in addition to soluble mitogenic stimuli , a number of leukocyte cell surface molecules have been shown to regulate integrin - mediated leukocyte adhesion . one particular receptor , cd31 , a platelet endothelial cell adhesion molecule ( pecam-1 ) ( newman 1997 ) , stimulates integrin - dependent adhesion in a wide variety of vascular cells , including leukocytes and platelets , via a process termed adhesion amplification ( tanaka et al . 1992 ; berman and muller 1995 ; berman et al . 1996 ; ranger et al . numerous studies have also demonstrated a critical role for cd31 in mediating integrin - dependent leukocyte chemotaxis and extravasation in vitro and in vivo ( berman et al . the molecular basis for this has remained obscure , but recent studies have demonstrated that , in addition to mediating homophilic cell adhesion ( newton et al . 1997 ) , cd31 acts as an adhesion - dependent signaling receptor ( newman 1997 ; newton et al . 1997 cd31 ligation stimulates tyrosine phosphorylation of cd31 , leading to its association with a number of cytoplasmic signaling proteins , including shp-1 , shp-2 , ship , and plc-1 , suggesting that cd31 functions as a dynamic modulator of vascular cell behavior ( newman 1997 ; newton et al . several reports suggest a role for ras family gtpases in the control of integrin - mediated adhesion . ras superfamily gtpases cycle between inactive gdp - bound forms and active gtp - bound forms , and exchange for gtp and hydrolysis of gtp to gdp are catalyzed by guanine nucleotide exchange factors ( gefs ) and gtpase activating proteins ( gaps ) , respectively ( bos 1997 , bos 1998 ) . use of active and dominant - negative mutants of h - ras and r - ras has revealed that these enzymes can regulate coupling of 1 and 2 integrin - dependent adhesion to t cell receptor ( tcr / cd3 ) , interleukin-3 , and chemokine receptor signaling in t lymphocytes and pro - b cell leukemic cell lines ( zhang et al . 1996 ; o'rourke et al . 1998 ; liu et al . 1999 ; shibayama et al . 1999 ; tanaka et al . we have examined whether cd31 stimulates integrin - dependent adhesion via an intracellular signaling pathway , and if so , whether members of the ras family were involved in this process . we find that the cytoplasmic tail of cd31 is essential for cd31-induced adhesion , and that rap1 is specifically and selectively involved in this process . hemagglutinin ( ha)-tagged rap1 ( rap ) , rapv12 , and h - rasv12 in the mammalian expression vector pmt2 ha , prsv , and prsv - rapn17 have been described previously ( zwartkruis et al . pmt2ha - r - rasv38 was generated by subcloning a full - length ncoi ( klenow - filled)/xhoi fragment of r - rasv38 into noti ( mung bean nuclease - digested)/xhoi - digested pmt2 ha . a sali / noti fragment encoding amino acids 200297 of human ralgds was amplified by pcr to generate pmt2ha - ralgds - rbd . pmt2-ha - rapgap was generated by subcloning full - length rapgap ( provided by dr . paul polakis , onyx pharmaceuticals , richmond , ca ) as a sali / bglii fragment into pmt2 ha . catalytically inactive rapgap lig was generated by mutating rapgap amino acids 284286 ( rkr ) to lig using the stratagene quick site - directed mutagenesis kit . pcaggs - c3 g and psr - his - tagged spa1 were provided by drs . michiyuki matsuda ( national institutes of health , tokyo , japan ) and masakazu hattori ( kyoto university , kyoto , japan ) , respectively . jurkat t cell lines were maintained as described previously ( reedquist and bos 1998 ) . the jurkat cell line jhm1 2.2 was provided by dr . doreen cantrell ( imperial cancer research fund , london , uk ) , with kind permission of dr . art weiss ( university of california at san francisco , san francisco , ca ) . jurkat cells used to generate cd31 wild - type ( wt ) , cd31 glycosylphosphatidylinositol ( gpi ) , and y663/686f stable transfectants were provided by dr . cd31 and cd31 variants were established by six rounds of macs - sorting of parental jurkat cells with anti - cd31 antibody 10b9 . 1997 ) , and stable polyclonal lines established by selection in g418 ( 1 mg / ml ) and facs sorting . jurkat cells ( 1.2  10 cells / ml in 0.4 ml complete media ) were pulsed at 250 v and 960 f with 5 g tk - luciferase plasmid dna , construct plasmids as indicated in figure legends , and added vector plasmid to keep dna amounts constant . 