Unmodified Prolactin (PRL) and S179D PRL-Initiated Bioluminescence Resonance Energy Transfer between...

Unmodified Prolactin (PRL) and S179D PRL-InitiatedBioluminescence Resonance Energy Transferbetween Homo- and Hetero-Pairs of Long and ShortHuman PRL Receptors in Living Human Cells

Dunyong Tan, David A. Johnson, Wei Wu, Lingfang Zeng, Yen Hao Chen, Wen Y. Chen,Barbara K. Vonderhaar, and Ameae M. Walker

Division of Biomedical Sciences (D.T., D.A.J., W.W., L.Z., Y.H.C., A.M.W.), University of California,Riverside, Riverside, California 92521-0121; Mammary Biology and Tumorigenesis Laboratory(B.K.V.), National Cancer Institute, Bethesda, Maryland 20892; and Oncology Research Institute(W.Y.C.), Clemson University, Greenville, South Carolina 29605

We have used bioluminescence resonance energytransfer (BRET) to examine the interaction betweenhuman prolactins (PRLs) and the long (LF) and twoshort isoforms (SF1a and SF1b) of the human PRLreceptor in living cells. cDNA sequences encodingthe LF, SF1a, and SF1b were subcloned intocodon-humanized vectors containing cDNAs foreither Renilla reniformis luciferase (Rluc) or agreen fluorescent protein (GFP2) with a 12- or 13-amino acid linker connecting the parts of the fusionproteins. Transfection into human embryonic kid-ney 293 cells demonstrated maintained function ofRluc and GFP2 when linked to the receptors, andconfocal microscopy demonstrated the localiza-tion of tagged receptors in the plasma membraneby 48 h after transfection. All three tagged recep-tors transduced a signal, with the LF and SF1astimulating, and SF1b inhibiting, promoter activity

of an approximately 2.4-kb �-casein-luc con-struct. Both unmodified PRL (U-PRL) and the mo-lecular mimic of phosphorylated PRL, S179DPRL, induced BRET with all combinations of longand short receptor isoforms except SF1a plusSF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the firstdemonstration, 1) that species homologous PRLpromotes both homo- and hetero-interaction ofmost long and short PRLR pairs in living cells, 2)that both U-PRL and S179D PRL are active in thisregard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular het-ero-receptor pairing. In addition, we concludethat preferential pairing of different receptor iso-forms is not the explanation for the differentsignaling initiated by U-PRL and S179D PRL.(Molecular Endocrinology 19: 1291–1303, 2005)

PRLRs [PROLACTIN (PRL) RECEPTORS] are mem-bers of the class I cytokine superfamily and, as

such, share a number of structural features with moreclassical cytokine receptors (1–3). The structural fea-tures of this superfamily include: 1) all are single trans-membrane domain receptors that associate with ef-fector kinases rather than having intrinsic kinaseactivity and 2) homo- or hetero-receptor isoform mul-timerization is thought to be initiated by ligand binding,which in turn leads to transphosphorylation of the

associated kinase and initiation of several intracellularsignaling cascades (4, 5). For the PRLR, dimerizationis considered sufficient to initiate signaling (3). Thismodel of dimerization activation of the PRLR was orig-inally developed by analogy to closely related hor-mones and is supported by studies showing the for-mation of a ternary complex between a relatedhormone, ovine placental lactogen, and two copies ofthe extracellular domain (ECD) of the rat PRLR (6),functional analysis of mutations in PRL (7), solutionanalysis of species heterologous PRL-PRLR-ECD in-teractions (8), ligand-receptor cross-linking studies (9)and, most recently, the demonstration of placentallactogen-induced heterodimerization of PRLRs withGH receptors using fluorescence resonance energytransfer (10). To date, however, there is no evidencefor species-homologous PRL-induced PRLR dimer-ization because all attempts to demonstrate this havefailed. The ability of species heterologous, but nothomologous PRL, to cause measurable dimerizationhas been attributed to a more stable interaction withthe second receptor (11). Thus, species heterologousinteractions may not be representative of species ho-

First Published Online February 3, 2005Abbreviations: B/F, Bioluminescence/fluorescence; BRET,

bioluminescence resonance energy transfer; DPBS, Dulbec-co’s PBS; ECD, extracellular domain; GFP, green fluorescentprotein; HEK293, human embryonic kidney 293 cells; LF,long isoform of the human PRLR in living cells; PRL, prolactin;PRLR, PRL receptor; Rluc, Renilla reniformis luciferase; SF1aand SF1b, short isoforms of the human PRLR in living cells;Stat, of signal transducer and activator of transcription; U-PRL, unmodified PRL.

Molecular Endocrinology is published monthly by TheEndocrine Society (http://www.endo-society.org), theforemost professional society serving the endocrinecommunity.

0888-8809/05/$15.00/0 Molecular Endocrinology 19(5):1291–1303Printed in U.S.A. Copyright © 2005 by The Endocrine Society

doi: 10.1210/me.2004-0304

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mologous ones. In this regard, a number of mutations,which have no influence on biological effect generatedin heterologous ligand-receptor interactions, havebeen demonstrated to have marked effects in homol-ogous ligand-receptor interactions (12). Likewise, bi-ological activity in a heterologous system can be dif-ferent from that in a homologous system (12, 13). Fromthe work published to date, therefore, it remains pos-sible that dimerization is an artifact of either the use ofspecies heterologous PRL or the use of cross-linkingand immunoprecipitation protocols.

