4 x 105 Jurkat T cells (2 x 106/ml in RPMI 1640, 1% FCS) were loaded in the upper chamber. Plates were incubated for 3 h at 37°C in a humidified atmosphere containing 5% CO2, and cell migration assessed by counting cells in the lower chamber on a hemocytometer. Each experiment was performed in triplicate. The same procedure was used to assess migration of Ro2-transfected Jurkat cells in response to 1 µM spiradoline.
To assess wound healing, A431 cells transfected with empty vector, PLIC-1, or PLIC-2 were grown to confluency. The monolayers were wounded with a pipette tip, washed, and the distance between wound edges measured using a micrometer at specifically marked points along the wound. After 5 h at 37°C in a humidified atmosphere containing 5% CO2, the distance between wound edges was measured again at the same sites, and the distance covered by migrated cells determined. At least three different points were used to determine the average distance migrated along the wound edge. In some experiments, confluent monolayers were pretreated overnight with 100 ng/ml PTX, or for 30 min with 4 µM U73122 or 25 µM BAPTA before wounding.
Intracellular calcium measurement
Jurkat T cells (2 x 107 cells/ml) in growth medium were loaded with Fura-2 as described previously (Rebres et al., 2001a,b). Cells were washed once with complete medium and twice with ice-cold Ca2+ buffer (25 mM Hepes, pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, and 0.1% glucose), resuspended at 3 x 106 cells/ml in Ca2+ buffer, and kept on ice until use. Before stimulation, cells were transferred into cuvettes, prewarmed to 37°C, and placed in a spectrofluorimeter (model F-4500; Hitachi Instruments) after which SDF-1 was added to the stirred cell suspension to a final concentration of 2 µg/ml. To assess the Ca2+ response to TCR cross-linking, cells were incubated with OKT3 (American Type Culture Collection) for 30 min on ice, washed twice, and 10 µg/ml antimouse IgG was added to the cuvette to cross-link bound antibody in place of SDF-1
. In Jurkat cells transfected with the M3 muscarinic receptor, changes in [Ca2+]i were measured after stimulation with 100 µM carbachol. Fluorescence was monitored as described previously (Green et al., 1997) and [Ca2+]i was calculated by the method of Grynkiewicz et al. (1985).
Measurement of intracellular IP3 level
Intracellular IP3 concentration was determined using a radioreceptor assay (Amersham Biosciences). Jurkat T cells were stimulated with 2 µg/ml SDF-1 for various times, and the reaction ended by addition of 0.2 vol ice-cold 20% perchloric acid. The samples were handled, IP3 was measured, and IP3 concentration was calculated exactly as per the manufacturer's instructions.
Measurement of CXCR4 endocytosis
Vector or PLIC-1 transfected Jurkat cells were stimulated with 500 ng/ml SDF-1 for 0 or 5 min. At end point, cells were chilled, washed with cold PBS, and incubated with 10 µg/ml a CXCR4 antibody (Prosciences) for 30 min at 4°C, and subsequently with Alexa 488coupled antimouse IgG (Molecular Probes). Cells were fixed and fluorescence of individual cells was measured by flow cytometry (Coulter Epics).
Immunostaining of PLICs and Gß
Murine 3656 fibroblasts plated onto coverslips were transfected with myc-tagged PLIC-1 or PLIC-2 and maintained in complete medium for 24 h. Cells were fixed with 3.7% PFA, briefly permeabilized with 0.1% Triton X-100, and stained with Alexa 488labeled anti-myc (clone 9E10; Upstate Biotechnology) and Alexa 594labeled anti-Gß (BD Transduction Laboratories).
Image acquisition
Images shown in Fig. 5 were acquired using a microscope (Axiovert 100TV; Carl Zeiss MicroImaging, Inc.), with a Plan-APOCHROMAT 63X inverted oil objective lens with an NA of 1.40. Cells were fixed and stained with specific antibodies coupled to Alexa 488 or Alexa 594 as described in Immunostaining of PLICs and Gß, and mounted in Prolong medium (Molecular Probes). Images were acquired with a CCD camera (Micromax; Princeton Instruments) using IPLabs software and subsequently merged using Adobe Photoshop.
GST pull down
GSTPLIC-1 and controls were induced by addition of 0.1 mM IPTG (16 h/RT) to bacterial cultures. Lysates were incubated with GSH-agarose beads (Amersham Biosciences) overnight at 4°C. The beads were washed with a buffer containing 20 mM Hepes, pH 7.4, 25 mM NaCl, 0.5% Triton X-100, 10% glycerol, 0.1% ßME, and extensively washed with 20 mM Hepes, pH 7, 150 mM KCl. Cell membranes were prepared by centrifugation of sonicated cells at 100,000 g for 1 h and solubilized in 0.1% Triton X-100 for 2 h at 4°C under constant rotation. Solubilized proteins were separated from insoluble pellet by centrifugation at 15,000 g, and incubated with the GST proteinloaded agarose beads overnight at 4°C. After washing, bead-bound protein was eluted in Laemmli buffer and analyzed by SDS-PAGE and Western blot. To assess potential Gi binding, the membrane lysate was incubated with 100 µM GTP
-S for 15 min at 30°C before incubation with fusion proteins.
To test direct binding of Gß, 12 ng of purified Gß
(Calbiochem) was incubated with the same amount of GST fusion proteins in PBS/0.1% Triton X-100 overnight at 4°C. Bead pellets were washed three times, solubilized in Laemmli buffer and analyzed by SDS-PAGE and Western blot.
Statistical analysis
Data were analyzed by t test.
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Acknowledgments |
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This work was supported by grants GM38330 and AI24674 from the National Institutes of Health to E.J. Brown.
Submitted: 25 July 2003
Accepted: 10 October 2003
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