24 h after transfection , cells were transferred to serum - free media and used 4248 h after transfection for adhesion assays . subconfluent a14 and cos-7 cells were transfected by calcium phosphate precipitation as described previously ( zwartkruis et al . antiintegrin 2 antibody l15 , activating antiintegrin 1 antibody ts2/16 , and activating antiintegrin 2 kim185 have been described previously ( van de wiel - van kemenade et al . mab 24 , recognizing a ligand - induced epitope of l , was kindly provided by dr . anti - cd31 antibodies , pecam 1.2 and pecam 1.3 , were generous gifts from dr . anti - cd31 antibodies 2h8 , wm59 , gi18 , and 9g11 have been described previously ( yan et al . other mabs used in these studies were anti - rap1 , rala , and ras antibodies ( transduction laboratories ) . purification of gst - rbd fusion proteins and their use in detecting activated rap1 , rap2 , ras , and r - ras by precipitation , sds - page , and immunoblotting have been described previously ( de rooij and bos 1997 ; franke et al . detection of bound radiolabeled gdp and gtp to rap1 was also described previously ( zwartkruis et al . for adhesion assays , transiently transfected jurkat cells were harvested , washed , and resuspended in tsm media ( 20 mm tris , ph 8 , 150 mm nacl , 1 mm cacl2 , 2 mm mgcl2 ) . 96-well costar maxisorp plates were coated overnight at 4c with goat anti  human igg antibodies ( jackson immunoresearch ; 4 g / ml in tsm ) , washed , blocked for 30 min at 37c with 1% bsa / tsm , followed by incubation 1 h at 37c with 50 ng / ml recombinant intercellular adhesion molecule ( icam)-1 or vascular cell adhesion molecule ( vcam)-1 human igg fc fusion proteins . the 50-l cell suspension was mixed with 50 l tsm or tsm with stimuli indicated . cells were allowed to adhere for 3060 min , and nonadherent cells removed with warm 0.5% bsa / tsm . adherent cells were lysed and subjected to luciferase assays as described previously ( medema et al . cells bound were calculated and were corrected for transfection efficiency and nonspecific effects of constructs by measuring luciferase activity of total input cells . comparison of effects of cdna construct overexpression on adhesion were made by paired t test or unpaired t test as appropriate . fluorescent labeling of stably transfected cells with 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy fluorescein acetoxymethyl ester ( molecular probes ) and measurement of adherent cells with a fluoroscan ascent fluorescent plate reader ( lab systems ) has been described previously ( newton et al . flow cytometric analysis of transfected cells was performed by cotransfecting jurkat jhmi cells with 1 g pcmv - egfp c1 plasmid ( promega ) and the indicated cdna constructs . after overnight serum starvation , cells were equilibrated in 0.5% bsa/1 mm cacl2/pbs ( facs buffer ) and left unstimulated , or stimulated for 30 min with plate - immobilized anti - cd31 antibody 2h8 ( 10 g / ml ) . cells were harvested , resuspended in facs buffer containing primary antibodies ( 10 g / ml ) , and incubated for 30 min on ice , or 37c for mab 24 epitope expression . for mab 24 staining , additional sets of transfected cells were coincubated with 400 m mncl2 during primary antibody staining . fluorescence intensity of egfp - transfected cells was determined using a facs caliber flow cytometer and cellquest software ( both from becton dickinson ) . previously , cd31 has been demonstrated to stimulate t lymphocyte adhesion to icam and vcam via t cell lfa-1 ( l2 integrin ) and vla-4 ( 41 integrin ) , respectively ( tanaka et al . 1992 ) . to examine whether cd31-stimulated integrin - dependent adhesion in t cells was regulated by signals generated through the cd31 cytoplasmic tail or via interactions using the extracellular domain of cd31 ( hemler 1998 ) , we made use of jurkat cells stably expressing full - length cd31 ( cd31 wt ) , a gpi - anchored cd31 construct , previously shown to mediate cd31 homophilic binding ( newton et al . 1997 ) , but lacking the cd31 cytoplasmic tail , or full - length cd31 containing two tyrosine - to - phenylalanine mutations ( y663/686f ) in the major tyrosine - phosphorylation sites of the cd31 cytoplasmic tail ( pumphrey et al . cd31 expression of parental , cd31- and cd31-selected variants , and stable transfectants is shown in fig . 1 a , and was equivalent in cd31 wt , cd31 gpi , and y663/686f lines . all cell lines also expressed similar levels of l , 4 , 5 , 1 , and 2 integrins ( data not shown ) . although all cell lines adhered to icam-1 and vcam-1 when stimulated by pma ( data not shown ) , only cd31 wt transfectants , but not cd31 gpi and y663/686f cells , adhered to icam-1 or vcam-1 after stimulation with anti - cd31 antibodies ( fig . this suggested that cd31-induced , integrin - mediated adhesion requires intracellular signaling pathways generated by the cytoplasmic tail of cd31 , specifically tyrosine phosphorylation of tyrosine residues 663 and/or 686 , but not signaling pathways mediated by cis - interactions of the cd31 extracellular or transmembrane domains . to address which ras family members , if any , might participate in cd31 signaling , we precipitated endogenous gtpases from lysates of cd31-stimulated jurkat cells with gst fusion proteins of ras family - binding domains ( rbds ) of raf and ralgds , which bind with high selectivity and specificty to activated ras and rap gtpases , respectively ( de rooij and bos 1997 ; franke et al . immunoblotting of precipitated gtpases provides a qualitative representation of gtpase activation status after cell stimulation , corresponding to quantitative changes detected using classical gdp / gtp - binding ratio techniques . time course analyses of jurkat cells treated with anti - cd31 antibodies or tpa ( fig . 