Alternatively spliced PRLRs, possessing differentcytoplasmic tails, have been recognized in rodent spe-cies for some time (11, 14) but have only recently beencloned from normal human tissue. In the case of hu-man PRLR, two short isoforms have been identified(15, 16), which are in addition to a long form and anintermediate form cloned from human breast cancercells (17, 18). The designations as long, intermediate,and short isoforms are a little confusing when referringto the human receptors, because one of the shortforms is actually longer than the intermediate form.Thus, the short 1a (SF1a) isoform is 352 amino acids,the short 1b (SF1b) isoform is 264 amino acids, theintermediate form is 325 amino acids, and the longisoform (LF) is 598 amino acids (15, 16). In addition tothe question of whether hormone-induced dimeriza-tion occurs with species homologous PRL and PRLRs,it is unclear whether human PRL induces pairing of allcombinations of PRLR isoforms, i.e. homo- and het-ero-pairs of receptors. Also unclear is whether bothunmodified PRL (U-PRL) and a molecular mimic ofphosphorylated PRL (S179D PRL) cause the samehomo- and hetero-pair interactions because these two

forms of human PRL initiate different intracellular sig-nals (19). To address these issues, we assessed theability of human PRL forms to induce a biolumines-cence resonance energy transfer (BRET) signal usingvarious combinations of the LF and SF1a and SF1bisoforms of human PRLRs. Chimers of Renilla renifor-mis luciferase (Rluc) or a variant of green fluorescentprotein (GFP2) linked to the LF, SF1a, and SF1b iso-forms of the PRLR were engineered. We found specieshomologous PRL-dependent BRET as a result of ho-mo- and hetero-pairing of all receptor isoform pairsexcept the SF1a-SF1b hetero-pair in living cells. Thisoccurred with both U-PRL and S179D PRL, indicatingthat pairing of specific receptors is not what producesthe different signaling activities of these two formsof the hormone. Additionally, no pairing occurred withthe mutant PRL, G129R, engineered to eliminate bind-ing to the second PRLR (20), demonstrating that eachPRL molecule must interact with two receptors toinduce BRET.

RESULTS

Biological Characterization of Rluc- and GFP2-Tagged PRLRs in Response to U-PRL

To assess the ability of the cells transfected with theRluc- and GFP2-tagged receptors (Fig. 1) to produce aPRL-induced intracellular signal, U-PRL-dependentactivation of the �-casein gene promoter was mea-sured with a �-casein promoter-luciferase assay. Fig-ure 2 (upper panel), shows that the �-casein luciferaseactivity was significantly (P � 0.05) increased in cells

Fig. 1. Vector Map for Rluc and GFP2 Receptor ConstructscDNA sequences encoding LF, SF1a, and SF1b were PCR-amplified from original plasmid, PEF6-LF, PEF4-SF1a, and

pCR2.1–1b, respectively, by using forward and reverse primers harboring unique MluI and KpnI sites and subcloned into acodon-humanized expression vector containing cDNAs for either the bioluminescent Rluc protein or GFP2 such that a 12- or13-amino acid linker would occur between the two parts of the resultant fusion proteins. The amino acid sequence of the linkerin the GFP2- and Rluc-tagged receptor are Trp-Tyr-Arg-Gly-Pro-Gly-Ile-Pro-Pro-Val-Ala-Thr and Trp-Tyr-Arg-Gly-Pro-Gly-Ile-Pro-Pro-Ala-Arg-Ala-Thr, respectively. The sizes were 6171 bp for LF-GFP2, 5443 bp for SF1a-GFP2, 5178 bp for SF1b-GFP2,6810 bp for LF-Rluc, 6082 bp for SF1a-Rluc, and 5817 bp for SF1b-Rluc. SV40, Simian virus 40; Zeo, zeocin; Kan, kanamycin;amp, ampicillin; cmv, cytomegalovirus; TK, thymidine kinase; Mlu I, restriction enzyme from Micrococcus luteus; Kpn I, restrictionenzyme from Klebsiella pneumoniae.

1292 Mol Endocrinol, May 2005, 19(5):1291–1303 Tan et al. • Homo- and Hetero-Pairing of PRLR Isoforms

cotransfected with the construct combinations of LF-GFP2/�-casein-luciferase and SF1a-GFP2/�-casein-luciferase. The relative luciferase activity units wereincreased from 3.8 � 0.7 � 103 in the control to 15.4 �1.3 � 103 and 6.6 � 0.3 � 103, respectively, demon-strating that receptors tagged with GFP2 are capableof transducing a signal and are therefore functional. Inthe cells cotransfected with SF1b-GFP2 and �-casein-luciferase, the relative luciferase activity units weredecreased from the control value to 2.8 � 0.4 � 103,indicating, in agreement with our previously publishedobservation (15), that SF1b acts as a negative regula-tor of baseline �-casein activity. Similar results wereobserved in the cells cotransfected with LF-Rluc and

�-casein-luciferase, SF1a-Rluc and �-casein-lucif-erase and SF1b-Rluc and �-casein-luciferase (Fig. 2,lower panel). The ability of the SF1a form to increasepromoter activity was not due to the attachment ofRluc or GFP because it also occurred with receptorswithout attached fusion proteins (data not shown).There is no way to accurately determine the activity ofhetero-pairs because transfection with two forms ofthe receptor does not guarantee that the observedsignal derives from heterodimerization. Instead, itcould arise from the mean of multiple homodimeriza-tion and heterodimerization events. Bearing in mindthis caveat, however, one can observe a reduced sig-nal to �-casein luciferase in cells cotransfected withthe LF-GFP2 and either SF1a-GFP2 or SF1b-GFP2 vs.those transfected with only the LF-GFP2 (Fig. 2, upperpanel). This occurs for the LF-SF1a pair despite thefact that the SF1a-GFP2 form alone signals weakly to�-casein. Cotransfection of SF1a-GFP2 and SF1b-GFP2 produced no increase in �-casein promoter ac-tivity in response to U-PRL (data not shown). Inclusionof a control vector normalized for total DNA trans-fected. This vector coded for a nonspecific protein andtherefore also controlled for levels of competing pro-tein expression.

