2.1.

Preparation of CRHR-YFP/CFP and VT2R-YFP/CFP Constructs

VT2R and CRHR fusion proteins were produced as described previously¹⁷ by cloning either the VT2R or CRHR into pEYFP and pECFP plasmids to obtain a total of four fusion constructs, VT2R-CFP, VT2R-YFP, CRHR-CFP, and CRHR-YFP. Each plasmid was transformed into XL10 Gold E. coli cells (Stratagene Inc., La Jolla, CA) and cultured on Luria-Bertani (LB) agar plates with $30\mathrm{\mu g}∕\mathrm{ml}$ kanamycin to select for positive transformants. Single colonies of each fusion were then propagated by inoculating $500\mathrm{ml}$ LB broth containing $30\mathrm{\mu g}∕\mathrm{ml}$ kanamycin and culturing at $37°\mathrm{C}$ with shaking. VT2R and CRHR fusion constructs were purified from E. coli cells using a QiaFilter Plasmid Maxi Kit (Qiagen Inc., Valencia, CA) and DNA concentrations were measured spectrophotometrically. Fusion plasmids were screened by BamHI and EcoRI (Promega Corp., Madison, WI) restriction endonuclease digestion for proper insertion of AVTR and CRHR DNA sequences.

2.2.

Site-Directed Mutagenesis

A single point mutation, A206K, in the fluorescent part of the fusion proteins was performed as described previously.¹⁷ Mutations of ${\mathrm{Ala}}^{206}$ to Lys in the fluorescent part of pECFP-N1 and pEYFP-N1 expression vectors were made by using the Quick Change site-directed mutagenesis kit (Stratagene), a iCycler Thermal Cycler (BIO-RAD Laboratories, Inc.), and PfuTurbo DNA polymerase (Stratagene). The $50\mathrm{\mu }\mathrm{l}$ reaction mixtures each contained approximately $40\mathrm{ng}$ plasmid template, $125\mathrm{ng}$ each oligonucleotide primer sense A206K top: $5^{\prime }$ -CAG TCC $\underset{̱}{\mathrm{AAG}}$ CTG AGC AAA GAC CCC AAC GAG AAG CGC GAT CAC- $3^{\prime }$ and antisense A206K bottom: $5^{\prime }$ -GTG ATC GCG CTT CTC GTT GGG GTC TTT GCT CAG $\underset{̱}{\mathrm{CTT}}$ GGA CTG- $3^{\prime }$ ¹⁹ (Integrated DNA Technologies), each deoxynucleoside triphosphate (Stratagene) in a final concentration of $0.05\mathrm{mM}$ , and $2.5\mathrm{U}$ PfuTurbo DNA polymerase. After a 1-min hot-start at $95°\mathrm{C}$ , 16 cycles of the following program were run: denaturation for $30\sec$ at $95°\mathrm{C}$ , primer annealing for $1\min$ at $55°\mathrm{C}$ , and polymerization for $7\min$ at $68°\mathrm{C}$ . The PCR product was digested for $1\mathrm{h}$ at $37°\mathrm{C}$ with restriction enzyme DpnI (Stratagene) to eliminate the original template and thereby increase mutation efficiency. The DNA sequences of the mutated plasmids were verified by sequencing.

2.3.

Preparation of VT2R/ ${\beta }_2$ AR Chimeras

As described in Fig. 2 , a $383\mathrm{bp}$ sequence containing TM4 of the ${\beta }_2\mathrm{AR}$ , flanked on both $5^{\prime }$ and $3^{\prime }$ ends with the VT2R sequence to incorporate endogenous SacII and KpnI restriction sites, was synthesized and cloned into a pUC57 vector (EZ Biolabs, Westfield, IN). The pUC57-VT2R/ ${\beta }_2$ AR-TM4 vector was digested with SacII and KpnI restriction enzymes (New England Biolabs, Ipswich, MA) and the digestion product was separated by gel electrophoresis on a 1% TBE agarose gel to obtain the now $380\mathrm{bp}$ fragment with the ${\beta }_2\mathrm{AR}$ TM4 insertion, flanked on both sides with VT2R sequence. This $380\mathrm{bp}$ fragment was gel-purified via a QIAquick Gel Extraction Kit (Qiagen). VT2R-YFP/CFP fusion plasmids were digested with KpnI and SacII and the digestion products were separated on a 1% agarose gel to obtain the $5985\mathrm{bp}$ pEYFP/pECFP fragments, which were gel-purified. Complimentary gel-purified DNA fragments were then ligated (Fig. 2) with T4 DNA ligase in 2X rapid ligation buffer (Promega), and transformed into XL10 Gold E. coli. Cells were grown on agarose plates with $30\mathrm{\mu g}∕\mathrm{ml}$ kanamycin. Positive transformants were screened for proper insertion by StuI (Invitrogen) digestion and sequenced to further confirm that the ${\beta }_2$ AR-TM4 insertion was present in pEYFP/CFP-VT2R plasmids, and to ensure that no mutations had incurred in either the VT2R, CFP, or YFP sequences.