2 a ) surprisingly revealed a rapid activation of the ras - related gtpase , rap1 , by cd31 , which was maximal 25 min after stimulation and decreased slowly toward basal levels after 20 min . in contrast , whereas both rap1 and ras were activated by tpa , cd31-dependent stimulation of rap1 was highly selective and no activation of rap2 , ras , or r - ras was noted at any point during this time course . both rap1 and ras , but not the other gtpases tested , were also activated by tcr stimulation ( data not shown ) . gtp - bound r - ras was detected in raf - rbd pull - downs only after longer exposures of films , reflecting the relatively low levels of r - ras protein expression in jurkat cells . r - ras protein expression was at least tenfold lower in jurkat cells as compared with a431 fibroblasts , while rap1 , rap2 , and ras levels were relatively equivalent ( fig . 2 a , right , last two lanes ) . functional anti - cd31 antibodies against cd31 extracellular domains 1 and 6 ( pecam 1.2 , pecam 1.3 , and 2h8 ) , activated rap1 more potently than nonfunctional antibodies ( wm59 , 9g11 , and gi18 ; fig . 2 b ) , correlating with the ability of these antibodies to induce cd31-dependent adhesion ( newton et al . 1997 ) . additionally , activation of rap1 by anti - cd31 antibodies was observed in cd31 wt jurkat cell transfectants , but not cd31 gpi or y663/686f transfectants ( fig . 2 c ) , demonstrating a correlative requirement for the cd31 cytoplasmic tail in the activation of rap1 and induction of adhesion . as cd31 stimulation selectively activated rap1 , and rap1 activation correlated with cd31-dependent adhesion to icam and vcam , we next addressed whether constitutive activation of rap1 was sufficient to induce adhesion . jurkat t cells transiently transfected with a luciferase reporter plasmid and indicated cdna constructs were allowed to bind to immobilized recombinant icam and adhering transfected cells detected by measurement of luciferase activity . t cells expressing constitutively active rap1 ( rapv12 ) or the rap1 gef c3 g ( tanaka et al . 1994 ) displayed 3.5- and 3-fold increases in adhesion to icam , respectively ( fig . c3g - induced spreading and adhesion on fibronectin has been observed previously in 32-d cells , although involvement of rap1 was not examined and ras , r - ras , or other gtpases were implicated ( arai et al . 1999 ) . expression of either the active form of ras ( rasv12 ) or r - ras ( r - rasv38 ) resulted in a lower , but detectable induction of adhesion ( 1.52-fold ) . however , rasv12-stimulated adhesion was not statistically significant , rasv12 stimulated adhesion in only two of five experiments , and rapv12-induced adhesion was always higher than that induced by rasv12 and r - rasv38 , even though the latter two constructs were expressed at significantly higher levels than rapv12 ( fig . 3 a , left inset ) . introduction of a rapgap mutant ( rapgap - lig ; rubinfeld et al . 1991 ) , containing a substitution of amino acids 284286 in the rapgap arginine finger critical for rapgap catalytic activity ( rkr , mutated to lig ) , which , unlike wild - type rapgap , displays no gap activity toward rap1 ( fig . 3 b ) , increased the number of icam - bound cells to a similar level as observed with rapv12 and c3 g . rapgap - lig may perturb rap1 signaling by displacing endogenous rapgap from a subcellular localization required for downregulating rap1 or may constitutively associate with basally activated rap1 and serve as an effector protein linking rap1 to an as yet unidentified target . therefore , we introduced a putative dominant - negative mutant of rap1 , rapn17 , into cells . rapn17 did not affect basal adhesion of jurkat cells to icam , but completely abolished cd31-dependent increases in adhesion ( fig . 3 c ) . although the rapn17 mutation was modeled on the corresponding dominant - negative mutation in h - ras ( medema et al . 1991 ) , rapn17 is not considered to function as a strict dominant - negative protein . whereas rasn17 blocks ras signaling by sequestering ras gefs , rapn17 fails to bind to rap1 gefs in vitro ( van den berghe et al . 