Cellular Expression of the GFP2 and Rluc-Tagged PRLR

The cells were cotransfected with equimolar amountsof cDNA in various combinations of GFP2- and Rluc-tagged receptors. A GFP2-Rluc fusion construct, inwhich the GFP2 protein was directly linked to the Rlucprotein, once expressed, was used as a control fromwhich a reference bioluminescence/fluorescence (B/F)ratio, indicative of equimolar expression of GFP2 andRluc, was derived. For all experiments shown in Fig. 3,the longer-lived and more economical substrate ofRluc, coelenterazine h, was used for analyses. Lumi-nescence peaks at 475 with this substrate. Coelen-terazine h is suitable for this analysis, but because ofits spectral properties is less suitable for the BRETanalyses presented (see Figs. 6–8 and 10) for whichthe DeepBlueC (PerkinElmer, Wellesley, MA) substratewas used. It should also be noted that the spectralproperties of GFP2, with excitation at 405 nm and peakemission at 500–520 nm, are different from those ofthe more commonly used EGFP. As shown in Fig. 3,both the fluorescence (Fig. 3A) and bioluminescence(Fig. 3B) intensities of GFP2- and Rluc-tagged recep-tors were lower than that of GFP2-Rluc, indicating ahigher expression level of the cytoplasmically ex-pressed control construct relative to any of the PRLRreceptors. The peak fluorescence intensities for GFP2-Rluc, LF-GFP2/LF-Rluc, SF1a-GFP2/SF1a-Rluc, andSF1b-GFP2/SF1b-Rluc transfected cells were 1.8 �107, 1.1 � 107, 0.75 � 107 and 1 � 107, respectively,and the bioluminescence peak intensities were 13.6 �103, 9.2 � 103, 4.7 � 103, and 7.2 � 103, respectively,resulting in similar ratios of B/F for all the construct

Fig. 2. Ability of the GFP2- and Rluc-Tagged Receptors toActivate �-Casein Promoter in Response to U-PRL

HEK 293 cells were cotransfected with fusion constructs(GFP2-PRLRs or pRluc-PRLRs) and �-casein-luc, which con-tains a �-casein gene promoter and a luciferase coding cDNAsequence. Twenty-four hours after transfection, the mediumwas changed to DMEM without serum containing 45.7 nM

(final concentration) of U-PRL. After a further 24 h, the cellswere washed with DPBS three times, lysed with the reporterlysis buffer and the luciferase activity was measured using aMonolight 2010 Luminometer (Analytical Luminescence). ForPRLR-GFP2 constructs (upper panel), the LF cDNA was thesame with and without any SF cDNA. Total DNA was keptconstant by using empty vector. Data are the mean of threeto six determinations � SEM. *, Different from control with P �0.05; **, different from control with P � 0.01; ##, differentfrom LF-GFP2 or LF-Rluc with P � 0.01. RLU, Relative lumi-nescence unit. Data presented are relative �-casein promoteractivity in the absence vs. presence of the receptor isoformindicated. All cultures were treated with U-PRL as indicatedabove.

Tan et al. • Homo- and Hetero-Pairing of PRLR Isoforms Mol Endocrinol, May 2005, 19(5):1291–1303 1293

pairs. These were 8 � 10�4, 8 � 10�4, 6.3 � 10�4 and7.3 � 10�4, respectively (Fig. 3C), strongly suggestingthat equimolar expression of donor and acceptor wasachieved by equimolar concentrations of plasmid DNAused for the transfections. Different receptors, how-ever, were expressed at different levels. The SF1aisoform, whether tagged with Rluc or GFP, was ex-pressed at lower levels than either the SF1b or LFisoforms. This is in agreement with data from theDufau lab (16), which also showed a decreased ex-pression of the SF1a and is likely therefore a functionof the receptor per se and not just the fusion protein.

It is also important to note that the spectral proper-ties of luciferase or GFP were not altered by theirlinkage to the different receptors.

Subcellular localization of LF-GFP2 (Fig. 4A), SF1a-GFP2

(Fig. 4B) and SF1b-GFP2 (Fig. 4C) was investigated. PanelsA–C of Fig. 4 show single confocal images from 48 h aftertransfection. Panel D shows an autofluorescence control.At this time point, the confocal imaging showed that asubstantial number of PRLRs were present in the region ofthe plasma membrane (white arrows), although there was,of course, intracellular staining consistent with synthesis inthe rough endoplasmic reticulum and movement throughthe Golgi. Plasma membrane localization of the receptorswas confirmed by the production of a BRET signal in re-sponse to added PRL (see below). Large quantities of re-ceptor were sequestered in a juxtanuclear compartment.Sequestration of receptors within a juxtanuclear compart-

ment, such as the Golgi apparatus, is typical of a widevariety of cells because receptors mature through the Golgiand are recycled through the Golgi (21–23). SubstantialGolgi localization is also typical in cells overexpressing re-ceptors (24).

To be sure that the levels of expression of theconstructs used in this study did not produce anonspecific BRET signal due to overexpression anda resultant decrease in the average distance be-tween expressed proteins, levels of transfectingDNA of half and double those used to measureBRET (see below) were examined. We reasoned thatif nonspecific BRET occurs, increasing concentra-tions of transfecting DNA should increase BRET.Cells were therefore cotransfected with GFP2 andRluc plasmids at either half (2 �g/well) or double (8�g/well) the amount used for the subsequent BRETexperiments. Figure 5 shows that, although the bi-oluminescence from the Rluc increases with theconcentration of transfecting DNA, no detectableGFP emission and therefore no BRET was observed,suggesting that the concentrations of plasmid wewere using for the GFP2- and Rluc-tagged PRLRsshould not produce nonspecific BRET. This doesnot mean that further increases in transfecting DNAwould not have produced ligand-independentBRET. The GFP2-Rluc fusion construct was used asa positive control to indicate where a BRET signalwould have been observed, had one occurred.

Fig. 3. Fluorescence and Bioluminescence Measurements with the ConstructsHEK293 cells were transfected with equal moles (0.48 pmol) of GFP2-Rluc, LF-GFP2/LF-Rluc, SF1a-GFP2/SF1a-Rluc, and SF1b-GFP2/

SF1b-Rluc, respectively. The spectra were examined within 48 h after the transfection. Samples were excited at 405 nm. For bioluminescencemeasurements, coelenterazine h was present at a concentration of 5 �M. A, Fluorescence; B, bioluminescence; C, luminescence/fluores-cence (L/F) ratio.


Spectral Properties of the Rluc- and GFP2-Tagged PRLR Constructs

The addition of DeepBlueC (5 �M) to all the Rluc-transfected cells was associated with a biolumines-cence emission peak at 395 nm, a value identical withwhat has been reported for the untagged Rluc(PerkinElmer Application notes). The fluorescence ex-citation and emission maxima of all the GFP2-taggedPRLRs were also the same at 405 nm and 515 nm,respectively (data not shown).

