selection and characterization of small random transmembrane...

Selection and Characterization of Small RandomTransmembrane Proteins that Bind and Activate thePlatelet-derived Growth Factor b Receptor

Lisa L. Freeman-Cook1, Ann M. Dixon2, Jennifer B. Frank1, Yu Xia2

Lara Ely1, Mark Gerstein2, Donald M. Engelman2 and Daniel DiMaio1*

1Department of Genetics, YaleUniversity School of MedicineNew Haven, CT 06510, USA

2Department of MolecularBiophysics and BiochemistryYale University School ofMedicine, New Haven, CT06510, USA

Growth factor receptors are typically activated by the binding of solubleligands to the extracellular domain of the receptor, but certain viral trans-membrane proteins can induce growth factor receptor activation by bind-ing to the receptor transmembrane domain. For example, homodimersof the transmembrane 44-amino acid bovine papillomavirus E5 proteinbind the transmembrane region of the PDGF b receptor tyrosine kinase,causing receptor dimerization, phosphorylation, and cell transformation.To determine whether it is possible to select novel biologically activetransmembrane proteins that can activate growth factor receptors,we constructed and identified small proteins with random hydrophobictransmembrane domains that can bind and activate the PDGF b receptor.Remarkably, cell transformation was induced by approximately 10% ofthe clones in a library in which 15 transmembrane amino acid residuesof the E5 protein were replaced with random hydrophobic sequences.The transformation-competent transmembrane proteins formed dimersand stably bound and activated the PDGF b receptor. Genetic studiesdemonstrated that the biological activity of the transformation-competentproteins depended on specific interactions with the transmembranedomain of the PDGF b receptor. A consensus sequence distinct from thewild-type E5 sequence was identified that restored transforming activityto a non-transforming poly-leucine transmembrane sequence, indicatingthat divergent transmembrane sequence motifs can activate the PDGF breceptor. Molecular modeling suggested that diverse transformingsequences shared similar protein structure, including the same homo-dimer interface as the wild-type E5 protein. These experiments haveidentified novel proteins with transmembrane sequences distinct fromthe E5 protein that can activate the PDGF b receptor and transform cells.More generally, this approach may allow the creation and identificationof small proteins that modulate the activity of a variety of cellulartransmembrane proteins.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: cell transformation; oncogene; TOXCAT; bovine papillomavirus;E5 protein*Corresponding author

Introduction

Receptor tyrosine kinases are normally activated

by soluble ligands that bind to the extracellulardomain of the receptor and induce receptor dimer-ization. Certain viral transmembrane proteins thatdo not resemble these ligands can also activateendogenous receptor signaling pathways andinduce tumorigenic transformation by interactingwith the transmembrane domain of growth factorreceptors.1 For example, the gp55 protein of theFriend leukemia virus binds and activates theerythropoietin receptor, and the E5 protein of

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

Supplementary data associated with this article can befound at doi: 10.1016/j.jmb.2004.03.044

E-mail address of the corresponding author:[email protected]

Abbreviations used: PDGF, platelet-derived growthfactor; CAT, chloramphenicol acetyltransferase.

doi:10.1016/j.jmb.2004.03.044 J. Mol. Biol. (2004) 338, 907–920

bovine papillomavirus (BPV) binds and activatesthe platelet-derived growth factor b (PDGF b)receptor.1 In both of these cases the interactionsdriving receptor activation occur primarily withinthe lipid bilayer. To test the possibility that side-by-side protein–protein interactions involvingdiverse transmembrane segments can inducegrowth factor receptor activation, we devised astrategy to identify novel transmembrane domainsthat can recognize the transmembrane domainof the PDGF b receptor and induce receptoractivation and cell transformation.

We used the 44-amino acid BPV E5 protein asa scaffold to identify small transmembraneproteins that activate the PDGF b receptor. The E5protein, which induces transformation of culturedrodent fibroblasts,2 has a very hydrophobictransmembrane domain that is interrupted onlyby a glutamine at position 17.3 In transformedcells, disulfide-linked homodimers of the E5 pro-tein appear to be localized largely to the mem-branes of the endoplasmic reticulum and Golgiapparatus in a type II transmembraneorientation.3 – 5 Transformation is inhibited bymutations at Gln17, Asp33, and the carboxy-terminal cysteine residues required for homodimerformation, but the transforming activity of the E5protein is not impaired by a variety of singlehydrophobic substitution mutations in the centralhydrophobic domain.6 – 12 In limited surveys, asmall number of E5 mutants with apparently ran-dom transmembrane domains have been identifiedthat can transform cells, but the mechanism oftransformation by these mutants has not beenexplored.9,13,14

Although the wild-type E5 protein interacts withseveral cellular proteins,10,15 – 17 its primary trans-forming function appears to be mediated by acti-vation of the cellular PDGF b receptor, a type Itransmembrane receptor tyrosine kinase.8,18 – 21 TheE5 dimer is present in a stable complex with twotyrosine-phosphorylated forms of the PDGF breceptor in transformed cells, a mature form andan intracellular form with immaturecarbohydrates.16,21 – 23 Complex formation resultsin the ligand-independent dimerization of twomolecules of the PDGF b receptor, trans-phosphorylation of each receptor in the dimericcomplex, and constitutive association with SH2domain-containing signaling proteins.8,18,21,23 – 25

The interaction between the E5 protein and thePDGF b receptor is mediated by transmembraneand juxtamembrane interactions and is quitespecific, in that the E5 protein can bind andactivate the wild-type PDGF b receptor but notPDGF b receptor mutants with a foreign trans-membrane domain or other receptor tyrosinekinases.18,19,22,26 – 28 Furthermore, the E5 protein isable to bind and activate mutant PDGF b receptorsthat lack the extracellular ligand binding domain,and it can bind the transmembrane segment of thePDGF b receptor linked to very little intracellularor extracellular sequence.18,27

We proposed that the E5 homodimer transits themembrane as a symmetric left-handed coiled-coilwith an interface consisting of Leu14, Gln17,Leu21, Leu24, and Phe28,5,11 although an alterna-tive model has been proposed.29 Gln17 stabilizesthe E5 dimer, presumably by making interhelicalhydrogen bonds.5,8 Extensive mutagenesis experi-ments also suggest the existence of a hydrogenbond between Gln17 of the E5 protein and Thr513of the receptor, and a salt bridge between Asp33of the E5 protein and Lys499 in the PDGF breceptor.7 – 10,12,28 Although Thr513 and Lys499 areboth required for the interaction between the E5protein and the PDGF b receptor, many otherpoint mutations in the PDGF b receptor trans-membrane domain also impair transformation,implying that there are additional contactsbetween the PDGF b receptor and the E5 pro-tein28,30 (and L.E., D. Mattoon & D.D., unpublishedresults).