1997 ) , and fails to inhibit egf - dependent activation of cotransfected wild - type rap1 in cos-7 cells ( wolthuis , r.m.f . , and j.l . bos , unpublished observation ) . because of uncertainties in the mechanism by which rapn17 might block cd31-stimulated adhesion , we sought independent confirmation that rap1 was required for cd31-induced adhesion of jurkat cells to icam . transient expression of the rap1-specific gap , rapgap , resulted in a strong reduction of basal adhesion to icam , and significantly reduced cd31-dependent adhesion ( fig . this result is compatible with the notion that a rap1gap should decrease levels of gtp - bound rap1 ( see fig . finally , we used the rap1 binding domain of ralgds , rbd , which binds with high affinity and specificity to gtp - bound rap1 in vitro ( franke et al . thus , using three independent strategies for interfering with rap1 signaling , overexpression of inactive rap1 , rap1-specific rapgaps , and an isolated rbd of a rap - binding protein , these experiments demonstrate a critical role for rap1 in mediating cd31-induced adhesion to icam . to address whether rap1 selectively regulated 2 integrins ( lfa-1 ) on jurkat cells , or if it was also coupled to the regulation of 1 integrins ( vla-4 ) , we examined the effects of activating and inactivating mutants of rap signaling on jurkat cell adhesion to vcam . stimulation of cd31 resulted in a smaller induction of adhesion to vcam ( 1.52-fold increase ; fig . 4 a ) than observed to icam , in part due to the higher basal level of adhesion observed on vcam ( typically 3050% , compared with 1020% on icam ) . a similar increase was also observed in cells expressing rapv12 , c3 g , and mutant rapgap - lig , but not rasv12 or r - rasv38 . conversely , rapn17 , rapgap , and a second gap for rap1 , spa1 ( kurachi et al . 1997 ) , significantly reduced basal jurkat cell adhesion to vcam , and strongly blocked cd31-dependent adhesion ( fig . 4 b ) . it has been reported previously that ras regulates tcr - stimulated lymphocyte adhesion to icam ( o'rourke et al . 1998 ; tanaka et al . 1999 ) . as we failed to detect ras activation by cd31 , and rasv12 did not induce a clear increase in adhesion to icam or vcam , it is unlikely that ras mediates the main signaling pathway from cd31 stimulation to integrin - mediated adhesion . however , overexpression of rasn17 did inhibit cd31-dependent adhesion to vcam ( data not shown ) , suggesting that ras might also contribute to adhesion responses . integrin - dependent adhesion can be regulated by changes in integrin surface expression , integrin surface distribution ( avidity ) , or induction of conformational changes that increase integrin ligand affinity ( stewart and hogg 1996 ) . to investigate at which level rap1 might influence cell adhesion , we first examined if rap signaling affected lfa-1 expression levels . jurkat cells were cotransfected with egfp as a reporter marker , along with indicated rap1 signaling pathway cdna constructs . staining of transfected cells with anti-2 integrin antibody revealed that overexpression of rapv12 , rapn17 , rbd , rapgap , or rapgap lig failed to influence surface expression levels of lfa-1 ( fig . 5 a ) . similarly , rapv12 , rapgap lig , rapgap , and rapn17 failed to induce lfa-1 surface clustering , as detected with the nki - l16 antibody ( data not shown ) , although active rasv12 induced clustering of lfa-1 , as previously reported ( tanaka et al . the anti-l integrin antibody mab 24 recognizes a conformation - dependent ligand - induced epitope on lfa-1 , and induction of the mab 24 epitope correlates with increased lfa-1 affinity for icam ( dransfield and hogg 1989 ) . tcr and tpa - induced mab 24 epitope expression previously have been found to be dependent on the presence of icam , suggesting that signals generated by tcr and tpa facilitate weak basal interactions between lfa-1 and icam , stabilizing lfa-1 in a high - affinity conformation ( cabanas and hogg 1993 ) . this conformational change can also be induced by lfa-1 exposure to mn , independently of  inside - out  signaling . we found that anti - cd31 stimulation of jurkat cells , or jurkat cells transfected with gfp alone , resulted in an 1.5-fold increase in mab 24 expression ( fig . 