elucidation of protein  protein interactions contributes to the characterization of the function of novel proteins and hence the genes that encode them .
methods for investigation of protein  protein interactions include biophysical , computational , biochemical and genetic approaches . for the identification of multi - protein complexes and to determine their association and dissociation rates together with sites of interactions , methods based on surface plasmon resonance ( spr ) ( 5 ) and different types of mass spectrometry ( ms ) have been employed ( 68 ) .
these methods are proven to be useful , although purification , sequencing and identification of novel proteins can be limiting especially when present only in small quantities .
computational methods based on various principles , including correlated changes of amino acid sequence between interacting protein domains ( 9 ) , properties related to interface topology , solvent accessible area ( asa ) ( 10,11 ) that estimates sites of interaction and primary structure and associated physiochemical properties ( 12 ) , each can predict interactions . while these predictive methodologies are informative in so much as they provide an indication of the affinity of a given protein for another protein , they all require further experimental validation
this system exploits the fact that transcription factors are comprised of two functional domains , a dna binding domain and a transcription activation domain .
the dna binding domain recognizes a specific dna sequence whilst the activation domain facilitates the recruitment of polymerase ii associated transcription complex and initiates transcription of the downstream gene . in this system
the protein domains are separated from each other until brought together by two interacting proteins .
this system has been widely used to screen pray - expression libraries for proteins that interact with a bait protein .
the system is prone to contamination by false positives ( interactions that are difficult to validate ) and false negatives ( interactions that are not detected ) .
bait proteins that alone activate or repress the expression of the reporter gene can also prove problematic . to counter a number of inefficiencies associated with the system
proteins of interest are fused to c- and n - terminal domains of ubiquitin . in the event of protein  protein interactions an active ubiquitin
ubiquitin is recognized by ubiquitin specific protease , which leads to the release of reporter protein .
additional technologies , based on the yeast two - hybrid screen are being developed all the time .
these include systems based on signalling ( 15 ) and dual - bait ( 16 ) .
each of these modifications has a major limitation as many mammalian proteins are exposed to significant post - translational modifications , important in protein 
protein complex formation and function . in an attempt to overcome these difficulties mammalian versions of the two - hybrid screen
have been developed ( 1720 ) but these have been single reporter functions and are hence susceptible to variation between samples of levels of transfection and transcription .
we have developed a dual reporter assay system based on yeast two - hybrid screen , which comprised two autonomous units of gene expression .
the upstream unit is expressed regardless of protein  protein interactions . in the absence of interacting proteins
the downstream unit is switched off . in the event of an interaction , the downstream expression unit
thus , the ratio of the two reporter activities can be used as a reference value to detect mutations or trans - acting factors that might affect protein  protein interactions .
construction of the reporter plasmids ( ptn114 and ptn110 ) was based on pbluga ( 21 ) , which contains reading frames for the -galactosidase and luciferase genes . to construct ptn114 , a deactivation  activation
( d / a ) unit was cloned into xhoi / sali and bglii / bamhi sites of pbpluga .
the unit comprises a translation termination signal for the upstream reporter followed by a 3 end trimming and polya tail addition site ( aataaa ) , an upstream activation sequence of saccharomyces cerevisiae ( 22 ) linked to a tata box sequence from adenovirus e1b minimal promoter ( 23 ) , six copies of binding sites for gal4 transcription factor ( 24 ) and a synthetic mrna splicing signal for effective expression of the downstream reporter , a 5-utr and an in - frame start codon for the downstream reporter . a dna fragment containing the activation sequences , tata box , gal4 binding site and the splicing signal that comprised nucleotides 1535 of pgal / lacz ( invitrogen )
was amplified using uasf(xho ) 5-ccgctcgagggtgaaataaagtcgacccgagctcttacgcggg-3 and uasr(bglii ) 5-gaaagatcttgccatgtcttcgatctgcagaattcc-3. for cloning purposes the internal xhoi site was replaced by a bamhi site using site directed mutagenesis ( 25 ) .
the construct ptn111 was made by cloning the gal4 dna binding domain ( residues 1147 ) ( 26 ) with a n - terminal t7 epitope tag into aflii and kpni sites of pcdna3.1 ( invitrogen ) .
the coding region of limk1 gene ( nm_002314 ) was sub - cloned into kpni and xbai sites of ptn111 .
construct ptn112 was constructed by cloning the vp16 transcriptional activation domain ( 27 ) into xbai and apai sites of pcdna3.1 ( invitrogen ) .