The experiments summarized above revealedthat receptor tyrosine kinases can be activated bytransmembrane proteins unrelated to their normalligand. In order to isolate novel proteins that canrecognize and bind cellular transmembraneproteins, we developed a method to select smallrandomized transmembrane proteins that can acti-vate the PDGF b receptor. We identified numeroustransmembrane sequences quite distinct from theE5 protein that can activate the PDGF b receptor,suggesting an unexpectedly wide range ofsequences capable of participating in productiveinteractions with the transmembrane domain of agrowth factor receptor.

Results

Experimental strategy

In order to isolate novel small transmembraneproteins that activated the PDGF b receptor, weconstructed a library in which 15 transmembraneamino acid residues of the 44-amino acid E5 pro-tein were replaced with random hydrophobicsequences. The transmembrane glutamine atposition 17 was retained in this library becauseit plays a role both in homodimerization of E5monomers and in the interaction between the E5protein and the PDGF b receptor. Degenerateoligonucleotides were synthesized that encoded arandom mixture of Leu, Val, Ile, Met, and a lowlevel of Ser, Thr, Pro, and Ala. The leucine-richcomposition of the encoded amino acids waschosen to reflect the abundance of leucine in trans-membrane domains, and particularly in papilloma-virus E5 proteins. In addition, we excludedphenylalanine from the library, even though thereare conserved transmembrane phenylalanineresidues in the E5 proteins from variousfibropapillomaviruses,31 in an attempt to identifynovel sequence motifs able to interact with thePDGF b receptor transmembrane domain. The

908 PDGF Receptor-binding Transmembrane Proteins

oligonucleotides were converted to double-stranded form, amplified by PCR, and used toreplace the central portion of the wild-type E5gene in a retroviral vector.

Murine C127 fibroblasts, which expressendogenous PDGF b receptor, were infected withthe resulting library, and infected cells were incu-bated at confluence to allow focus formation(Figure 1(b)). We estimate that 1 £ 105 viruses werescreened for transforming activity, which representa small fraction of possible transmembranesequences, given the number of positions random-ized. Individual transformed foci were picked andexpanded, and transforming virus was recoveredby infecting transformed cells with a helper virusto supply viral replication functions. The rescuedvirus was used in a second round of infection ofnaıve C127 cells, and transformed cells were againselected. The retroviral inserts were amplifiedfrom genomic DNA isolated from transformedcells, subcloned, sequenced, and reintroduced intonaıve C127 cells to confirm their ability to inducefocus formation.

Sequence analysis of the library clones

We sequenced 45 randomly sampled, unselectedclones from the library, which were shown to benon-transforming. These unselected clones hadthe expected amino acid composition andappeared to have a random distribution of thepossible amino acids at each position (see

Figure 3). Approximately 10% of the randomizedlibrary clones induced foci. We recovered 37 trans-forming clones from the library screen, and fouradditional transforming clones were identifiedwithin the set of unselected clones. Figure 2 listsrepresentative transforming and non-transformingclones, and their relative focus-forming activity.Figure 1S in the Supplementary Material showsthe sequences of all of the transforming and non-transforming clones.

The great majority of randomized sequences didnot support transformation, but a wide variety ofsmall transmembrane proteins were capable oftransforming cells. Inspection of the sequences ofthe transforming clones revealed that transform-ation did not require the wild-type E5 amino acidat any randomized position. In fact, nearly anytested hydrophobic amino acid was tolerated ateach position within the transmembrane domain.We used Sequence Logo to graphically representthe transmembrane sequences of the non-trans-forming and transforming proteins (Figure 3). Inthe Sequence Logo graphs, the frequencies of theamino acids at each position are represented bythe relative heights of the letters. For the non-trans-forming proteins, the composition at each positionwas quite similar and reflected the expectedfrequency of each amino acid based on the designof the library. The transforming proteins werestrikingly different, with a bias toward a particularamino acid at several positions. For example,position 24 was strongly biased toward valine,and position 25 was strongly biased towardleucine.

We used a x2 statistical test to quantify the differ-ence in amino acid distribution at each positionbetween the transforming and non-transformingproteins. Figure 4 shows the difference score calcu-lated for each position (see Materials and Methodsfor a definition of difference score). At positions18, 20, 24, and 28, the difference score is greaterthan 1.3, corresponding to a greater than 95% prob-ability that the distribution of amino acids at thesepositions is different between the transformingand non-transforming proteins. When thesepositions were plotted on a helical wheel with 3.5residue periodicity, consistent with previousevidence that the E5 dimer transits the membraneas a left-handed coiled-coil,5,11 they all fell on oneside of the helix, overlapping with the proposedE5 homodimer interface (Figure 5).