5 b and data not shown ) , whereas a 2.252.5-fold increase was observed in mn - treated cells . incubation of cells with mab 24 at 4c abolished cd31 and mn - induced mab 24 expression to background levels ( data not shown ) , consistent with the temperature sensitivity of the mab 24 epitope ( dransfield and hogg 1989 ) . although rapv12 expression had no statistically significant effect on basal or induced mab 24 epitope expression , rapn17 and rbd abolished induction of the mab 24 epitope by cd31 and mn . conversely , rapgap lig enhanced cd31- and mn - induced expression of the mab 24 epitope . rapn17 also inhibited the increase in the percentage of mab 24-reactive cells observed after cd31 or mn treatment ( fig . thus , our studies suggest that whereas rap does not directly stimulate activation of lfa-1 , signaling via rap1 , or basal activation of rap1 , appears to be a requisite factor for stabilization of the active conformation of lfa-1 . moreover , our finding that rapn17 blocks direct conformational changes induced by extracellular mn may suggest that inactive rap may act to lock lfa-1 in an inactive conformation . consistent with this model , overexpression of rapn17 , rapgap , or ralgds rbd significantly blocked mn - induced adhesion of jurkat cells to immobilized icam , whereas rapv12 augmented the induced cell adhesion ( fig . our report is the first example of requisite involvement of rap1 in coupling cell surface receptor stimulation to integrin - mediated adhesion . previously , it was shown that rap1 is activated after stimulation of a large variety of cell surface receptors , including b and t cell antigen receptors , chemokine and cytokine receptors ( including fmlp , paf , and gm - csf ) , and receptors for platelet agonists ( thrombin , thromboxane a2 , and adp ; reviewed in bos 1997 ) . many of these receptors have been implicated in the control of integrin - mediated adhesion . furthermore , the involvement of rap1 in the control of cell adhesion recently has been suggested by studies in which spa1 was found to inhibit g - csf also , fcri - mediated adhesion of rbl cells to culture dishes is inhibited by expression of rapgap ( m'rabet 1999 ) . therefore , it is plausible to propose that one of the functions of rap1 is to control integrin - mediated adhesion . the mechanism by which rap1 signaling modulates integrin function is unknown , although our data suggest that rap1 may regulate the ability of lfa-1 to undergo ligand - induced conformational changes . this effect could be direct , by binding of rap1 to integrins or integrin - associated complexes . interestingly , rap2 , a very close relative of rap1 , has been found in complex with the iib3 integrin ( torti et al . putative downstream signaling pathways of rap1 include b - raf / map kinase cascade and ral gtpase gefs ( bos 1997 , bos 1998 ) , although we have not detected cd31-dependent activation of map kinase or ral in jurkat cells ( data not shown ) . novel binding partners of activated rap1 recently identified in vitro , krit1 ( serebriiskii et al . 1997 ) and af6 ( linnemann et al . 1999 ) , are proteins lacking any obvious enzymatic activity and are thought to associate with cytoskeletal components . our identification of integrin - adhesion as a physiological target of rap1 should facilitate identification of rap1 effectors . finally , the unique coupling of cd31 , which is also expressed on vascular cells , to rap1 suggests a critical role for rap1 in mediating not only leukocyte adhesion , but other integrin - dependent events mediated by cd31 that require platelet and leukocyte interactions with vascular endothelial cells , including leukocyte transmigration and extravasation . interestingly , regulation of integrin adhesion might also explain the flat revertant phenotype of ras - transformed cells expressing rap1 ( kitayama et al .
integrin - mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli , including chemokines , antigen receptors , and adhesion receptors . how cellular signals from cd31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood . here 
 , we show that the cytoplasmic tail of cd31 , an important integrin adhesion amplifier , propagates signals that induce t cell adhesion via 1 ( vla-4 ) and 2 ( lfa-1 ) integrins . 
 we identify the small gtpase , rap1 , as a critical mediator of this effect . 
 importantly , cd31 selectively activated the small ras - related gtpase , rap1 , but not ras , r - ras , or rap2 . 
 an activated rap1 mutant stimulated t lymphocyte adhesion to intercellular adhesion molecule ( icam ) and vascular cell adhesion molecule ( vcam ) , as did the rap1 guanine nucleotide exchange factor c3 g and a catalytically inactive mutant of rapgap . 
 conversely , negative regulators of rap1 signaling blocked cd31-dependent adhesion . 
 these findings identify a novel important role for rap1 in regulating ligand - induced cell adhesion and suggest that rap1 may play a more general role in coordinating adhesion - dependent signals during leukocyte migration and extravasation . 
 our findings also suggest an alternative mechanism , distinct from interference with ras - proximal signaling , by which rap1 might mediate transformation reversion .