the coding sequences of bmpr - ii [ nm_001204 and nm_033346 for long ( lf ) and short forms ( sf ) , respectively ] and d485 g mutant were cloned into bamhi and ecori sites of ptn112 .
activation unit into xhoi and bamhi sites of pdsred - tn24-gfp ( siskoglou and nasim , unpublished data ) .
this vector was similar to ptn24 ( 28 ) except that the genes encoding -galactosidase and luciferase were replaced by genes encode for fluorescence proteins and the sv40 promoter was replaced by the cmv promoter .
the d / a unit is similar to that used in ptn114 except that an additional sv40 polya site comprising nucleotides 14981648 of the dsred - express - c1 ( clonetech ) was introduced at the 5 end of the upstream activation sequence .
( human embryonic kidney ) cells were transiently transfected with relevant plasmids using gene jammer ( stratagene ) . cells were harvested 48 h after transfection and -galactosidase and luciferase activities were measured using dual light system ( applied biosystems ) as described elsewhere ( 28,29 ) .
for live cell imaging , cells were grown as before and the images were taken under a fluorescence microscope ( te 300 , nikon ) .
for single - cell expression , cells expressing both green and red fluorescences were selected using a facscan flow cytometer ( becton dickinson ) and the fluorescence intensities were measured using cellquest software .
construction of the reporter plasmids ( ptn114 and ptn110 ) was based on pbluga ( 21 ) , which contains reading frames for the -galactosidase and luciferase genes . to construct ptn114 , a deactivation  activation
( d / a ) unit was cloned into xhoi / sali and bglii / bamhi sites of pbpluga .
the unit comprises a translation termination signal for the upstream reporter followed by a 3 end trimming and polya tail addition site ( aataaa ) , an upstream activation sequence of saccharomyces cerevisiae ( 22 ) linked to a tata box sequence from adenovirus e1b minimal promoter ( 23 ) , six copies of binding sites for gal4 transcription factor ( 24 ) and a synthetic mrna splicing signal for effective expression of the downstream reporter , a 5-utr and an in - frame start codon for the downstream reporter . a dna fragment containing the activation sequences , tata box , gal4 binding site and the splicing signal that comprised nucleotides 1535 of pgal / lacz ( invitrogen )
was amplified using uasf(xho ) 5-ccgctcgagggtgaaataaagtcgacccgagctcttacgcggg-3 and uasr(bglii ) 5-gaaagatcttgccatgtcttcgatctgcagaattcc-3. for cloning purposes the internal xhoi site was replaced by a bamhi site using site directed mutagenesis ( 25 ) .
the construct ptn111 was made by cloning the gal4 dna binding domain ( residues 1147 ) ( 26 ) with a n - terminal t7 epitope tag into aflii and kpni sites of pcdna3.1 ( invitrogen ) .
the coding region of limk1 gene ( nm_002314 ) was sub - cloned into kpni and xbai sites of ptn111 .
construct ptn112 was constructed by cloning the vp16 transcriptional activation domain ( 27 ) into xbai and apai sites of pcdna3.1 ( invitrogen ) .
the coding sequences of bmpr - ii [ nm_001204 and nm_033346 for long ( lf ) and short forms ( sf ) , respectively ] and d485 g mutant were cloned into bamhi and ecori sites of ptn112 .
activation unit into xhoi and bamhi sites of pdsred - tn24-gfp ( siskoglou and nasim , unpublished data ) .
this vector was similar to ptn24 ( 28 ) except that the genes encoding -galactosidase and luciferase were replaced by genes encode for fluorescence proteins and the sv40 promoter was replaced by the cmv promoter .
the d / a unit is similar to that used in ptn114 except that an additional sv40 polya site comprising nucleotides 14981648 of the dsred - express - c1 ( clonetech ) was introduced at the 5 end of the upstream activation sequence .
hek-293 ( human embryonic kidney ) cells were transiently transfected with relevant plasmids using gene jammer ( stratagene ) .
cells were harvested 48 h after transfection and -galactosidase and luciferase activities were measured using dual light system ( applied biosystems ) as described elsewhere ( 28,29 ) .
for live cell imaging , cells were grown as before and the images were taken under a fluorescence microscope ( te 300 , nikon ) .
the images were analysed using openlab software ( improvision ) . for single - cell expression ,
cells expressing both green and red fluorescences were selected using a facscan flow cytometer ( becton dickinson ) and the fluorescence intensities were measured using cellquest software .