Identification and testing of a consensussequence in the transforming proteins

We used a computational algorithm to identify aconsensus sequence that best discriminatedbetween the sequences of the transforming smalltransmembrane proteins and the sequences of thenon-transforming proteins. The optimal six-aminoacid residue sequence that met this criterion wasLeu18, Val20, Val24, Leu25, Val28, and Leu29.This consensus sequence has a t-score of 8.44,

Figure 1. (a) Library design. Fifteen transmembraneamino acids of the BPV E5 protein were replaced withrandom, predominantly hydrophobic sequencesencoded by NXR codons as described in Materials andMethods. A CAA codon encoding glutamine 17 wasmaintained. (b) Diagram depicting selection strategy.Rare foci arising after infection of C127 fibroblasts withthe randomized library were picked and expanded. Ahelper virus was used to rescue the transforming virus,which was then used to re-infect naıve C127 cells.Genomic DNA was recovered from resulting trans-formed cell lines, and the retroviral inserts wereamplified, subcloned, and sequenced.

PDGF Receptor-binding Transmembrane Proteins 909

Figure 2. Transmembranesequences of ten representativetransforming (top) and non-trans-forming proteins (bottom). The farright column lists the relativefocus-forming activity of the pro-teins. þþþþ Corresponds to.130% of wild-type E5 activity,þþþ to 70–130%, þþ to 30–70%,þ to 10–30%, and 2 to ,10% ofwild-type E5 activity. For eachprotein, the number of amino acidresidues that match the six-positionconsensus sequence is shown inthe column labeled CS.

Figure 3. Sequence logo46 representation of the trans-membrane sequence of 41 transforming (top) and 45non-transforming (bottom) proteins. The height of eachletter is proportional to the frequency of the amino acidat that position.

Figure 4. A graphical representation of the position-specific differences in amino acid distribution betweenthe transforming and non-transforming E5-like proteins.At each position, the difference score is defined as2 log10(Pi), where Pi is the probability that the two distri-butions are identical, as determined from the x2 value.


corresponding to a probability of less than 10215

that the transforming and non-transformingproteins have identical distributions of aminoacids at these six positions. Figure 6 is a histogramenumerating the number of positions at whicheach transforming and non-transforming proteinmatches the consensus. Greater than 70% of thetransforming proteins matched four or five of thesix consensus positions, and the matched positionsvaried from clone to clone. In contrast, less than10% of the non-transforming proteins matchedfour positions, and none matched more than four.There was no obvious correlation between thenumber of matches to the consensus and focus-forming activity in C127 cells (Figure 2 andSupplemental Figure 1S).

The consensus sequence identified above isdifferent from the sequence of the wild-type E5protein (Figure 7). In the wild-type sequence,there are phenylalanine residues at positions 23,

27, and 28, but the consensus sequence includedvaline residues at positions 20, 24, and 28. Notonly are two of the three positions of the non-leucine residues different in the phenylalanine-and valine-based motifs, but the chemical proper-ties of phenylalanine and valine are also quitedifferent. To determine if a simple transmembraneprotein containing the consensus sequence wascapable of inducing transformation, the transmem-brane domain of the E5 protein was replaced withthree sequences: (1) poly-leucine containing gluta-mine at position 17 (pL-Q), (2) poly-leucine withglutamine 17 and phenylalanine 23, 27, and 28(pL-QF), similar to the wild-type E5 sequence, and(3) poly-leucine with glutamine 17 and valine 20,24, and 28 (pL-QV), representing the consensussequence derived from the analysis of the activeproteins recovered from the library (Figure 7). ThepL-Q clone did not induce focus formation. Incontrast, either the phenylalanine or valine motifrestored efficient focus formation to the poly-leucine protein. Thus, both the phenylalaninemotif and the consensus valine motif supportedefficient cell transformation.

Productive interaction between the activetransmembrane proteins and the PDGFb receptor

To determine whether the transformation-competent small transmembrane proteins trans-formed cells by activating the PDGF b receptor,stable C127 cell lines expressing proteins recoveredfrom the library or the poly-leucine proteins wereanalyzed. All cell lines expressed similar levels ofthe endogenous PDGF b receptor (Figure 8(a) and(b), top panel). Cell extracts were also immuno-precipitated with an antibody specific for thePDGF b receptor and then immunoblotted with amonoclonal antibody that recognizes phosphotyro-sine (Figure 8(a), second panel). Wild-type E5 pro-tein (far right) induced marked phosphorylationof both the mature and precursor forms of thereceptor, whereas the empty vector control (C)showed weak background phosphorylation of themature form only. The mature form of the PDGF breceptor was phosphorylated significantly morethan the vector control in cells transformed by thesmall transmembrane proteins, but there was nosignificant increase in receptor phosphorylationabove background levels in the three non-trans-formed cell lines tested. The pL-Q protein inducedweak phosphorylation of the receptor, the pL-QVprotein induced significant phosphorylation, pre-dominantly on the mature form of the receptor,and the pL-QF protein induced dramatic phos-phorylation of both the mature and precursorforms of the receptor (Figure 8(b), second panel).

To determine if the small transmembraneproteins formed a stable complex with the PDGFb receptor, cell extracts were immunoprecipitatedwith an antibody that recognizes the C terminusof the E5 protein, a segment that was not

Figure 5. Helical wheel diagram of transmembraneprotein dimers. This representation assumes the sym-metric left-handed coiled-coil structure predicted for thewild-type E5 protein. The positions in bold, 18, 20, 24,and 28, are those positions where there is a greater than95% chance that the distribution of amino acids isdifferent between the transforming and non-transform-ing proteins. The most abundant amino acid in the trans-forming proteins is shown at these positions. Also shownin bold is the glutamine at position 17. The residues atvertices a and d form the proposed homodimer interface.

Figure 6. Analysis of the consensus sequence. Thehistogram shows the percentage of non-transforming(gray) and transforming (black) proteins that match theindicated number of positions in the consensus sequenceshown at the top.


randomized in the library construction. Theimmunoprecipitates were immunoblotted with anantibody specific for the PDGF b receptor. Thewild-type E5 protein and all tested transformingproteins, including the active poly-leucineproteins, formed a stable complex with both themature and immature forms of the PDGF breceptor, although the strength of the signal andthe relative abundance of the two receptor formsdiffered among the various transformed cell lines(Figure 8(a) and (b), third panel). However, thenon-transforming proteins recovered fromthe library failed to associate significantly with thereceptor. Interestingly, although the pL-Q proteinwas not capable of inducing transformation, itwas able to interact with the PDGF b receptor at alevel similar to the transforming pL-QV protein. Itis possible that the interaction between pL-Q andthe PDGF b receptor was aberrant, causing phos-phorylation to occur at a set of sites that were notsufficient to activate the downstream signalingcascades required for cellular transformation.