Unmodified PRL Induces BRET between Homo-Pairs of PRLRs

Shown in Fig. 6 are a set of representative biolumi-nescence scans of suspensions of LF-Rluc and LF-GFP2 cotransfected and nontransfected human em-bryonic kidney 293 (HEK293) cells. In the absence ofU-PRL, primarily Rluc emission (peak �395 nm) wasobserved with barely detectable amounts of GFP2

emission (�515 nm; BRET) (Fig. 6A). Addition of U-PRL, however, was associated with a large increase inGFP2 emission (Fig. 6B), indicative of a large increasein BRET and therefore a reduction in the distancebetween donor and acceptor, each attached to the

cytoplasmic tails of paired receptors. Figure 6 alsoillustrates the averaged effects of U-PRL on the BRETratio of cells transfected with RLuc and GFP2 taggedto the same receptor isoforms (homo-pairs): LF (Fig.6C), SF1a (Fig. 6D), and SF1b (Fig. 6E). PRL increasedthe BRET ratio of LF, SF1a, and SF1b homo-pairs by9-, 4-, and 4-fold respectively.

Unmodified PRL Induced Hetero-Pairingof PRLRs

To assess the ability of U-PRL to induce pairing ofhetero-receptor isoforms, HEK293 cells were cotrans-fected with equal amounts of LF-GFP2 plus SF1a-Rluc, LF-GFP2 plus SF1b-Rluc, or SF1a-GFP2 plusSF1b-Rluc plasmids. As illustrated in Fig. 7, U-PRLincreased the BRET ratios of cells cotransfected withSF1a-Rluc plus LF-GFP2 (11-fold) and SF1b-Rluc plusLF-GFP2 (5-fold) plasmids, but no significant BRETwas observed with SF1b-Rluc plus SF1a-GFP2. Sim-ilar results were also observed in the reverse combi-nations of the constructs including LF-Rluc plus SF1a-GFP2, LF-Rluc plus SF1b-GFP2, and SF1a-Rluc plusSF1b-GFP2, showing that it made no difference whichreceptors were used as energy donors or acceptors(data not shown).

Fig. 4. Cellular Localization of the GFP2-Tagged ReceptorsHEK 293 cells, cultured on a coverslip (� � 12 mm), were transfected as described in Materials and Methods. Forty-eight hours

after transfection, the cellular localization of the expressed fusion proteins of LF-GFP2 (A), SF1a-GFP2 (B), and SF1b-GFP2 (C)were examined with a confocal fluorescence microscope (Zeiss 510) using filter sets designed for regular GFP. This was notoptimal but was sufficient to demonstrate subcellular localization. D, Mock transfected control with no measurable fluorescence.White arrows, Fluorescence in the region of the plasma membrane. Bars in all panels, 10 �m.


S179D PRL and G129R PRL Effects on Homo-and Hetero-Pairing of PRLRs and Activation ofthe �-Casein Promoter

When similar experiments were conducted with themolecular mimic of phosphorylated PRL, S179D PRL,the same results were obtained. Figure 8A illustrateshomo-pairing and Fig. 8B, hetero-pairing. S179D PRLincreased the BRET ratio of LF, SF1a and SF1b homo-pairs by 8-, 3-, and 3-fold, respectively. S179D PRL,like U-PRL, induced hetero-pairing between LF-SF1aand LF-SF1b, but not between SF1a and SF1b. TheS179D PRL-induced increases in BRET were 3-fold forLF-SF1a and 5-fold for LF-SF1b. The only significantdifference between the effects of U-PRL and S179DPRL was that S179D PRL was less efficient at produc-ing BRET with the LF-SF1a hetero-pair (3-fold vs. 11-fold for U-PRL).

As was true for U-PRL, interaction of S179D PRLwith both LF and SF1a homo-pairs, and LF-SF1ahetero-pairs stimulated �-casein promoter activity(Fig. 9). S179D PRL was an equivalent stimulator withLF homo-pairs, and a better stimulator of promoteractivity with SF1a homo-pairs, than U-PRL (comparethe fold control in Figs. 2 and 9). In contrast to U-PRL,there was no measurable dominant-negative activitywhen LF-SF1a hetero-pairs were engaged by S179DPRL. Like U-PRL, however, S179D PRL still caused adominant-negative effect with LF-SF1b. No effect ofS179D PRL on promoter activity was seen with SF1bhomo-pairs or SF1a-SF1b hetero-pairs (data for thelatter not presented).

G129R PRL is a mutant human PRL, engineered toeliminate binding to the second PRLR (20). This formof human PRL was unable to induce BRET with anyhomo- or hetero-receptor pairs (data mostly notshown). Data for the LF homo-pair are shown in Fig.10. Figure 10 also shows that G129R PRL competed ina dose-related fashion with U-PRL for the generationof a BRET signal by LF homo-pairs of the receptor.

This result is not due just to increasing concentrationsof PRL and the undoing of dimers because in otherexperiments 90 nM U-PRL produced an equivalentBRET signal to that seen with 45 nM U-PRL (data notshown). G129R PRL also failed to increase �-caseinpromoter activity with any form of the receptor (Fig. 9).

DISCUSSION

PRL is usually described as inducing PRLR dimeriza-tion through a sequential process that includes initialhigh-affinity binding to one receptor followed by loweraffinity binding to a second, recruited receptor (25).The lower affinity interaction with the second receptorhas the potential to be undone by excess ligand be-cause higher affinity 1:1 ligand-receptor interactionswould prevail in a competitive process. Because re-ceptor dimerization has been linked to one of the mainsignaling pathways, excess ligand would therefore bepredicted to result in at least some decreased biolog-ical responses, a prediction in keeping with the long-recognized biphasic dose response curves observedfor PRL (26, 27). The lower affinity binding of the sec-ond receptor to the PRL-PRLR binary complex hasprevented any demonstration of PRL-induced dimer-ization with homologous PRL and PRLRs because thelower affinity interaction easily dissociates during sam-ple processing. Heterologous PRL-PRLR interactions,on the other hand, have been shown to have a higheraffinity for the second receptor (11), and this has al-lowed cross-linking and immunoprecipitation studiesto produce some evidence of dimerization. This phe-nomenon has also made it possible to demonstrate a1:2 ratio between PRL and the ECD of PRLRs usingsolution analysis or surface plasmon resonance (8).What is missing, however, is evidence of effectivepairing of PRLRs with the biologically relevant ligand inliving cells in real time.

