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Molecular Endocrinology, doi:10.1210/me.2007-0063
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Molecular Endocrinology 21 (8): 1997-2008
Copyright © 2007 by The Endocrine Society

Mapping the Architecture of Secretin Receptors with Intramolecular Fluorescence Resonance Energy Transfer Using Acousto-Optic Tunable Filter-Based Spectral Imaging

Cayle S. Lisenbee, Kaleeckal G. Harikumar and Laurence J. Miller

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259

Address all correspondence and requests for reprints to: Laurence J. Miller, M.D., Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, Arizona 85259. E-mail: miller{at}mayo.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production. A similar CFP insertion into the third intracellular loop resulted in a plasma membrane-localized receptor that bound secretin and signaled normally. This fully active third-loop variant exhibited a significant decrease in fluorescence resonance energy transfer signals that were recorded with an acousto-optic tunable filter microscope after exposure to secretin agonist but not to a receptor antagonist. These data demonstrate changes in the relative positions of intracellular structures that support a model for secretin receptor activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G PROTEIN-COUPLED RECEPTORS (GPCRs) are heptahelical, integral plasma membrane proteins that are activated by a diverse set of pharmacologically important molecules to initiate intracellular signaling cascades. Several members of this superfamily have become important targets for therapeutic intervention in disease. Drugs that act at these receptors target their natural ligand-binding sites, as well as other recognized sites for allosteric drug action. Although current approaches to drug discovery have been successful, much remains to be learned of the conformational changes that occur during receptor activation. It is hoped that such an understanding might open new pathways for the development and refinement of receptor-active drugs.

Data from spin labeling, photoaffinity labeling, and receptor mutagenesis approaches have led to a proposal for a common molecular mechanism of GPCR activation (1, 2). Best understood for rhodopsin and other class I receptors, this mechanism involves the communication of ligand-induced conformational information across the transmembrane (TM) core for the activation of G protein-mediated guanine nucleotide exchange at the intracellular face of the molecule. In rhodopsin, this communication process includes documented shifts in the relative positions of certain membrane-spanning helices (3). Some of these same movements have been inferred for class II receptors, such as the shift in the relative positions of helices 3 and 6 in the PTH receptor (4). However, structural differences between class I and II receptors suggest that these rearrangements may not be extrapolated to all heptahelical GPCRs because the helical bundle of class II receptors is predicted to be structurally divergent from that of class I receptors (5).

Several members of this small subset of GPCRs, including the glucagon-like peptide and PTH receptors, have garnered interest as drug targets for the treatment of diabetes and bone disorders. The SecR is a prototypic member of this family that stimulates pancreaticobiliary ductular bicarbonate secretion during binding a 27-residue peptide agonist (6). Ligand-binding studies have demonstrated spatial approximations of secretin agonist analogs with the receptor amino-terminal domain (7, 8, 9, 10, 11, 12) and with the top of TM6 (13, 14). The amino terminus is thought to provide a docking platform for natural peptide ligands that changes conformation during agonist binding to expose an endogenous, agonist ligand that can interact with the receptor core (15). Comprising as little as three residues within a short loop sequence of the amino-terminal domain of the receptor, this secondary ligand acts with full agonist potency through the top of TM6, in which it is presumed to induce changes in the helical bundle, which then stimulates signaling through Gs-mediated activation of adenylate cyclase (15). However, the precise means by which these changes are propagated to the intracellular face of the receptor for G protein coupling, stimulation, and turnover are poorly understood.

Current understanding of the activation of class II GPCRs has come from photoaffinity labeling and biophysical studies performed with peptide agonist analogs containing photolabile or fluorescent residues (7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18). Because these interactions have provided snapshots of receptor structure only from the perspective of the extracellular environment, our goal in the present work was to develop new tools that would allow for real-time monitoring of the intracellular architecture of the receptor in living cells. Therefore, we constructed and characterized a series of dual reporter-tagged SecR variants in which enhanced cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) tags were incorporated strategically within functionally relevant intracellular loop and carboxyl-terminal domains of the SecR. Integration of these tags effectively placed positional markers for two different receptor domains within the same receptor molecule, in which it was predicted that conformational rearrangements could be detected as changes in fluorescence resonance energy transfer (FRET) between the fluorescent reporters. This approach was introduced in kinetic analyses of the activation of PTH and {alpha}2A adrenergic receptors (19, 20), but that work used a limited number of receptor variants for which the energy transfer changes at best represented only 20% of the total signal.