we have constructed a set of reporter constructs in which genes encoding -galactosidase and luciferase are fused with a recombinant fragment containing the translation termination signal for the upstream reporter , a polyadenylation site , an upstream activation sequence linked to a tata box sequence from adenovirus e1b minimal promoter , six copies of binding sites for gal4 transcription factor , a pre - mrna splicing signal , a 5-untranslated region ( 5-utr ) and in - frame start codon for the downstream reporter .
upon transfection into the mammalian cells , transcription from the sv40 promoter leads to the production of a pre - mrna which contains a translation termination signal and a polyadenylation signal .
efficient processing and termination ( transcription and translation ) would result in the production of -galactosidase protein . in the event of protein 
protein interactions , transcription of the luciferase gene would be activated , the mrna would be exported to the cytoplasm resulting in the production of luciferase protein .
since luciferase activity would be produced only after protein  protein interactions , whereas -galactosidase is expressed constantly , the ratio of luciferase and -galactosidase activities would indicate the proportion of cytoplasmic rna derived from protein  protein interactions . to test the ability of the assay system ,
hek 293 cells were transfected with a reporter plasmid ptn114 . a second plasmid ( ptn110 )
was generated as identical to ptn114 except for the absence of signals for polyadenylation and splicing .
cells transfected with either plasmid produced only -galactosidase activity ( figure 2b ) . upon co -
transfection with plasmids encode for the murine p53 ( pcr2.1/p53 produced a gal4-p53 fusion protein ) and sv 40 large t antigen ( pcr2.1/lgt produced a vp16-lgt fusion protein ) , the plasmid ptn114 was able to produce both -galactosidase and luciferase activities , whereas ptn110 failed to show any luciferase activity ( figure 2a and b ) .
co - transfection with plasmids containing the p53 and the polyoma viral coat protein ( cp ) ( pcr2.1/vp16-cp generated by invitrogen produced a vp16-cp fusion protein ) , failed to produce a significant amount of luciferase activity ( figure 2a ) .
the luciferase and -galactosidase activities were highly variable between experiments whereas when the data was expressed as the ratio of luc  gal activities , the variability was significantly reduced ( figure 2c ) .
the presence of luciferase protein was confirmed by western blotting using luciferase hrp conjugate antibody ( ab cam , uk ) ( data not shown ) .
mutations in the type ii receptors for bone morphogenetic protein ( bmpr - ii ) , a member of the tgf- receptor family underlie the majority of inherited forms of primary pulmonary hypertension ( pph ) ( 30 ) and familial pulmonary arterial hypertension ( 31 ) .
bmpr - ii is a multi - domain protein containing extracellular , transmembrane , kinase and cytoplasmic tail domains .
the mutations are dispersed all over the functional domains and are likely to impinge upon receptor mediated function . a number of proteins have been shown to interact with bmpr - ii ( 8) . the interactions between lim kinase 1 ( limk1 ) and bmpr - ii were initially discovered by yeast two - hybrid screening and confirmed by immunoprecipitation in mammalian cells ( 32 ) .
we wished to examine whether we could employ the reporter system to examine the interactions of limk1 and bmpr - ii .
this would provide the basis of a rapid assay system to investigate the effects of mutations in the bmpr2 on receptor - mediated function .
we employed the system to screen mutants of bmpr - ii where protein  protein interactions might be abrogated .
we constructed a series of constructs where the coding sequence of lim kinase 1 gene was fused with a n - terminal dbd and the bmpr2 gene was fused with an activation domain at the c - terminal end .
both proteins were either t7 or myc tagged so that they could be easily purified .
co - transfection of plasmids encoding both proteins along with the reporter plasmid ( ptn114 ) into the mammalian cells enabled both proteins to interact with each other giving rise to a luc  gal ratio ( figure 2d ) .
co - transfection of plasmids encode for the limk1 and sf of the bmpr2 or missense mutation produced significantly reduced luc  gal ratio ( figure 2d ) .
the expression of limk1 and bmpr - ii proteins was confirmed using antit7 and anti - myc antibodies , respectively , by means of western blotting ( data not shown ) . in order to establish a system for high - throughput screening of libraries ( chemical , peptide , cdna , etc . )
a dual fluorescence reporter was constructed using genes encoding red ( dsred express ) and green fluorescence proteins ( gfp ) .