Fig. 2

Construction of chimeric plasmids. (a) Amino acid alignment of VT2R TM4 and ${\beta }_2$ AR TM4 (* most conservative amino acids) obtained with SIM-alignment tool for protein sequence (http://www.expasy.ch): (b) Gel electrophoresis of chimeric $\mathrm{VT}2\mathrm{R}∕{\beta }_2\mathrm{AR}$ plasmids following digestion with StuI to yield expected 3312 bp and 2673 bp fragments. Lane 1: $\mathrm{VT}2∕{\beta }_2$ AR-YFP chimeric plasmid. Lane 2: $\mathrm{VT}2∕{\beta }_2$ AR-CFP chimeric plasmid. (c) Sequence of the ${\beta }_2\mathrm{AR}$ TM4 domain (bold) with flanking VT2R sequence and the restriction enzyme sites underlined.

2.4.

Transfection of HeLa Cells with CRHR-CFP/YFP, VT2R-CFP/YFP Fusion Plasmids, and VT2R/ ${\beta }_2$ AR-CFP/YFP Chimeras

HeLa cells were cultured in a 95% air/5% ${\mathrm{CO}}_2$ incubator at $37°\mathrm{C}$ and plated on sterile coverslips in a six-well plate at a density of 40,000–60,000 cells/well in $2\mathrm{ml}$ Eagle minimal essential media (MEM, ATCC, Mannassas, VA) supplemented with 10% fetal bovine serum (FBS). Liposomes were created by diluting $2\mathrm{\mu }\mathrm{g}$ of respective fusion or chimeric receptor DNA with $250\mathrm{\mu }\mathrm{l}$ MEM, and combining with $10\mathrm{\mu }\mathrm{l}$ of Lipofectamine 2000 (Invitrogen) which had been diluted in $250\mathrm{\mu }\mathrm{l}$ MEM, gently shaken, and incubated for $5\min$ at room temperature. The receptor DNA/Lipofectamine mixture was incubated $20\min$ at room temperature to allow for liposome formation, after which $500\mathrm{\mu }\mathrm{l}$ was added to the appropriate well to reach a total volume of $2.5\mathrm{ml}$ . HeLa cells were cultured for $4\mathrm{h}$ , after which time the media was changed with fresh MEM-10% FBS media. The cells were then cultured additionally for $12\mathrm{h}$ and visualized with confocal microscopy to localize the receptor.

2.5.

Visualization of Chimeric Receptors

Cells were visualized by mounting the cover slips from each well of each respective transfection on slides and viewing with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, New York, NY). A 514-nm laser was used to excite YFP-tagged receptors, while a 458-nm laser was applied to excite CFP-tagged receptors. Images were adjusted to attain maximal resolution. A digital transformation of images was not applied.

2.6.

Hormonal Treatment

Treatment of cells with AVT or CRH was performed as described previously.¹⁷ Briefly, AVT and CRH were diluted into modified receptor buffer ( $10\mathrm{mM}$ $\mathrm{KCl}$ , $10\mathrm{mM}$ ${\mathrm{MgCl}}_2$ , $2\mathrm{mM}$ EGTA, $20\mathrm{mM}$ Hepes, $120\mathrm{mM}$ $\mathrm{NaCl}$ , $1\mathrm{mg}∕\mathrm{ml}$ BSA, pH 7.4) as previously suggested.²⁰ HeLa cells transiently transfected with CRHR-, VT2R-, and VT2/ ${\beta }_2$ AR-fusion protein constructs were incubated with $100\mathrm{nM}$ human CRH and $100\mathrm{nM}$ (Arg8)-vasotocin for $20\min$ at $37°\mathrm{C}$ .