We attempted to expand the scope of that previous study by incorporating CFP systematically into each of the three intracellular loops of the SecR. These mutants comprised a unique set of receptors that included fully functional variants, as well as those in which biological activity was uncoupled from high-affinity binding. In its first application, an acousto-optic tunable filter (AOTF) system was used in conjunction with standard epifluorescence microscopy for the collection of wavelength-registered serial fluorescence emission micrographs. Emission intensities from these images were compiled into pixel-specific spectra that, through careful correction and analysis, provided enhanced flexibility over fixed-bandwidth filter methods for calculating FRET ratios at the single-cell level. Moreover, FRET signals that were localized to the plasma membrane in a fully functional dual reporter receptor construct were used for analysis of changes that might occur after agonist binding. These data support a prediction of how the intracellular third loop and carboxyl-terminal domains of this receptor move apart during ligand binding.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction, Expression, and Subcellular Localization of SecR Constructs
Primary amino acid sequences of three SecRs and seven closely related vasoactive intestinal polypeptide receptors were aligned with the CLUSTALW algorithm to locate candidate sites within the predicted intracellular loop domains of the SecR that may tolerate the insertion of a CFP reporter. Figure 1Go shows these alignments and the positions that were chosen based on the following criteria: 1) the relative conservations of specific loop residues, 2) the need to avoid the less well-defined boundaries between the predicted loop and TM domains, and 3) the demonstration of a successful insertion point in another receptor. All but one of the first-loop residues were fully conserved and thus forced attempted insertions into the middle two positions of the loop. The residues predicted to comprise the second loop were less well conserved than those of the shorter first loop, but only two of these amino acids provided favorable insertion points near the middle of the loop. The third and longest loop exhibited the most variability; here, the insertion site was chosen to match that used in the construction of a dual reporter variant of the PTH receptor (19). CFP insertions into each of these sites within carboxyl terminally tagged SecR-YFP yielded a series of dual fluorescent reporter protein-tagged constructs for the current studies (Fig. 1Go). Analogous insertions within an untagged, wild-type SecR yielded the appropriate donor-only controls.


Figure 1
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  		Fig. 1. Structural Representations of Membrane-Bound SecR and Dual Reporter-Tagged SecR Constructs

The diagrams on the left compared with an unmodified wild-type receptor (A), three groups of receptor constructs having CFP in the first (two positions) (B), second (two positions) (C), or third (one position) (D) intracellular loops, and YFP at the carboxyl terminus. The predicted amino acid sequences on the right correspond to the intracellular loops (bold) of human SecR and their degrees of similarity to other secretin and closely related vasoactive intestinal polypeptide receptors (*, fully conserved residue; :, conservation of strong groups; ., conservation of weak groups; space, no consensus). Numbers refer to the nucleotide positions after which the open reading frame of the CFP reporter was inserted (arrowheads). Donor- and acceptor-only control constructs lacked the appropriate YFP or CFP moieties, respectively (data not shown). Diagrams modified from Vilardaga et al. (19 ).

 
Each of the new dual reporter receptor constructs was expressed individually in COS cells for microscopic assessment of subcellular localization. Figure 2AGo shows a single optical section of a representative cell that possessed intracellular and plasma membrane fluorescence attributable to transiently expressed SecR-YFP within biosynthetic and cell-surface compartments. Similar localization patterns were observed via YFP fluorescence for all three second- and third-loop receptor constructs, although larger proportions of the total fluorescence signal appeared to originate from receptors in the endoplasmic reticulum and Golgi. The two first-loop receptor constructs were completely retained within these organelles, as well as within endoplasmic reticulum-associated aggregates at the cell periphery, and were not detected in the plasma membrane. Summarized in Table 1Go, these data confirm that the addition of YFP to the carboxyl terminus of SecR had no apparent effect on plasma membrane localization and that receptors having CFP insertions in the second and third intracellular loops, but not the first loop, retained their ability to sort to the cell surface.