dsred express and gfp were chosen as their excitation peaks are 488 and 557 nm , respectively and therefore they can be easily distinguished under a fluorescence microscope . in addition , each is a highly stable protein , exhibiting no detectable photoinstability with high signal - to - noise ratio ( 33 ) and a shorter maturation time than that of other fluorescence proteins including dsred .
upon transfection into the mammalian cells the reporter plasmid produced only the upstream reporter ( dsred express ) ( figure 3a ) .
co - transfections of plamids containing either p53 or lgt gene were unable to initiate transcription of the green fluorescence protein .
overexpression of both genes ( in this case p53 and lgt ) initiated transcription of the gfp .
quantification of the fluorescence ratio indicated that the extent of interactions was uniform in a population of cells ( figure 3b ) .
significant amount of green fluorescence intensity was observed when cells were transfected with both p53 and lgt genes but not with the lgt alone ( figure 4a and b ) , a finding consistent with the data obtained from fluorescence microscopy .
since the conventional methods based on the yeast two - hybrid system for assaying protein  protein interactions in mammalian cells are susceptible to numerous variables , we developed a system that bypassed the variables that affect single reporter assays .
one of the important features of this system is that a synthetic deactivation  activation unit is created which comprises signals for transcription , polyadenylation , processing and translation .
signals for polyadenylation , transcription and translation termination are introduced such that the upstream reporter is expressed . upon protein 
protein interactions the pol ii transcription machinery is recruited upstream of the second reporter , hence enabling the production of the second reporter .
we have previously showed that there is a good correlation between the level of transcript and luciferase  galactosidase ratio ( 28 ) .
we have analysed the inter - experiment variability for expression of the reporter proteins and found substantial scatter of the data points .
however , by taking advantage of constitutive expression of reporter a , we can correct the expression of reporter b to this reference standard .
hence , the ratio of two reporter activities show marked reduction in the signal variability ( figure 2c ) . we have tested the dual - light reporter in several ways to show that it reflects the efficiency of protein  protein interactions
. the highest ratio of luciferase and galatosidase activities was observed when co - transfection was carried out using plasmids encoding the p53 with lgt and the bmpr2 with limk1 ( figure 2c and 2d ) , as these proteins interact with each other ( 32,34,35 ) .
the lowest ratio was observed when either the reporter ( ptn114 ) was transfected alone or co - transfected with plasmids encoding the p53 with cp and the limk1 with short or mutant form of bmpr2 .
we observed that the interaction between limk1 and bmpr - ii was weaker than that of lgt and p53 , possibly due to mislocalization of bmpr - ii .
luciferase activity was completely abolished when the polyadenylation and splicing signals were ablated ( ptn110 ) .
co - transfections of the p53 and lgt had no discernible effects possibly due to transcriptional interference , a process that is known to affect the expression of downstream gene ( 36 ) .
in this case , upon transcription from the sv40 promoter a gal  luc fusion rna was produced .
the presence of translation termination signal at the end of -galactosidase gene prompted the production of -galactosidase protein .
we would assume that in the case of ptn114 , transcription termination occurred at poly a sites located at the end of both reporter genes ( 37 ) .
both rnas were polyadenylated , bound by poly a binding protein ( pabp ) , circularized by interacting with translation initiation complex ( 38 ) and thereby acted as autonomous units of expression .
a potential complication of the approach was that of transcriptional interference , which might affect recruitment of transcription complex for the second reporter .
this could be easily addressed by inserting multiple termination sites ( 36 ) at the end of first reporter as transcription termination occurs through a non - processive mechanism ( 39 ) .
we have not determined the level of promoter suppression by which the expression of the downstream reporter could be affected but again this could be tackled by interposing mar / sar elements ( 40 ) between the adjacent units .
although we have not encountered the problems of autoactivation of library screening ( 41 ) , it is unlikely to affect the usefulness of the method , as long as a second independent method ( i.e. pull - down , immunoprecipitation ) is used to validate the most interesting candidates that emerge from a screen .
identification of a small molecule that dimerizes two proteins can be used for induction of cell proliferation ( 42 ) and apoptosis ( 43 ) . on the contrary
, small molecule antagonists of proteins can be useful in developing antibiotic , antiviral and antiparasitic drugs ( 44 ) .
the advantages of the dual fluorescence reporter are that the assay is measured directly on the intact cell and therefore does not require cell wall disruption or addition of a substrate and the sensitivity of this assay is comparable with that reported for luc  gal assay .