To test whether the non-transforming smalltransmembrane proteins fail to activate or associatewith the receptor because they are poorlyexpressed, cell extracts were immunoprecipitatedand immunoblotted with the E5 antibody. Thewild-type E5 protein and the transforming proteinswere highly expressed, as were the non-transform-ing LFC1-NT10 and LFC1-NT16 proteins(Figure 8(a), bottom panel, and data not shown).All three poly-leucine proteins were highlyexpressed (Figure 8(b), bottom panel), includingpL-Q, which is transformation-defective. The non-transforming proteins LFC1-NT12 and -NT18were expressed at a lower but detectable level,which presumably contributes to our inability todetect receptor activation and complex formation.

Role of the PDGF b receptor in transformationby the small transmembrane proteins

As a further test of the mechanism of transform-ation, stable C127 cell lines expressing the wild-type E5 protein or one of several transformingsmall transmembrane proteins were grown withand without the specific PDGF receptor kinaseinhibitor AG1295 (Figure 9, and data not shown).In the absence of AG1295, the wild-type E5 protein

Figure 7. Amino acid sequenceand transforming activity of poly-leucine-based proteins. The aminoacid sequence of the trans-membrane domain of the wild-typeE5 protein, the poly-leucine proteincontaining Gln17 (pL-Q), the poly-leucine protein with Gln17 and thephenylalanine motif (pL-QF), andthe poly-leucine protein with Gln17and the valine motif (pL-QV) are

shown. Non-leucine residues are bold. The column on the right lists the relative focus formation activity of each ofthe proteins, scored as in Figure 2.

Figure 8. Biochemical analysis of the small trans-membrane proteins. Cell extracts were immunoprecipi-tated and blotted with the indicated antibodies in orderto detect expression of the endogenous PDGF b receptor(top), receptor tyrosine phosphorylation (second panel),association between the small transmembrane proteinsand the PDGF b receptor (third panel), and expressionof the small transmembrane proteins (bottom). The anti-bodies used for immunoprecipitation (IP) and Westernblotting (blot) are indicated: E5, E5 C terminus; PR,PDGF b receptor; PY, phosphotyrosine. Bands corre-sponding to the mature (m) and precursor (p) forms ofthe receptor are indicated by the arrows on the right.(a) Analysis of cells expressing the indicated transform-ing proteins (LFC1-T), an empty vector control (C), theindicated non-transforming proteins (LFC1-NT), and thewild-type E5 protein (E5). (b) Analysis of cells expressingthe controls in (a) or the poly-leucine proteins with gluta-mine alone (pL-Q), or the valine (pL-QV) or phenyl-alanine (pL-QF) motif.


and each of six transforming proteins tested grewto much higher density and acquired a trans-formed appearance (LFC1-T8 is shown as a repre-sentative example). The transformed cells revertedto a non-transformed morphology in the presenceof AG1295, indicating that the transformed pheno-type was due to the activity of the PDGF breceptor. As expected, the drug had no effect onthe morphology of cell lines expressing an emptyretroviral vector (top panels), non-transformingsmall transmembrane proteins, or cells that weretransformed with the unrelated oncogenic neureceptor tyrosine kinase (bottom panels, and datanot shown). AG1295 also reverted the transformedphenotype induced by the poly-leucine-QV andpoly-leucine-QF proteins (data not shown).

We used a transient reporter gene assay tomeasure the ability of the small transmembraneproteins to signal through the PDGF b receptor,and to determine whether the active proteins inter-acted specifically with the PDGF b receptor trans-membrane domain. CV1 monkey kidney cells,which do not express endogenous PDGF b recep-tor, were co-transfected with four plasmids. Allcells received a constitutively expressed Renillaluciferase plasmid to control for transfectionefficiency and a reporter plasmid containing threetandem copies of the GAS element driving a fireflyluciferase reporter gene. PDGF b receptor

activation initiates the STAT signaling cascade,which activates the GAS elements, resulting inincreased expression of firefly luciferase. Inaddition, cells received an empty vector, RVY, oran RVY-based plasmid encoding either a smalltransmembrane protein from the library, the wild-type E5 protein, or the v-sis PDGF homolog, aswell as a plasmid encoding either the wild-typePDGF b receptor or a PDGF b receptor mutant inwhich the transmembrane threonine 513 has beenreplaced with a leucine. This mutation blocks theinteraction with the wild-type E5 protein.28

Normalized firefly luciferase activity wasmeasured two days after introduction of each com-bination of plasmids and was expressed as a per-centage of activity induced by co-expression of thewild-type E5 gene and the wild-type PDGF breceptor.

As shown in Figure 10, there was low basalactivity in cells co-transfected with RVY and aplasmid encoding either receptor. As expected, thewild-type E5 protein activated the wild-typereceptor but not the T513L mutant, whereas v-sisactivated both receptors. Similarly, the transform-ation-competent proteins LFC1-T1 and -T2activated the wild-type receptor but not the T513Lmutant receptor, indicating that they interactedwith the PDGF b receptor via its transmembranedomain to initiate signaling. The non-transformingproteins LFC1-NT10 and -NT18 did not activateeither receptor, confirming their lack of biologicalactivity. Taken together, the results presented inthis and the preceding section indicate that most,if not all, of the active small transmembrane pro-teins transformed cells by activating the PDGF breceptor via transmembrane interactions, whereasthe non-transforming proteins did not interactproductively with the PDGF b receptor.