Figure 2
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  		Fig. 2. Fluorescence Localizations of SecR Constructs

Representative confocal micrographs (single optical sections) of formaldehyde-fixed COS cells exhibiting plasma membrane and/or intracellular fluorescence attributable to direct excitation of the YFP reporter of receptor constructs. Unlike the YFP-tagged wild-type receptor (A), the first-loop variants SecR(CFP\#2303;510)-YFP (B) and SecR(CFP\#2303;513)-YFP (C) did not sort to the plasma membrane and instead remained trapped within the endoplasmic reticulum and reticulum-associated aggregates. All other receptor variants sorted normally to the plasma membrane, including the second-loop constructs SecR(CFP&2303;732)-YFP (D) and SecR(CFP\#2303;735)-YFP (E), and the third-loop construct SecR(CFP\#2303;993)-YFP (F). Scale bar, 25 µm.

 

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  		Table 1. Functional Characteristics of SecR Mutants

 
Functional Characterization of Receptor Ligand Binding and Signaling
The dual reporter receptor constructs capable of providing the most relevant structure activity information would be those that exhibit binding and signaling characteristics similar to wild-type receptors. The intact cell binding assay results shown in Fig. 3Go indicate that both the second- and third-loop variants satisfied this metric for ligand binding. Specifically, all four receptors that possessed a CFP reporter in either of two positions within the second intracellular loop bound secretin saturably and with high affinity. The dissociation constant (Ki) values listed in Table 1Go show that their affinities were comparable with the nanomolar affinities typical of wild-type SecR, although the second-loop variants bound only one fourth the level of radiolabeled secretin that was bound by wild-type receptors. Similar results were observed for the third-loop variant, with this receptor binding with nanomolar affinity to that of wild-type receptor (Table 1Go). Neither of the first-loop variants exhibited saturable binding in intact COS cells, even at micromolar concentrations of ligand, consistent with the absence of these receptors within the plasma membrane. Cumulatively, these data suggest that the second and third intracellular loops of SecR tolerate reporter protein insertions and fold correctly for the maintenance of ligand binding function.


Figure 3
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  		Fig. 3. Ligand Binding Capabilities of Donor-Only and Dual Reporter SecRs

Each panel demonstrates the abilities of increasing concentrations of natural secretin agonist to compete for the binding of a radioiodinated [125I-Tyr10]secretin analog to intact COS cells expressing the first-loop (A), second-loop (B), or third-loop (C) receptor variants indicated. Data represent saturable binding as percentages of the maximal responses achieved in the absence of competitor (means ± SEM from three or more experiments performed in duplicate).

 
Figure 4Go shows in graphical form the results of biological activity analyses. Unlike fully functional wild-type and carboxyl-terminal YFP-tagged receptors, variants having a CFP moiety within the first intracellular loop were incapable of generating cAMP during treatment of intact cells with stimulatory concentrations of secretin. As shown in Table 1Go, these receptors produced cAMP levels that were at or below the background levels (≤1 pmol) recorded in the absence of agonist. Receptors with second-loop CFP insertions also were unable to elicit a secretin-stimulated cAMP response, despite their presence on the cell surface for ligand binding that was comparable with wild-type receptors. Conversely, the third-loop variant stimulated levels of cAMP representing more than half the maximal response exhibited by wild-type receptor. Addition of a carboxyl-terminal YFP to either untagged or third-loop CFP receptors allowed at least three fourths the maximal output of cAMP observed in wild-type receptors. The insertion of CFP within the third intracellular loop allowed for two thirds maximal signaling and secretin efficacy to within two orders of magnitude compared with wild-type receptor (Table 1Go). Together with the sorting and binding analyses, these results demonstrate that the first-, second-, and third-loop variants provide nonfunctional, partially functional, and fully functional examples, respectively, of single- and dual-tagged SecRs for structure activity studies. Based on these characterizations, only the functional third-loop variant was used for FRET studies.