in addition the dual fluorescence assay could be performed in 96- or 386-well format and would be useful to screen chemical or peptide libraries to identify dimerizers or antagonists that affect specific protein  protein interactions . in this case
it would be advantageous to set up a stable mammalian cell line by incorporating the fluorescence reporter .
the maturation time of the dsred express is shorter than that of its dsred counterpart , which makes the reporter system useful in investigating protein interactions in the early stages of developing embryos .
although we have only shown that this system would be useful in screening mutations of bmpr - ii that are involved in limk interactions , there are a number of other human diseases for which it would be beneficial to isolate drugs capable of targeting selected protein  protein interactions .
this system in its present form would be useful , for example , in identifying proteins that interact with selenoprotein n. mutations in the selenoprotein n gene have been implicated in muscular dystrophy ( 45 ) .
the classical two - hybrid screen to identify the interacting proteins is not suitable because yeast is known to contain no selenocysteine incorporation machinery .
other methods , such as pull - down or immunoprecipitation , would be difficult due to the fact that selenocysteine incorporation in mammalian cell line is an inefficient process ( 46 ) .
we earlier developed a dual - reporter based assay system ( 28 ) to determine rna processing efficiency in mammalian cells . in this system ,
two reporter activities fused with splicing signals were linked to single gene expression unit . in the event of splicing , the expression unit produced a fusion protein with two reporter functions whereas inefficient splicing produced a protein with single reporter function .
the system presented here is based on two autonomous gene expression units where one expression unit is expressed regardless of the expression of the other unit .
we have shown here the usefulness of this principle in upgrading a classical two - hybrid screen .
we suggest that this principle may be beneficial for other systems where the activities are limited to single reporter functions .
the dual - light reporter system for determining protein  protein interactions is based on two reporter genes , which are fused via a recombinant fragment containing a synthetic deactivaion / activation ( d / a ) unit .
the unit comprises polyadenylation signal(s ) , six copies of gal4 dna binding site , a tata box , a synthetic splicing unit .
upon transfection into mammalian cells the reporter a is transcribed under the control of sv40 promoter , while transcription of the reporter b is switched off . in the event of protein  protein interactions , transcription of the reporter b
analyses of the gal  luc reporter system to determine protein  protein interactions in mammalian cells .
the reporter construct along with relevant plasmids was co - transfected into hek 293 cells and activities of luciferase ( a ) and -galactosidase ( b ) were measured .
the ratio of both reporter activities was normalized to a value of 100 ( c ) with p53 and lgt and ( d ) with limk1 and bmpr2 .
( a ) the expression of gfp was suppressed in the absence of interacting proteins as shown , where cells were transfected with ( i ) reporter alone , or co - transfected with ( ii ) p53 and ( iii ) lgt .
the expression of the gfp is activated when cells were co - transfected with plasmids encoding the p53 and cp ( iv and v ) .
( b ) fluorescence intensities of both proteins were measured and normalized as mentioned in figure 2 . mean and standard deviations were derived from a pool of 30 to 40 randomly selected cells .
cells with both fluorescence activities were selected using a fluorescence activated cell sorter and their intensities were determined ( a ) and normalized ( b ) . the mean and standard deviations were derived from 300 to 400 cells .
 methods for determining protein  protein interactions in mammalian cells typically rely on single reporter functions and are susceptible to variations between samples particularly in regard to levels of transcription , processing and translation . 
 a method has been developed for determining protein  protein interactions in mammalian cells , which bypasses these variables confounding single reporter assays . 
 the approach utilizes two units of gene expression linked to reporter functions that are interposed by a deactivation  activation unit in such a way that the downstream expression unit is switched off . 
 hence upstream expression occurs regardless of protein  
 protein interaction , leading to the production of the upstream reporter . in the event of protein  
 protein interactions , the downstream expression unit is switched on leading to dual reporter read outs . 
 thus , the ratio of the two reporter activities provides a measure to determine the efficiency of protein  protein interactions . to access the system we screened a mutant of bmpr2 where the interaction between bmpr - ii and limk is abrogated . 
 bmpr - ii is a type ii receptor of the tgf superfamily and plays a key role in the pathogenesis of familial pulmonary arterial hypertension . 
 this system has potential for high - throughput screening of libraries ( peptide , chemical , cdna , etc . ) to isolate agents that are capable of interfering with highly selective protein  protein interaction . 
INTRODUCTION
MATERIALS AND METHODS
Plasmid construction
Gene transfer, cell culture, enzymatic assay, fluorescence microscopy and flow cytometry
RESULTS
DISCUSSION
Figures and Tables