Small transmembrane segments dimerizein membranes

The wild-type E5 protein must form a dimer tointeract with the PDGF b receptor and causecellular transformation. We used the TOXCATassay to assess the ability of the transmembranesegments recovered from the library to dimerizein membranes.32 This assay employs a chimericfusion protein in which the transmembranedomain of interest is inserted between theN-terminal DNA binding domain of ToxR, atranscriptional activator that functions in an oligo-merization-dependent fashion, and maltose-binding protein, which acts as a periplasmicanchor protein. The fusion protein is expressed inEscherichia coli together with a chloramphenicolacetyltransferase (CAT) reporter gene under thecontrol of a ToxR-responsive ctx promoter. Oligo-merization of the transmembrane domains withinthe bacterial inner membrane results in oligo-merization of the linked ToxR domain, transcrip-tional activation of the ctx promoter, and CATexpression. We chose this approach to monitor

Figure 9. Effect of the PDGF receptor kinase inhibitorAG1295 on transformation of C127 cells. Photo-micrographs of C127 cells stably expressing an emptyRVY vector, the wild-type E5 protein, the LFC1-T8 trans-forming protein, or the neu oncoprotein are shown inthe absence of AG1295 (left) and after four days oftreatment with the drug (right).


dimer formation because the level of CATexpression is proportional to the strength of thetransmembrane interaction. In addition, thisapproach allowed us to assess the intrinsic abilityof the transmembrane segments to oligomerize inthe absence of disulfide bond formation.

The transmembrane domains of the wild-type E5protein, a Q17L mutant which lacks transformingactivity and displays impaired dimerization,8 twotransforming proteins recovered from the library(LFC1-T8 and LFC1-T14), and three non-transforming proteins (LFC1-NT10, LFC1-NT12,and LFC1-NT18) were cloned into the TOXCATfusion construct. TOXCAT constructs containingtransmembrane domains of wild-type dimeriza-

tion-competent glycophorin A (GpA) and itsdimerization-defective mutant, G83I, were used ascontrols, and the CAT activities for all constructswere normalized to the values obtained for GpA.The TOXCAT chimera containing the trans-membrane domain of the wild-type E5 proteinresulted in high CAT activity (Figure 11), consistentwith published results,33 indicating that the E5transmembrane domain is capable of strong self-association in this system. Mutating Gln17 toleucine resulted in a 60% reduction in CAT activity,confirming the importance of Gln17 in E5 dimerformation. The chimeras containing the trans-membrane domains of the two transforming andthree non-transforming proteins all yielded similarCAT activity, comparable to that induced by thewild-type E5 transmembrane domain. Theseresults indicate that the transmembrane domainsof both the transforming and the non-transformingproteins are capable of forming strong homo-oligomers, presumably dimers, in membranes.

Structural models of transmembrane dimers

To determine whether the proteins recoveredfrom the library were capable of forming dimerswith structures similar to that proposed for thewild-type E5 protein, we generated structuralmodels of the wild-type E5 protein and several ofthe recovered proteins. Structural calculationswere carried out using the CNS searching of helixinteractions (CHI) method from the crystallo-graphic and NMR system (CNS).34 – 36

CHI simulations were performed on two parallela-helices containing the transmembrane sequenceof interest. The dimer models indicated that all ofthe proteins analyzed can form plausible left-handed coiled-coils. A model of the wild-type E5protein, in good agreement with previously

Figure 10. Activity of small trans-membrane proteins requires thePDGF b receptor transmembranedomain. The graph shows therelative firefly luciferase activityinduced by the wild-type E5 pro-tein (wt), an empty vector control(RVY), a viral PDGF homolog(v-sis), and two transforming andnon-transforming library proteinsco-expressed with the wild-typePDGF b receptor (gray) or theT513L mutant receptor (white). Theactivity of each combination isexpressed as a percentage of theactivity induced by co-transfectionof the wild-type E5 gene and thewild-type PDGF b receptor. Foreach experiment, three replicates ofeach transfection combination wereperformed. The average of two orthree such experiments is shown,with error bars representing thestandard deviation.

Figure 11. TOXCAT assay to determine the oligo-merization capacity of the small transmembrane proteinsin membranes. The relative CAT activity is shown for thewild-type E5 transmembrane domain as well as thetransmembrane domain of the dimerization-impairedE5 Q17L mutant, the indicated transforming proteins(LFC1-T), and the indicated non-transforming proteins(LFC1-NT). As controls, the CAT activity was deter-mined for the glycophorin A (GpA) protein and itsdimerization-defective G83I mutant, and the CATactivities for all constructs were normalized to the valuesobtained for GpA. The average of four or more indepen-dent experiments is shown with error bars.


published results,5 is shown together with modelsof the small transmembrane proteins in Figure 12.There are striking similarities between the modelsof the wild-type E5 protein (a), the smalltransforming proteins that closely conform to theconsensus sequence, namely LFC1-T14 (b) andLFC1-T8 (not shown), and the active poly-leucineproteins (not shown). In all of these models, thedimer interface consisted of well-packed residuesat positions 14, 21, 24 and 28 as well as the gluta-mine at position 17. Based on this analysis, all ofthe interfacial residues contributed to stabilizationof the dimer, even though many of these residuesin the small transmembrane proteins differed fromthe corresponding residues in the wild-type E5protein. Figure 12 also shows that the active pro-teins contained likely interhelical hydrogen bondsinvolving glutamine 17, defined in this analysiswhenever a hydrogen bond donor and acceptorwere within 3 A of one another. A model of anLFC1-T2 dimer was also generated (Figure 12(c)),because this transforming protein poorly matchedthe consensus sequence. Interestingly, the LFC1-T2dimer had a different dimer interface, containingresidues at positions 13, 20, 24 and 27, and yet stillcontained the critical glutamine 17 residue.Although glutamine 17 was present at the dimerinterface in this model, it did not appear to partici-pate in interhelical hydrogen bonding.

Models were also generated for three non-transforming proteins, LFC1-NT10 and -NT12 (notshown) and LFC1-NT18 (Figure 12(d)). The LFC1-NT10 and -NT12 proteins dimerized with an inter-face very similar to the wild-type E5 protein,while the interface of the LFC1-NT18 dimer wascompletely divergent, lacking even glutamine 17.