Fig. 5. Overexpression of the Constructs Does Not Result in a BRET Signal from Random CollisionsCells were cotransfected with GFP2-N1 and Rluc-N1 plasmids at either half (2 �g/well) or double (8 �g/well) the usual amount

for each construct. The bioluminescence and BRET2 signal were examined within 48 h after the transfection in the presence of5 �M of DeepBlueC.


In the current study, we used BRET to examine therapid interaction of homologous PRL with cell surfacereceptors in living cells. We observed very little BRETin the absence of ligand and increased BRET in re-sponse to ligand, thereby demonstrating that theBRET reflected events at the plasma membrane. Thetechnique, however, cannot distinguish betweenthe ability of a ligand to recruit monomers into dimersand a conformational change in the cytoplasmic re-gions of preexisting dimers. Either way, however, thetechnique measures close approximation (within 100Å) of the signal transducing portions of the receptorand hence an important interaction. Our goal in apply-ing BRET was to determine whether species homolo-gous ligands could produce such interactions. Also,we have examined the ability of three specific forms ofhuman PRL to cause interactions between the longand recently cloned short receptors, both as homo-and heterodimers.

To be sure that the different tagged PRLRs hadbiological activity, we examined �-casein expressionin response to U-PRL. Activation of the casein pro-moter has only been previously reported for the LF ofthe human PRLR; previous studies have failed to dem-onstrate activation of the casein promoter by eitherSF1a or SF1b after transient transfection of Chinesehamster ovary (15), COS-1 and HEK293 cells (16).However, we observed activation of �-casein by boththe LF and, to a lesser extent, the SF1a isoform of thereceptor. By contrast, the SF1b isoform reduced �-casein expression below that seen in the controls,consistent with a previously reported dominant-nega-tive activity (15, 16). The reason for the difference inactivity of the SF1a reported herein does not reflect itsattachment to the tags because untagged versionswere similarly active. Rather, it reflects the use of theapproximately 2.4 kb construct because there is noactivity with a shorter �344 to �1 region of the pro-

Fig. 6. U-PRL-Induced Dimerization of the Homo-Receptor PairsA, Bioluminescence from the DeepBlueC reaction without U-PRL; B, bioluminescence after the addition of U-PRL (45.7 nM).

Note the absence of a BRET signal in the absence of U-PRL. C (LF), D (SF1a), and E (SF1b) show the averaged results from fiveto nine separate determinations on different preparations of cells. The BRET ratio was calculated as (Emission500–520 nm �Background500–520 nm)/(Emission385–420 nm � Background385–420 nm). *, P � 0.05 for difference with and without PRL.


moter (data not shown). In this regard, Hu et al. (16) didnot define the size of the �-casein promoter used, andtheir experiments were conducted with a different re-ceptor construct isolated in their laboratory. In addi-tion, Hu et al. (16) report that the amino acid sequenceof the exon 11 portion of their SF1a results in morerapid turnover of the receptor. In similar studies, usingChinese hamster ovary cells and the construct iso-lated in our laboratory, conducted in the presence ofligand, we have been unable to demonstrate any sig-nificant difference in half-life of the LF and SF1a iso-forms (Long, G., and B. K. Vonderhaar, unpublished).A significant basal activity of the �-casein promoter inthe control suggests a significant degree of signaltransducer and activator of transcription (Stat) 5 acti-vation in the absence of PRL and the PRLR in thesecells, a phenomenon that was documented by immu-noprecipitation and Western blot analyses (data notshown). Thus, SF1a may increase activity at this longerpromoter by activating additional signaling cascadesthat can contribute to �-casein expression (19, 28).Although SF1a at 352 amino acids is the longer of theSFs, current consensus, based on PRLRs in otherspecies, would suggest that it is unable to efficientlyactivate Stat 5 (18, 29, 31). Whatever the details ofsignal transduction from the SF1a form turn out to be,however, the important issue for the current study isthat all tagged forms of the receptor are able to trans-duce a signal, as reflected either positively or nega-tively in �-casein promoter activity.

Analysis of the hetero-pairs for biological activity isproblematic because one cannot be sure that the ac-tivity derives from hetero-pairing. Instead, it could bethe average result of signals generated from two setsof homo-pairs and some hetero-pairs. That said, how-ever, it appears that cotransfection of either SF withthe LF reduces the signal to �-casein as comparedwith the LF alone when U-PRL is used as the ligand.This dominant-negative activity of the SFs for �-caseinexpression has been reported previously (15, 16). Be-cause the same amount of LF DNA was transfected in

the absence and presence of SF1a and SF1a trans-duces a signal to �-casein, one might have expectedthe LF-SF1a combination to have stimulated morepromoter activity than LF alone, but this was not thecase. Whether the dominant-negative activity of theSFs is caused by hetero-pairing, the generation ofalternate signals or interference with cell surface dis-play or synthesis of the more active LF remains to beestablished. This dominant-negative activity toward

Fig. 7. U-PRL-Induced Heterodimerization of PRLRsTransfections were carried out as described in Materials

and Methods and BRET2 assays were performed within 48 hafter transfection in the presence of DeepBlueC (DBC; 5 �M)with or without U-PRL (45.7 nM). **, P � 0.01 vs. for differencewith and without PRL.

Fig. 8. S179D PRL-Induced Homo- and Heterodimerizationof PRLRs

Transfections were carried out as described in Materialsand Methods and BRET2 assays were performed 48 h aftertransfection in the presence of DeepBlueC (DBC; 5 �M) withor without S179D PRL (45.7 nM). *, P � 0.05; **, P � 0.01

Fig. 9. Activation of the �-Casein Promoter by S179D PRLand G129R PRL with All Homo-Pairs and BRET-ProducingHetero-Pairs of the Receptor

This experiment was conducted as described in the legendfor Fig. 2 using S179D PRL and G129R PRL at 45.7 nM. **,P � 0.01 compared with control; ##, P � 0.01 comparedwith LF.