2.7.

Fura-2AM Calcium Mobilization Assay

HeLa cells were cultured in a 95% air/5% ${\mathrm{CO}}_2$ incubator at $37°\mathrm{C}$ and plated in twelve 10-cm dishes. Four plates were transfected with VT2R-YFP fusion plasmids, four plates were transfected with VT2R/ ${\beta }_2$ AR-YFP chimeras, and four plates were not transfected and served as a negative control. Liposomes were made by combining $24\mathrm{\mu }\mathrm{g}$ of respective DNA, diluted in $1.5\mathrm{ml}$ MEM, with $60\mathrm{\mu }\mathrm{l}$ Lipofectamine 2000 in $1.5\mathrm{ml}$ MEM, gently shaken, and incubated at room temperature for $5\min$ . Three ml of respective liposomes were then added to the appropriate plates, which contained $12\mathrm{ml}$ 10% FBS-MEM. Cells were cultured for $40\mathrm{h}$ at $37°\mathrm{C}$ in a humidified ${\mathrm{CO}}_2$ incubator. Fresh 10% FBS-MEM was added $4\mathrm{h}$ posttransfection, and 10% FBS-MEM with 1% penicillin-streptomycin fungizone (Invitrogen) was added $16\mathrm{h}$ posttransfection. Cells were harvested and washed twice with $10\mathrm{ml}$ phosphate buffered saline (PBS). Cells were then resuspended in Gey’s buffer and the Fura-2AM assay was performed as previously described.²¹

2.8.

FRET Analysis

As described above, HeLa cells were grown on cover slips placed on the bottom of 6-well plates for $24\mathrm{h}$ . Prior to transfection with CFP/YFP constructs, the cover slips were moved to new 6-well plates containing fresh media. Twenty-four hours after transfection, the fluorescent fusion proteins were visualized in intact cells. Images were collected by using Carl Zeiss 510 META laser scanning confocal microscope with a $40\times$ water immersion objective. LSM Image Browser Software was used for collection of images. CFP was excited at $457\mathrm{nm}$ (power $118\mathrm{\mu }\mathrm{W}$ ) and observed from $485∕30\mathrm{nm}$ . YFP was excited at $514\mathrm{nm}$ (power $29\mathrm{\mu }\mathrm{W}$ ), observed from $545∕40\mathrm{nm}$ . Images from all single-labeled (CFP or YFP fusion proteins samples) or double-labeled (CFP and YFP fusion proteins) specimens were taken under exactly the same conditions (PCM $1024\times 1024$ color, $2.3\times$ zoom, without processing). There are two contaminants in the FRET signal: donor cross talk and acceptor bleed through. We used the previously developed algorithm,²² which removes these contaminants pixel by pixel on the basis of matched fluorescence levels between the double-labeled specimen and a single-labeled reference specimen, using seven images: two single-labeled donor reference images (donor excitation/donor channel and acceptor channel); two single-labeled acceptor reference images (donor and acceptor excitation, both in the acceptor channel); and three double-labeled images (acceptor excitation/acceptor channel, and donor excitation/donor and acceptor channels). This algorithm allowed the capture of a pure FRET image with following E% analysis and distance calculation. With these values, the FRET data were corrected for cross talk and filter leaks as well as potential differences in the concentrations of the donor and acceptor. A background (region with no cells) was subtracted from the foreground value (region within the cell). Approximately five images were taken from each specimen; six to eight regions of interest (ROI) on cell membranes were chosen from each image.

3.1.

VT2R/ ${\beta }_2$ AR-YFP/CFP Chimera Construction

Proper insertion of the ${\beta }_2$ AR-TM4 sequence into VT2R-YFP and VT2R-CFP fusion proteins was confirmed by StuI digestion [Fig. 2b] and sequencing [Fig. 2c]. The sequencing identified that the TM4 of the chimeric receptors was represented by 19 amino acid from the ${\beta }_2$ -adrenergic receptor. As expected, the rest of the DNA sequences of each construct contained no other mutations.

3.2.