Figure 4
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  		Fig. 4. Biological Activities of Donor-Only and Dual Reporter SecRs

Each panel shows the relative levels of intracellular cAMP produced during stimulation with increasing concentrations of secretin of approximately 25,000 COS cells expressing the first-loop (A), second-loop (B), or third-loop (C) receptor variants indicated (means ± SEM of duplicate data points from three or more independent experiments).

 
Receptor FRET
The efficiency of energy transfer in FRET applications depends on both the distance between the donor and acceptor fluorophores and their orientation relative to each other. The latter typically is excluded as contributing to the FRET signal by demonstrating that the fluorophores are adequately mobile with sufficient rotational freedom. Therefore, it was important to measure fluorescence anisotropy in the same cellular expression system and conditions to be used for the FRET studies. For the third-loop dual reporter receptor construct expressed in intact cells, fluorescence anisotropy measurements supported adequate rotational freedom for both fluorophores, when both the receptor was unbound and it was occupied by secretin (Fig. 5Go). Anisotropy of the CFP donor was 0.21 ± 0.001 in the control state and 0.32 ± 0.003 after secretin binding. Similarly, anisotropy of the YFP acceptor was 0.01 ± 0.002 in the control state and 0.05 ± 0.004 after secretin binding. Of note, the anisotropy of both donor and acceptor increased after agonist binding, suggesting a decrease in mobility, although it was still adequate for valid FRET measurements.


Figure 5
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  		Fig. 5. Fluorescence Anisotropy of the Third-Loop Dual Reporter SecR Construct Expressed in CHO Cells

Anisotropy measurements were performed in intact cells with or without secretin treatment after excitation at 435 nm. Values are represented as mean ± SEM values of five different measurements.

 
The functional third-loop dual reporter receptor construct also was evaluated microscopically for its ability to exhibit resonance energy transfer during excitation of the donor fluorophore. FRET signals were determined from single cells by a spectral imaging method that included the use of an AOTF for the collection of a series of images at incremental emission wavelengths. As described in Materials and Methods, this procedure required the use of a blue-shifted filter for CFP excitation that limited bleedthrough attributable to direct excitation of the acceptor to no more than 1% of the signal recorded at the excitation maximum of YFP. Photobleaching also was negligible because of the rapid response time of the acoustically tuned emission filter; each image was acquired at millisecond exposure times that allowed for total capture durations of 10–45 sec for an entire image series. Figure 6AGo shows a representative series collected in 5-nm increments from 460–600 nm during excitation at 400 nm of a single living COS cell transiently expressing this receptor construct. As a whole, the fluorescence intensities of the images varied with acquisition wavelength according to the expected emission signatures of the incorporated CFP donor and YFP acceptor. Specifically, virtually no fluorescence was detected at the ends of the spectral region scanned from 460–480 and 580–600 nm, whereas peak intensities occurring at 520–550 nm fell within a range of prominent signals that were detected from 480–580 nm. Analogous data were acquired from cells expressing the appropriate control receptors tagged singly with only the donor or acceptor fluorophore. Although image quality was traded in these experiments for camera sensitivity, all receptors were observed routinely within the same intracellular and plasma membrane compartments that were seen in the confocal analyses shown in Fig. 2Go.


Figure 6
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  		Fig. 6. FRET Analyses of the Third-Loop SecR Construct

A, Fluorescence micrographs of a representative dual reporter receptor-expressing COS cell excited with a CFP-specific bandpass filter. Fluorescence emissions were selected with an AOTF and captured with a CCD camera set to collect images in 5-nm increments from 460 to 600 nm. The final panel shows the cellular and background ROI from which wavelength-specific intensity data were derived. Scale bar, 50 µm. B, Microscopically derived fluorescence emission spectra of living COS cells expressing the receptor variant. The spectra were generated by plotting as a function of the acquisition wavelength the background-subtracted, normalized fluorescence intensities of cellular ROIs similar to the example depicted in A. Cells expressing CFP donor-only ({lambda}ex of 400 nm) or YFP acceptor-only ({lambda}ex of 480 nm) control receptors exhibited peak emissions at 505 and 525/530 nm, respectively, whereas cells expressing dual reporter receptors ({lambda}ex of 400 nm) displayed donor-suppressed FRET emissions that peaked at wavelengths corresponding to the YFP acceptor. C, Fluorescence emission spectra of CHO cells expressing the dual reporter receptor before and after photobleaching at 525 nm for the noted period of time (minutes). The spectra represent average background-subtracted fluorescence intensities within the plasma membranes of four different cells excited at the donor-specific wavelength ({lambda}ex of 400 nm).