�-casein promoter activity with LF-SF1a was notpresent when S179D PRL was used as the ligand, aresult that may be related to the reduced BRET seenwith S179D PRL and this hetero-pairing vs. that seenwith U-PRL. In our experiments, U-PRL significantlyincreased the BRET ratio with all the homo-pairs of thetagged receptors and most of the hetero-pairs. Onlyreceptors at the cell surface contributed to the BRETsignal because there was very little signal in the ab-sence of ligand and measurements were taken imme-diately after addition of ligand. The combined SF1aand SF1b hetero-pair, however, did not produce aBRET signal. This implies that U-PRL has a muchgreater capacity to cause interactions between homo-pairs of either SF1a or SF1b isoforms than it doesbetween SF1a-SF1b hetero-pairs. Because the meth-odology cannot distinguish between actual dimeriza-tion and conformational change in the cytoplasmicregions of preexisting dimers, we cannot say thatSF1a-SF1b dimers don’t exist, but we can say that thesignal transducing parts of the receptor remain furtherapart than 100 Å upon addition of PRL. One mecha-nism by which added PRL could distinguish betweenSF homo- and heterodimers would be by differentconformations of their respective ECDs. The ECD ofeach of the receptors has the same amino acid se-quence, but a change in conformation could be theresult of attachment to the very different cytoplasmicdomains. The ability of the intracellular domain of thePRLR to influence the conformation of the extracellulardomain has been previously described (32–34). Onestudy, which examined the effect of deletion of 55 of57 of the amino acids in the intracellular domain

of the long receptor, demonstrated that this increasedthe affinity of the receptor when this was examinedby the binding of ovine PRL to microsomes derivedfrom transiently transfected cells (32). Another studydemonstrated that the PRL-binding protein present inrabbit milk, which is probably equivalent to the ECDof the receptor (15, 33), interacted differently witha monoclonal antibody when compared with themembrane receptor (34). This suggested that themembrane-embedded and free ECD had differentconformations. Finally, a mutant form of the rat recep-tor with a reduced cytoplasmic tail has been demon-strated to have a greater affinity for ligand than thefull-length version (35). For each of these examples,the amino acid sequence of the extracellular domain ofthe full-length and foreshortened version was thesame. Thus, it appears extremely likely that the lengthand conformation of the intracellular domain affectsthe conformation of the ECD, and hence that SF ho-modimers and SF heterodimers can have differentconformations such that the ligand cannot interactproperly with the SF hetero-pair. When S179D PRLwas used as the ligand, we found it to have the sameability to homo-pair all receptor forms and to hetero-pair all but the SF1a-SF1b pair. Thus, with two differ-ent ligands, this incompatibility is evident.

S179D PRL has been shown to act as an antag-onist to U-PRL-mediated cell proliferation and as-sociated Stat 5 activation (36) but has also beenshown to act as an agonist for �-casein expression[Ref. 19 and data herein (Fig. 9)]. Before the currentstudy, the easiest way to explain this would havebeen if antagonistic signals were brought about byblockade of specific receptor pairing, and agonisticproperties, by preferential pairing of other receptors.Our BRET results, however, indicate that S179DPRL interacts with all of the same receptor pairs asU-PRL and so this is not what accounts for thedifferent effects of the two ligands. Our currentworking model involves the generation of differentreceptor conformations with each ligand, resultingin inhibition and activation of different sets of sig-naling molecules. Indeed, the generation of differentsignals by S179D PRL and U-PRL has been dem-onstrated (19, 36). The idea that different receptorconformations would result in different signaling issupported by an elegant study analyzing the effectof stable one amino acid turns in the transmembranehelix of the erythropoietin receptor (37). In thisstudy, one conformation produced full Jak-Stat andERK 1/2 signaling, whereas another favored ERK 1/2signaling. In other words, one conformation pro-duced signals like U-PRL and another, signals likeS179D PRL (19). A mechanistic cartoon of differen-tial signaling in response to U-PRL and S179D PRLcan be found in a recent review (38). If there aredifferent conformations produced by U-PRL andS179D PRL, however, they are not large enough tosee absolute differences in BRET generated by eachligand. This suggests that approximation of the cy-

Fig. 10. Competition between G129R PRL and U-PRL forthe Generation of BRET at the LF Receptor

Cells transfected with the LF were exposed to either 45.7nM U-PRL (concentration used for all previous analyses), 45.7nM G129R PRL, or 45.7 nM U-PRL plus increasing concen-trations of G129R PRL between 4.57 and 45.7 nM and BRETproduced was measured. For the sake of clarity on the figure,45.7 nM was written as 45 nM and all proportions of 45.7 nM

were similarly represented. *, P � 0.05 compared with G129RPRL alone; #, P � 0.05 compared with U-PRL alone.


toplasmic domains of two receptors is important forall signaling. Some aspects of the different confor-mations, however, may be reflected in the degree ofBRET. The only significant BRET difference betweenthe two ligands uncovered in the present study wasthat S179D PRL was about one third as efficient asU-PRL at creating a BRET signal from LF-SF1a het-ero-pairs and, unlike U-PRL, did not show a domi-nant-negative effect with this hetero-pairing. It ispossible therefore that the reduced BRET generatedby the interaction of S179D PRL with LF-SF1a het-ero-pairs reflects an altered conformation vs. thatgenerated by U-PRL.

G129R PRL was designed to substantially lower theaffinity of binding to the second PRL receptor to blockternary complex formation (20) and, as such, would beexpected to be inactive in BRET assays. This wasconfirmed in the present study and is a useful controlindicating that the BRET signal required interaction ofthe ligand with both receptors in a pair and not just aconformational change in one of the two receptors.When titrated against a constant amount of U-PRL,G129R PRL competed with U-PRL such that a re-duced BRET signal was produced. This demonstratedthat G129R PRL, by occupying some receptors, inter-fered with the ability of U-PRL to form as many recep-tor pairs. The only surprising aspect of this experimentwas the inhibitory potency of G129R PRL when ana-lyzed in this manner because other work has sug-gested that more than a 1:1 ratio of G129R PRL toU-PRL is necessary to see significant antagonism (39).

Because activation of the �-casein promoter onlyreflects some of the signals generated by PRL at theLF and very little is yet known about signaling fromeither SF1a or SF1b, the current study cannot addressthe question of whether dimers can ever be formedwithout generation of a signal. What can be said, how-ever, is that any signal generated by dimerization ofthe SF1b form does not lead to activation of the �-casein promoter. For the LF, where we know thatsignaling results in activation of the �-casein pro-moter, we can also say that those forms of PRL thatproduce BRET, also activate the promoter, whereasthe one that does not produce BRET, does not.