Furthermore, neither LFC1-NT10 nor -NT18displayed evidence of interhelical hydrogen bonds.

We have proposed that aspartic acid 33 of the E5protein forms an essential salt-bridge with lysine499 in the PDGF b receptor.7 In the wild-type E5protein and all of the transforming proteinsmodeled, the side-chains of both aspartic acid 33residues pointed away from the dimer interfaceand were presumably accessible to interact withthe lysine of the receptor (Figure 12(a)–(c)). In con-trast, the dimer model of the non-transformingLFC1-NT18 protein shows aspartic acid 33 pointedtoward the dimer interface, preventing any favor-able interactions with the PDGF b receptor lysine499 (Figure 12(d)). Without the crucial interactionsbetween aspartic acid 33 and lysine 499, bindingof the LFC1-NT18 protein to the PDGF b receptormay be greatly reduced, consistent with itsinability to co-immunoprecipitate with the PDGFb receptor.

Discussion

Here, we selected and characterized novel, trans-formation-competent proteins from a library ofsmall proteins with random transmembranedomains. The inability of 90% of the random clonesto induce transformation indicates that there areimportant sequence constraints on activity. Never-theless, it is striking that so many differentsequences were compatible with transformation,even though fully one-third of the E5 protein wasrandomized. In fact, nearly any tested amino acidwas tolerated at each of the 15 randomized pos-itions within the transmembrane domain. Despite

Figure 12. Structural models of transmembrane dimers. CHI structural models of protein dimers are shown for thewild-type E5 protein, the LFC1-T14 and LFC1-T2 transforming proteins, and the LFC1-NT18 non-transforming protein.The residues predicted to lie in the interface of the protein dimers are colored and labeled, as is Asp33, and likely inter-helical hydrogen bonds are designated by dotted lines.


the diversity of active transmembrane sequences,all tested transforming proteins bound andactivated the PDGF b receptor via transmembraneinteractions and required receptor activity fortransformation and signaling. This sequencedegeneracy suggests a remarkable flexibility andfew absolute sequence requirements for recog-nition and activation of the PDGF b receptor.

Glutamine 17, aspartic acid 33, and theC-terminal cysteine residues were fixed in thelibrary. Thus, the specific residues known to berequired for E5 dimerization and PDGF b receptorbinding were present in all of the library clones,biasing any potential interactions toward thePDGF b receptor. We have not ruled out thepossibility that a subset of the small trans-membrane proteins interacts with other cellulartransmembrane proteins.

Statistical analysis revealed significant differ-ences between the amino acid residues present atspecific positions in the transforming compared tothe non-transforming transmembrane segments.These positions fall on one side of a left-handedcoiled-coil helical wheel, overlapping with theputative dimer interface, and they may form asecond interface on the transmembrane dimer thatis critical for an essential aspect of transformation,such as interaction with the PDGF b receptor. Ouranalysis also identified a consensus sequence thatdiscriminates strongly between the transformingand non-transforming proteins. However, some ofthe small transmembrane proteins transformedpoorly or not at all even though they conformedwell to the consensus, implying that there areadditional features of the transmembrane domainthat modulate transforming activity. The positionand identity of non-leucine residues in the con-sensus sequence were different from those of thewild-type E5 protein, as demanded by ouromission of phenylalanine residues from therandom segment. Nevertheless, a similar level oftransforming activity was conferred on a poly-leucine protein by both the valine motif identifiedas the consensus sequence and the phenylalaninemotif present in the wild-type E5 protein. Further-more, we recovered rare transforming proteinsthat differed from both the wild-type E5 proteinand the consensus sequence. Thus, multiple,different transmembrane sequence motifs arecapable of inducing transformation via activationof the PDGF b receptor.

Despite their divergent sequences, the trans-membrane domains of the random proteinsoligomerize in cell membranes. Dimerization ispresumably largely due to the presence of gluta-mine 17 in the dimer interface, because studieswith model peptides show that polar amino acidssuch as glutamine are able to induce dimerizationin hydrophobic environments, due to the for-mation of strong interhelical hydrogen bonds.37 – 40

In addition, the C-terminal cysteine residuespresumably favor disulfide bond-mediated dimerformation in mammalian cells and tend to align

the transmembrane monomers in the sameorientation as in the wild-type E5 dimer.

The CHI program was used to model thedimeric structure of the novel transmembranesegments. Two transforming proteins that were agood match to the consensus sequence (LFC1-T8and LFC1-T14) as well as the active poly-leucineproteins were predicted to form dimers with thesame interfacial positions as the wild-type E5protein.5,11 This interface allows glutamine 17 toform interhelical hydrogen bonds within the E5dimer as well as to the PDGF b receptor, and itorients aspartic acid 33 away from the homodimerinterface so that it is available to interact withlysine 499 of the PDGF b receptor. In contrast, thepredicted dimer interface of the transformation-competent LFC1-T2 protein, which is a poormatch to the consensus sequence, is shifted by onerotational position relative to the wild-type E5interface. Although the active homodimers appearto adopt similar overall structures, different aminoacids constitute the homodimer interface in thetransforming proteins. A variety of amino acidsequences are also tolerated outside of the homo-dimer interface, and some of these amino acidsmust contact the PDGF b receptor. These resultssuggest that both the homodimer interface and theinterface with the PDGF b receptor can accommo-date diverse sequences, implying that there is con-siderable sequence flexibility in these interactions.In fact, the nature of the interaction between someof the transforming proteins and the PDGF breceptor may be very different from the interactionwith the wild-type E5 protein. For example, asingle monomer of the LFC1-T2 protein contributesboth glutamine 17 and aspartic acid 33 to the sameface of the dimer (A.M.D., unpublished results),whereas each of the two intertwined monomers ofthe wild-type E5 coiled-coil contributes one ofthese residues to each face of the dimer.5

In order to transform cells, the small trans-membrane proteins must accumulate in cells, formstable homodimers, localize properly, and bind tothe PDGF b receptor to induce receptor dimeriza-tion in the proper orientation. It has been shownfor other receptor tyrosine kinases that the properorientation of receptor monomers within thedimer is essential for trans-phosphorylation andinitiation of the signaling cascade.41,42 The trans-membrane domains of the non-transforming smalltransmembrane proteins may lack specific residuesthat directly interact with the PDGF b receptor.Alternatively, some of the non-transformingproteins may not be expressed at a sufficient levelor may not form stable homodimers in mammaliancells able to interact productively with the receptor.