VT2R/ ${\beta }_2$ AR-YFP/CFP Chimera Localization and Functionality

In order to prove that function correlates with correct trafficking, two chimeras, VT2/ ${\beta }_2$ AR chimera tagged with the donor or acceptor and CRHR tagged with the acceptor or donor, were prepared. Following transfection of HeLa cells, both fusion proteins were localized mainly in cytoplasmic membrane (Fig. 3 ). We next determined whether the chimeric receptors were capable of responding to AVT by mobilizing intracellular ${\mathrm{Ca}}^{2+}$ and thus are functionally active. For this, only VT2R-YFP and VT2/ ${\beta }_2$ AR-YFP chimeric receptor constructs were used to prevent an overlap with the Fura-2 fluorescence that occurs with the CYP fluorochrome. Using nontransfected HeLa cells as a control, the addition of AVT did not cause any change in ${\mathrm{Ca}}^{2+}$ mobilization as measured using Fura-2 [Fig. 4a ]. In cells expressing either the VT2R or the VT2/ ${\beta }_2$ AR chimeric receptor, identical increase of Fura-2 fluorescence was observed indicating that ${\mathrm{Ca}}^{2+}$ mobilization was equal between the two expressed proteins [Fig. 4b and 4c]. These data suggest that the overexpressed chimera receptor was able to activate downstream signal transduction pathways and mobilize intracellular ${\mathrm{Ca}}^{2+}$ .

Fig. 3

Images for algorithm FRET method. Laser scanning confocal images in HeLa cells are shown for CRHR-CFP and VT2R-YFP (a, b, c, d), CRHR-CFP and $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-YFP chimeric receptor (f, g, h, i), and CRHR-YFP and $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-CFP chimeric receptor (k, l, m, n). The associated histograms are shown for CRHR-CFP and VT2R-YFP (e), CRHR-CFP and $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-YFP (j), and CRHR-YFP and $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-YFP (o). HeLa cells were labeled with CFP (donor, CRHR or $\mathrm{VT}2\mathrm{R}∕{\beta }_2\mathrm{AR}$ ) and YFP (acceptor, VT2R, $\mathrm{VT}2\mathrm{R}∕{\beta }_2\mathrm{AR}$ or CRFR). To set the parameters for quantitative FRET, the images of HeLa cells expressing only CRHR-CFP or $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-CFP and either VT2R-YFP or $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-YFP or CRFR-YFP were taken with donor and acceptor filter sets (images are not shown). Using confocal microscopy, processed FRET (PFRET) fluorescence signals (nonradioactive transfer of energy from CRHR or $\mathrm{VT}2\mathrm{R}∕{\beta }_2\mathrm{AR}$ donor fluorophore to VT2R or $\mathrm{VT}2\mathrm{R}∕{\beta }_2\mathrm{AR}$ or CRHR acceptor fluorophore) were collected in the double-labeled cells. Images are representative of five experiments. Images collected during double-labeled donor excitation, donor channel revealing the distribution of CRHR-CFP (a and f) or $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-CFP (k); double-labeled, donor excitation, acceptor channel (b, g, and l); double-labeled acceptor excitation, acceptor channel revealing the distribution of VT2R-YFP (c), $\mathrm{VT}2\mathrm{R}∕{\beta }_2$ AR-YFP (h), or CRHR-YFP (m). (d, i, and n) PFRET calculated with algorithm methods. Scale bar: $20\mathrm{\mu }\mathrm{M}$ .

Fig. 4

Calcium mobilization in HeLa cells expressing either the VT2R or the $\mathrm{VT}2∕{\beta }_2\mathrm{AR}$ chimeric receptor in response to AVT. HeLa cells were either not transfected or transfected with plasmids expressing VT2R-YFP or $\mathrm{VT}2∕{\beta }_2$ AR-YFP. Cells were subsequently treated with $100\mathrm{nM}$ AVT and changes in fura-2 fluorescence were used to monitor intracellular ${\mathrm{Ca}}^{2+}$ levels. (a) A representative tracing from nontransfected cells demonstrating nonresponsiveness to AVT. (b) Representative tracing from HeLa cells expressing VT2R-YFP demonstrating mobilization of intracellular ${\mathrm{Ca}}^{2+}$ upon addition of AVT. (c) Representative tracing from HeLa cells expressing the $\mathrm{VT}2∕{\beta }_2\mathrm{AR}$ chimeric receptor demonstrating mobilization of intracellular ${\mathrm{Ca}}^{2+}$ upon addition of AVT.