The conformation of the cytoplasmic domains of thePRLRs has not been examined but is unlikely to belinear. It is interesting in this regard that the BRET ratiogenerated with either U-PRL or S179D PRL is verysimilar whether a LF-LF dimer or a LF-SF1b dimer isformed, despite the 334-amino acid difference inlength between the LF and S1b receptor. Thus, itseems that with folding, the intracellular tags in alllikelihood are not as far apart as the length of theamino acid chain would imply

In summary, in our present study, U-PRL- andS179D PRL-induced BRET was observed with allcombinations of long- and short-receptor isoforms ex-cept the combination of SF1a plus SF1b isoforms.This is the first demonstration that human PRL pro-motes homo- and heterodimerization of most long and

short human PRLR pairs in living cells and that thisoccurs using species homologous hormone and re-ceptors. The results also highlight the incompatibilityof SF1a and SF1b and exclude the formation of dif-ferent receptor pairs as an explanation for the differentsignaling abilities of U-PRL and S179D PRL.

MATERIALS AND METHODS

Materials

The codon-humanized pGFP2-N1 (h) and pRluc-N1 (h) vec-tors, containing multiple cloning sites upstream of the GFP2

gene or Rluc gene, and the luciferase substrate, DeepBlueC,were purchased from PerkinElmer. Coelenterazine h, anothersubstrate for Rluc was purchased from Assay Designs, Inc.(Ann Arbor, MI). The PRLR cDNAs, encoding LF, SF1a andSF1b cloned into pEF6C, pEF4C, and pCRscript were estab-lished as described previously (13). A �-casein-Luc plasmid,which contains 2.4 kb of the �-casein promoter upstream ofa luciferase reporter gene (40), was a generous gift from Dr.Linda Schuler (Madison, WI) and Jeffrey Rosen (Baylor, TX).U-PRL and S179D PRL were purified from Escherichia coli(DH5�) as described previously (30). G129R PRL was purifiedfrom E. coli as described by Zhang et al. (39).

BRET Assay

In a BRET assay, excited-state electron energy generated byan Rluc substrate oxidation reaction is transferred to an en-ergy acceptor molecule when it is within about 100 Å. Theexcited acceptor molecule then emits light at its specificemission wavelength. The BRET2 technique we used im-proves upon the original BRET by the use of a new substrate,DeepBlueC, and a new acceptor fluorophore, a modifiedGFP, GFP2. In the original BRET, bioluminescence from Rlucusing the older coelenterazine h substrate peaks between475 nm and 480 nm and the fluorescence from the acceptor,yellow fluorescent protein between 520 nm and 530 nm,resulting in poor peak resolution (only 45–55 nm) betweendonor and acceptor emission. In addition, the emission fromRluc using coelenterazine h is very broad, and overlaps withyellow fluorescent protein emission. The DeepBlueC sub-strate, by contrast, has a peak emission at 390–405 nm andthe GFP2 acceptor has a peak emission at 500–520 nm. Thus,peak resolution is increased to about 115 nm and overlappingdonor and acceptor emission is markedly reduced, leading togreater BRET sensitivity. Also in the current study, we used ascanning spectrofluorometer so as to record whole spectralscans with each set of cells. This controlled for two things:coexpression of Rluc- and GFP2-tagged receptors so that wecould properly interpret a lack of GFP2 emission, and spectralshifts that may have resulted from association or dissociationof other proteins from the receptors upon initiation of signal-ing. As it turned out, no significant spectral shifts were ob-served and so future experiments can be conducted by mon-itoring only peak wavelengths.

Construction of PRLR Eukaryotic Expression VectorsTagged with GFP2 and Rluc

To prepare PRLR tagged with GFP2 (LF-GFP2, SF1a-GFP2,SF1b-GFP2) and Rluc (LF-Rluc, SF1a-Rluc, SF1b-Rluc) at thecarboxy terminus, the entire coding sequences of the LF(1866 bp), SF1a (1128 bp) and SF1b (864 bp) receptorswithout their stop codons were amplified by PCR from theoriginal plasmids, pEF6-LF, pEF4-SF1a and pCR2.1-SF1b,


respectively, by using Taq DNA polymerase (Promega, Mad-ison, WI) and sense/antisense primers harboring unique MluI(at sense) and KpnI (at antisense) restriction sites. The extraDNA base pairs corresponding to MluI (ACGCGT) and KpnI(GGTACC) were designed to be upstream of the initiatorcodon on the sense and to replace the stop codon on theantisense. An extra triplet ACC was also designed to beimmediately before the initiator codon, ATG, to facilitate andtherefore increase the transcription rate in transfected mam-malian cells. The primers (sense/antisense) were as follows(cleavage sites for MluI and KpnI are shown in boldface):

LF: 5-GAC ACGCGT ACC ATG AAG GAA AAT GTG-3(sense)/5-AAC GGTACC A GTG AAA GGA GTG TGT-3(antisense).

SF1a: 5-GAC ACGCGT ACC ATG AAG GAA AAT GTG-3(sense)/5-AAC GGTACCA CTG GAC TGT GGTCAA-3(antisense).

SF1b: 5-GAC ACGCGT ACC ATG AAG GAA AAT GTG-3(sense)/5-AAC GGTACCA AGG GGT CAC CTC CAA-3(antisense).

GFP2-N1 and Rluc-N1, which have a cytomegalovirusearly promoter and a polyadenylation site, as well as thelinearized PCR product of PRLR cDNA, were then digestedwith MluI and KpnI and purified from an agarose preparativegel. The fragments then were subcloned in-frame into theMluI/KpnI site of the GFP2-N1 and Rluc-N1 vectors to givefusion plasmids of LF-pGFP2N1, SF1a-pGFP2N1, SF1b-pGFP2N1, LF-pRlucN1, SF1a-RlucN1 and SF1b-pRlucN1.Sequence analyses were performed to verify the correct ori-entation and open reading frame of the newly made con-structs. The fusion proteins contain a linker peptide of 12 and13 amino acids in GFP2- and Rluc-tagged receptor, respec-tively (Fig. 1).