 
Wavelength-registered intensity information included in the images in Fig. 6AGo was extracted from appropriate pixels of interest for the subsequent correction and quantification of spectral relationships, including FRET. In these experiments, "appropriate" pixels were those within custom-shaped regions of interest (ROIs) defined by thresholding total cellular fluorescence that included both intracellular and plasma membrane organelles. Figure 6BGo shows the spectral profiles of the average pixel intensities within these regions, as well as within similar regions from image sets of representative controls, after background subtraction and normalization. Cells expressing the CFP-only third-loop control receptor gave two peaks of fluorescence at 480 and 505 nm, whereas those expressing the YFP-only carboxyl-terminal control receptor gave a single peak of fluorescence at 525 nm. The broad emission spectrum of cells expressing the dual-tagged third-loop construct included emissions that coincided with the sharper fluorescence peaks of the corresponding donor-only and acceptor-only controls. The normalized intensities of the CFP portion of this signal were approximately one fifth of the intensities of the YFP portion, suggesting that the former was suppressed by donor quenching attributable to FRET. All fluorescence signals were confirmed independently in spectrofluorometry assays that used suspensions of live, receptor-expressing COS cells (data not shown).

Intensity data from the third-loop dual reporter receptor and its corresponding donor-only control were integrated across CFP- and YFP-specific wavelengths to remove the large proportion of donor signal that was observed in the acceptor portion of the spectrum (Fig. 6BGo). This method for correction of donor cross-talk was verified by quantification of donor fluorescence after acceptor photobleaching, as described by Miyawaki and Tsien (21). These experiments resulted in the expected dequenching of the donor signal, as shown in Fig. 6CGo.

Spectral Microscopic Analyses of Stably Expressed, Plasma Membrane-Localized Third-Loop Receptor Constructs in Ligand-Treated Cells
The FRET signal observed between the third intracellular loop and carboxyl terminus of the functional third-loop construct provided an opportunity to study the predicted conformational changes of these domains during ligand-mediated receptor activation. Stable transfections were used to minimize experimental differences associated with different levels of expression that could come from transient transfection experiments. In the Chinese hamster ovary (CHO) cell system, these receptors bound secretin saturably (Ki of 1.89 ± 0.15 nM) and with an affinity comparable with that recorded from a well-characterized cell line that stably expresses wild-type human SecR (Ki of 0.71 ± 0.06 nM). Similar results were obtained for a separate CHO cell line prepared to express the SecR(CFP 993) third-loop control receptor (Ki of 1.02 ± 0.20 nM).