In summary, we have developed a geneticmethod to identify small transmembrane proteinsthat bind and activate the PDGF b receptor, result-ing in cell transformation. A surprisingly largefraction of transmembrane sequences are active inthis assay. This approach may not only help todefine the rules of engagement with the


transmembrane domain of the PDGF b receptor,but may also provide an approach to modulatethe activity of a variety of transmembrane proteins.For example, variations of this method may be use-ful in constructing and identifying small trans-membrane proteins that activate other receptortyrosine kinases. Receptor tyrosine kinases areessential for growth and development, andaberrant activation of these kinases is associatedwith many human diseases, including cancers.Thus, a general strategy to modulate receptoractivity and the oligomerization state of trans-membrane proteins is of great interest.

Materials and Methods

Library construction

A PCR-based approach was used to construct a libraryof small transmembrane proteins. The upstream 50

degenerate oligonucleotide had a fixed 50 end derivedfrom the E5 sequence, including a Spe I restrictionenzyme site, followed by three NXR codons where N isan equal mix of A, T, C, and G, X is a 5 : 0.1 mixture ofT : C, and R is an equal mix of A and G; a CAA codonencoding glutamine; 12 more NXR codons; and finally afixed 30end derived from the E5 sequence (Figure 1(a)).The downstream oligonucleotide was non-degenerateand contained a Bam HI restriction enzyme site at the 50

end followed by the antisense sequence of the 30 end ofthe wild-type E5 gene. The 30 end of this primer wascomplementary to the 30 end of the upstream primer.The two oligonucleotides were annealed and extendedto create double-stranded products with a randomizedcentral sequence. Two short primers that annealed tothe extreme fixed ends of the double-stranded productswere then used to amplify the library for 30 cycles. ThePCR products were purified, digested with Spe I andBam HI, and subcloned into the RVY retroviral vector togenerate a library of genes in which 15 transmembranecodons of the E5 gene were replaced with a randommix of L : V : I : M : S : T : A : P codons in the followingexpected ratio 40 : 20 : 10 : 10 : 2 : 2 : 2 : 2. Cloning detailsare available from the authors upon request.

Cell lines and tissue culture

The retroviral packaging cell line, helper virus pro-ducer line, C127, and CV1 cells were maintained inDulbecco’s modified Eagle’s medium supplementedwith 10% (v/v) fetal bovine serum and antibiotics(DMEM-10). Retroviral stocks were prepared in GP2-293cells as described.43 To assay focus-forming activity,C127 cells were infected with retrovirus, and the numberof foci was counted three weeks after infection. Inparallel, infected cells were selected in medium contain-ing 300 mg/ml of hygromycin B. Focus formationefficiency was calculated by normalizing the number offoci to the number of hygromycin B-resistant coloniesarising in the same infection. Cell lines were establishedfrom pools of drug-resistant colonies.

Helper virus rescue of transforming library clones

Helper virus was harvested from NIH 3T3 TK-cellstransfected with a pMLV-K plasmid construct to produce

Moloney MLV (obtained from A. D. Miller, FredHutchinson Cancer Research Center).44 Individual C127foci were isolated using cloning cylinders, transferredinto 24 well plates, and after 24 hours were infectedwith helper virus in medium with 4 mg/ml of Polybrene.The media was changed to DMEM-10 with 4 mg/ml ofpolybrene after four to five hours, and approximately16 hours later the infected cells were passed into T25flasks in 5 ml DMEM-10 with 4 mg/ml of polybrene.After four days, the supernatant was harvested, filteredthrough a 0.45 mm syringe filter, and used to infectnaıve C127 cells as described.8

Recovery of transforming library clones

Hygromycin-resistant colonies that grew after infec-tion of C127 cells with the rescued virus were pooledand expanded, and genomic DNA was harvested byusing a DNeasy kit (Qiagen). A Hot-Start PCR protocolwith rTthXL polymerase (Applied Biosystems) wasused essentially as described45 to amplify the retroviralinsert. Briefly, the PCR mixture containing the polymer-ase, 1 mg of genomic DNA, and primers flanking the ran-domized segment was preheated to 80 8C for 60 secondsprior to addition of magnesium acetate at a final concen-tration of 1.0 mM. We used 75 cycles of amplification,rapidly cooling from the 93 8C denaturing step to 70 8C,before slowly cooling to the extension temperature of60 8C. The cycling parameters and preheating reducednon-specific priming. In some cases, nested PCR wasperformed using primers internal to the primers used inthe Hot-Start reaction. Detailed PCR protocols are avail-able from the authors upon request. The PCR productswere digested with Spe I and Bam HI and subclonedback into a retroviral vector to generate individualgenes encoding the random transmembrane sequences.The sequence of the complete DNA insert was obtainedfor each clone.

PDGF b receptor inhibitor studies

C127 cells were plated at ,70% confluence in 12 wellplates and grown in the presence or absence of 50 mMof AG1295 (Calbiochem) in DMEM-10. Cells were photo-graphed after four days in the presence of the PDGFreceptor inhibitor or in its absence.

Biochemical analysis

Cell lines at near confluence were serum-starved for24 hours. Cell extracts were made using modified RIPAbuffer containing protease and phosphatase inhibitorsas described.18 Immunoprecipitation and immuno-blotting were carried out essentially as described.8,18 Foranti-phosphotyrosine blots, a 1 : 500 dilution of P-Tyr-100 (Cell Signaling Technology) in 5% (w/v) BSA inTBST was used.