Cell Culture and Transfection

HEK293 cells were maintained in DMEM (Invitrogen, Carls-bad, CA) containing high glucose, 1 mM sodium pyruvate,and pyridoxine hydrochloride, 2 mM L-glutamine, supple-mented with 10% fetal bovine serum, 100 U/ml penicillin, and0.7 �M streptomycin. The cells were seeded at a density of5 � 105 cells per well of six-well (or as indicated) tissueculture dishes. Transient transfections were performed thefollowing day when the cells were 90–95% confluent usingLipofectamine 2000 (Invitrogen) in accordance with the pro-tocol provided by the vendor. Briefly, 3.5–4.5 �g DNA wasused per well. The DNA was initially incubated in 250 �l ofOpti-MEM I Reduced Serum Medium (Invitrogen) (withoutserum and antibiotics) and mixed gently. A 1:25 dilution ofLipofectamine 2000 in 250 �l of the same medium was thenprepared. After a 5-min incubation at room temperature, thediluted DNA and Lipofectamine 2000 solutions were mixedand incubated at room temperature for 20 min to allow theDNA-Lipofectamine 2000 complexes to form. The complexeswere then added to the cells (in medium with serum withoutantibiotics) and mixed by rocking the plate back and forth.After a 48-h incubation of the cells at 37 C in a CO2 incubator,the cells were subjected to bioluminescence, fluorescence,and BRET2 analyses.

PRL-Induced Expression of �-Casein-Luciferase Assay

The biological activities of tagged PRLRs were analyzed us-ing a functional bioassay based on cotransfection with anapproximately 2.4-kb portion of the �-casein gene promoterfused to a luciferase reporter. For the analysis of het-erodimers, equal amounts of LF cDNA were transfected in theLF alone and LF plus SF. Total transfected DNA was keptconstant by adjustment with a control vector. Twenty-fourhours after transfection, the medium was changed to DMEMwithout serum containing 45.7 nM recombinant PRLs. After afurther 24 h, the cells were washed three times with Dulbec-

co’s PBS (DPBS), and then reporter lysis buffer (15 �l/cm2)was added to the plate. To ensure complete lysis, a freezethaw was performed followed by a 15-min shake at roomtemperature. The lysed cells were scraped off the dish andcentrifuged (12,000 � g) for 5 min. Twenty microliters of thesupernatant were added into luminometer tubes containing50 �l of luciferase assay reagent containing luciferin, and therelative luminescence signal was measured using a Mono-light 2010 luminometer (Analytical Luminescence, San Diego,CA). For the activity assay of Rluc-PRLRs, three separatetransfections including LF-Rluc only, SF1a-Rluc only andSF1b-Rluc only were performed and the normalized countswere subtracted from normalized LF-Rluc/�-casein-Luc,SF1a-�-casein-Luc, and SF1b-�-casein-Luc, respectively.

Expression Level of GFP2- and Rluc-Tagged Receptors

The relative expression levels of GFP2- and Rluc-taggedPRLR constructs in the cells were estimated by comparisonof the fluorescence and bioluminescence from cells cotrans-fected with tagged PRLRs to cells transfected with a GFP2-Rluc fusion plasmid. The fluorescence emitted by GFP2 wasmeasured with a FluorMax II spectrofluorometer (JY/Horiba,Edison, NJ) with excitation at 405 nm. Bioluminescence ofthe transfected cells was measured after the addition ofcoelenterazine h (5 �M) using the same spectrofluorometerbut with a black card placed in the excitation-beam path.Coelenterazine h and not DeepBlueC was used in thesemeasurements because coelenterazine h oxidation by Rluc isslower than DeepBlueC and thus provides the necessaryextra time for more complete spectroscopic measurements.Also, the much longer emission wavelength associated withcoelenterazine h oxidation (475 nm) reduces the possibility ofdonor quenching and increases the accuracy of themeasurements.

Confocal Imaging

Confocal microscopy (Zeiss 510; Zeiss, Jena, Germany) wasapplied to check the cellular expression and localization ofGFP2-tagged PRLRs. HEK293 cells were plated at a densityof 5 � 105 cells/well on polylysine-coated coverslips (� � 12mm) and cultured in DMEM as described above. One dayafter plating, when the cells reached about 90% confluence,the cells were transfected with 0.8 �g DNA/35-mm well usingLipofectamine 2000 as described above. At various timesafter transfection, microscopic observation was performed.

Fluorescence and BRET2 Measurements

Transfected cells were harvested within 48 h of transfection bywashing with DPBS (three times), detachment with DPBS con-taining 2 mM EDTA centrifugation at 800 rpm and resuspensionin BRET2 buffer (DPBS containing 0.9 mM CaCl2, 0.5 mM

MgCl2�6H2O and 5.5 mM D-glucose) at a density of 2 � 106/ml.Before the BRET2 measurements, the cells were incubated at37 C for at least 1 h. For each measurement, 0.5 ml cell sus-pension was loaded into a 0.5-cm2 quartz cuvette. Fluores-cence and bioluminescence spectral scanning were performedby using a FluoroMax-2 spectrofluorometer (Jobin Yvon Inc.,Edison, NJ). Bioluminescence scanning and BRET2 signaldetection were carried out immediately after the addition ofthe ligand and 5 �M of the cell permeant luciferase substrate,DeepBlueC, with a black card in the path of the excitationlight beam. Data were collected with the slits set at 5 nm,datum points collected every 5 nm, and signal integration for0.5 sec per datum point). Energy transfer was defined as theBRET ratio (Emission500 nm-520 nm-background500 nm-520 nm)/(Emission385 nm-420 nm-background385 nm-420 nm). The signalsobtained from the nontransfected cells were consideredbackground.


Acknowledgments

We are grateful to Dr. Linda Schuler and Jeffrey Rosen forproviding the approximately 2.4 kb �-casein-luciferase re-porter construct and to Erika Ginsburg for providing detailedinformation about the receptor constructs and for technicalassistance.

Received July 28, 2004. Accepted January 25, 2005.Address all correspondence and requests for reprints to:

Ameae M. Walker, Division of Biomedical Sciences, Univer-sity of California, Riverside, Riverside, California 92521. E-mail: [email protected].

This work was supported by the United States PublicHealth Service Grant DK61005 (to A.M.W.).

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