3.3.

FRET Analysis

It is known that dimers can be formed between two EFP parts regardless of the existence of any other dimerization of the proteins fused to these fluorescent proteins.¹⁹ A single point mutation, A206K, in the fluorescent part of any fusion protein has been shown to prevent the formation of nonspecific dimerization.¹⁹ To demonstrate that a FRET signal results from a specific interaction between the CRHR and VT2R rather than a nonspecific interaction occurring between the fluorophores, the site-directed mutation of ${\mathrm{Ala}}^{206}$ to Lys was performed in fluorescent parts of the CRHR-CFP, VT2R-CFP, ${\mathrm{VT}}_2$ R-YFP fusion proteins, VT2R/ ${\beta }_2$ AR-CFP, and VT2R/ ${\beta }_2$ AR-YFP. The mutated variants of the fusion proteins were expressed in the same cellular compartments as wild-type receptors (data not shown). All FRET experiments were obtained with mutated A206K variants of fusion proteins.

The ability to form heterodimers between CRHR and VT2 receptor and between CRHR and the chimera VT2/ ${\beta }_2$ AR receptor was studied in transfected HeLa cells using FRET analysis. This method detects the transfer of excitation energy from donor fluorophore to a nearby acceptor that can occur only over a distance of about $3–7\mathrm{nm}$ .^{22, 23, 24} In order to obtain a quantitative measure of heterodimer formation, image data were processed using PFRET software obtained from the W. K. Keck Center for Cellular Imaging (University of Virginia, Charlottesville, VA, www.kcci.virginia.edu).^{22, 25} Quantitative FRET analysis as described previously¹⁷ demonstrated that cotransfection of HeLa cells with CRHR-CFP and VT2R-YFP resulted in a constitutive resonance energy transfer between the fluorescence donor and acceptor. Addition of either CRH or AVT alone resulted in a slight, but not a statistically significant, enhancement of FRET efficiency, whereas simultaneous addition of the two hormones resulted in a significant $(p<0.001)$ increase in FRET efficiency (Fig. 5 ). The FRET efficiency reached 29–36%, and the distance between subunits was $6–7\mathrm{nm}$ .

Fig. 5

FRET efficiency in CRHR and either VT2R or VT2R/ ${\beta }_2$ AR-expressing HeLa cells following treatment with CRH and AVT. HeLa cells were either transfected with plasmids expressing CRHR-CFP and VT2R-YFP, CRHR-CFP and $\mathrm{VT}2∕{\beta }_2$ AR-YFP, or with $\mathrm{VT}2∕{\beta }_2$ AR-CFP and CRHR-YFP and $24\mathrm{h}$ later were treated with either vehicle or $100\mathrm{n}\mathrm{M}$ CRH and $100\mathrm{n}\mathrm{M}$ AVT together. FRET measurements were taken $30\min$ after hormone addition. Data are the means $\pm \mathrm{SEM}$ $(n=5)$ . ^*FRET efficiency was significantly different $(p<0.011)$ than that measured in HeLa cells expressing CRHR-CFP and VT2R that were not treated with the hormones. ^**FRET efficiency was significantly different $(p<0.008)$ than that measured in HeLa cells expressing CRHR-CFP and $\mathrm{VT}2∕{\beta }_2$ AR-YFP that were not treated with the hormones. ^*** FRET efficiency was significantly different $(p<0.02)$ than that measured in HeLa cells expressing CRHR-YFP and $\mathrm{VT}2∕{\beta }_2$ AR-CFP that were not treated with the hormones.

Quantitative FRET analysis demonstrated that substitution of TM4 from the VT2R with TM4 from the ${\beta }_2\mathrm{AR}$ affects the interaction between the two receptor proteins (Fig. 5). FRET analysis of HeLa cells cotransfected with CRHR-CFP and VT2R/ ${\beta }_2$ AR-YFP (Figs. 3 and 5) after separate or simultaneous addition of CRH and AVP hormones demonstrated that the FRET efficiency was 8–12% and the distance increased to $9–12\mathrm{nm}$ . As with VT2/ ${\beta }_2$ AR-CFP and CRHR-YFP receptors, FRET efficiency was 9–13% and the distance increased to $10–12\mathrm{nm}$ .