Transient assay for PDGF receptor signaling

2 £ 105 CV1 cells were plated into six well plates.Approximately 16 hours later, the cells were transfectedwith 10 ng of the pRL-SV40 Renilla luciferase plasmid,750 ng of the pGAS-luciferase reporter plasmid (obtainedfrom X. -Y. Fu, Yale University), 0.5 mg of an LXSN-basedplasmid encoding the wild-type or mutant PDGF breceptor, and 0.5 mg of an RVY-based plasmid encodingthe E5 protein, a small transmembrane protein from the


library, or v-sis. The DNA was added to 791 ml of Opti-MEM (Gibco) and 9 ml of 2 mg/ml lipofectamine(Invitrogen). The transfection mix was incubated atroom temperature for 20 minutes before adding it to thecells that had been washed twice with 1 ml of PBS andonce with 1 ml of Opti-MEM. After five hours, themedium was changed to 3 ml DMEM-10. After 24hours, the cells were incubated in medium withoutserum supplementation in order to decrease backgroundactivation of the PDGF receptor, and the cells wereharvested 24 hours later. A dual-luciferase assay wascarried out using the dual-luciferase reporter assaysystem (Promega), according to the manufacturer’sprotocol. Samples were read using a Turner DesignsTD-20/20 luminometer. The transfections were done intriplicate, and the results were normalized to theexpression of the Renilla luciferase transfection control.

Computational analysis of E5-like sequences

The sequence logo46 representation was created usingWebLogo†.47 At each position, the size of each letter isproportional to the frequency of the correspondingamino acid in that position. The total height of all theletters in the position is proportional to the conservationof the position.

To quantify the difference in amino acid distributionsbetween the transforming and non-transforming clones,we calculated a x2 value for each position after groupingthe amino acid residues into five categories: L (Leu), M(Met), V (Val), I (Ile), and O (Others).48 We then per-formed a chi-square test with four degrees of freedom(one less than the number of amino acid categories),and we derived a difference score for each position,where the difference score is the -log (probability of thedistributions being identical). Details are given in theSupplementary Material. The ranking of all positionswas the same when the difference score was computedby using other methods based on information theory,such as the Jensen-Shannon divergence (data notshown). We also developed an automated procedure toidentify an optimal consensus sequence. For each trialconsensus sequence, a similarity score was computedfor each clone by counting the number of matchesbetween the consensus sequence and the clone. Thisresulted in two sets of similarity scores: {xA} for trans-forming clones and {xB} for non-transforming clones.The performance of the trial consensus sequence wasthen evaluated by the t-score, a measure of the separ-ation of the means between these two sets of scores.48

An iterated simulated annealing procedure was thenused to find consensus sequences with optimalperformance.49 The details of the method are presentedin the Supplementary Material.

Construction and characterization of TOXCATchimera and CAT assays

Double-stranded oligonucleotides encoding the trans-membrane domains (residues L8–H34) of the wild-typeE5 protein, the E5 Q17L mutant, and the library proteinswere ligated into the Nhe I and Bam HI sites in thepccKAN plasmid.32 The resulting plasmids wereexpressed in E. coli, and equivalent expression levels ofthe chimeric proteins were verified by Western blotting.

Membrane insertion of the chimeric proteins was veri-fied using 0.1 M NaOH washes.50 Whole cell, membrane,and soluble fractions were electrophoresed on SDS-PAGE, followed by Western analysis using antibodiesagainst maltose binding protein (MBP) (New EnglandBiolabs). The correct orientation of the TOXCATconstructs in the membrane was confirmed throughprotease sensitivity in a spheroplast assay, describedelsewhere.32 Acetylation of chloramphenicol by bacteriallysates was measured in an enzymatic assay by bio-chemical fractionation and scintillation counting (seeSupplementary Material for details).

Computational searches using CHI

Details of the CHI computational search method havebeen described.34,35,36 All structural calculations were car-ried out in vacuo on a Silicon Graphics O2 workstation.Using CHI, two canonical a-helices containing residues8–34 of BPV E5 and its mutants were built. The startinggeometries incorporated both right-handed (2258) andleft-handed (þ258) crossing angles and a distancebetween the two helices ranging from 10.5–11.0 A. In afull search of all pairwise interactions, the two heliceswere independently rotated about their central axis in458 increments from 08 to 3608. Molecular dynamics(MD) simulations were performed using simulatedannealing of atomic coordinates at each pair of rotations.Four different MD simulations were performed for eachstarting geometry. Energy minimization of structureswas carried out both before and after MD simulations,and a total of 512 structures were obtained from eachsearch. Groups of ten or more final structures with abackbone RMSD of 1 A or less were placed into clusters,followed by calculation of an average structure for eachcluster and energy minimization.

Acknowledgements

We thank Joshua Drumm and Kristin Yates forassistance recovering and testing library clones;Inigo San Gil of the Yale core facility for bio-informatics for help with initial sequence analysis;Louis Alexander for assistance with PCR protocols;Dusty Miller and Xin-Yuan Fu for essentialreagents; Dawn Mattoon for critical reading of themanuscript; and Jan Zulkeski for assistance inpreparation of the manuscript. L.L.F.-C. wassupported by an Anna Fuller postdoctoral fellow-ship and an NIH postdoctoral training grant(CA09159). A.M.D. was supported by a post-doctoral fellowship from the American CancerSociety (PFCSM-0301701). J.B.F. was supported byan NIH predoctoral training grant (GM07499).D.M.E., Y.X. and M.G. were supported by a grantfrom the NIH (P01 GM054160). This work wassupported by a grant from the National CancerInstitute to D.D. (CA37157).

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Edited by G. von Heijne

(Received 6 February 2004; received in revised form13 March 2004; accepted 13 March 2004)

Supplementary Material comprising analysis ofclones, CAT assays and one Figure is available onScience Direct


selection and characterization of small random transmembrane...

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