Figure 7AGo shows two micrographs from a spectral dataset that depict fluorescence emissions at 460 and 525 nm from a representative CHO cell stably expressing the third-loop receptor construct. Like the COS cell example in Fig. 6AGo, the emission intensity varied with wavelength according to the predicted outputs of the CFP and YFP reporters. Specifically, the prominent signal at 525 nm included plasma membrane labeling that was essentially absent at 460 nm but was consistent with the localization of this receptor in COS cells (Fig. 2FGo). These and similar data acquired from the same cells after 30-min treatments with SecR ligands were used to generate the emission spectra shown in Fig. 7BGo, which represent intensity information from ROIs that included only fluorescence attributable to receptors localized within the plasma membrane. The spectra show that stimulatory concentrations of secretin (0.1 µM) caused a marked decrease in plasma membrane YFP fluorescence at 525 nm relative to the CFP signal at 480 nm. These conditions did not change the broad CFP emission profiles of donor-only control receptors (Fig. 6BGo). Shown in Fig. 7CGo, this difference corresponded to a two thirds decrease in FRET that was not observed in cells that were treated with the SecR antagonist PG97-269 [Ac-His', D-Phe2, K15, R16, L27]VIP(3–7)/GRF(8–27) peptide. All treatments were performed at or near 4 C to minimize the downstream processes of internalization and desensitization over and subsequent to the 30-min incubation period. The FRET decrease was not attributable to cell-to-cell variations in expression level or overall photobleaching during image acquisition, because the data were normalized on a cell-by-cell basis against the steady-state signals achieved before ligand applications. In addition, bleedthrough corrections were performed separately using coefficients calculated both before and after treatments to ensure that changes in the FRET signal were not attributable to donor quenching that was independent of the FRET response. Finally, it is unlikely that the observed FRET signals were derived from intermolecular energy transfer between stably oligomerized receptors, because secretin has been shown to have no effect on intermolecular bioluminescence resonance energy transfer signals recorded from tagged SecRs in agonist-stimulated cells (22). Because wild-type fluorescent proteins in the GFP family are known to have a propensity to dimerize, it would be ideal to use the A206K variant of these proteins in this type of study in the future, to be most confident that the signal is as specific as possible (23). Nevertheless, the observation in the current work that a significant decrease in FRET was observed only during agonist treatment suggests that it reflects a translational and/or rotational response of the incorporated fluorophores as a measurable characteristic of secretin binding.


Figure 7
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  		Fig. 7. FRET Analyses of Third-Loop SecR Constructs Expressed Stably in CHO Cells

A, Representative stack of wavelength-registered fluorescence micrographs showing SecR(CFP 993)-YFP receptors localized to the plasma membranes and biosynthetic organelles of living cells in culture. AOTF-selected emissions were collected from the same cells incubated at 4 C before and after treatment with secretin agonist or PG97-269 antagonist. The two squares mark ROIs over noncellular background and plasma membrane portions of the images from which intensities were calculated for generating emission spectra and FRET ratios. Scale bar, 5 µm. B, Fluorescence emission spectra of living CHO cells expressing the indicated third-loop donor-only control or dual reporter receptors in the absence or presence of 0.1 µM secretin. The spectra represent average background-subtracted and normalized intensities within plasma membrane ROIs of cells excited at donor-specific wavelengths ({lambda}ex of 400 nm). C, Plasma membrane-specific FRET ratio of ligand-treated CHO cells. FRET signals were determined for the same cells untreated and treated with agonist and antagonist. The data represent signals obtained from approximately 15 cells in at least five spectral datasets. **, P < 0.001; the values represented by the first and third bars were not significantly different from each other (P > 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Resonance energy transfer techniques represent powerful approaches for elucidating various aspects of GPCR structure (24, 25, 26, 27). Applied to soluble nuclear receptors, this approach has been used successfully in the demonstration of a hormone-induced conformational change in androgen receptors that had been appended at their amino and carboxyl termini with CFP and YFP reporters (28). However, this method has not been reported often for complex membrane receptors, most likely because the inclusion of multiple bulky fluorophores leads to defects in receptor sorting and/or function. This certainly was the case in the present study (see below), as it was in a previous report of intramolecular FRET in GPCRs, a closely related study that determined the kinetics of conformational changes within dual CFP/YFP-tagged constructs of the {alpha}2A adrenergic and PTH receptors (19). Like the present report, only third-loop constructs were analyzed in that study because all others exhibited functional defects and were determined to be unfit for kinetic analyses. A recent report attempted to address these concerns by targeting a much smaller biarsenical fluorescein acceptor to a tetracysteine motif that had been incorporated into a CFP-tagged receptor (29).

Although the dual reporter SecRs prepared for the current study were designed anticipating possible application to intramolecular FRET, their characterizations provided valuable information on the functional importance of the intracellular loop and carboxyl-terminal domains of the receptor. For example, appending SecR with a carboxyl-terminal YFP had no measurable effect on the plasma membrane sorting, ligand binding, or cAMP signaling capabilities of the modified receptor. Located at the end of what is presumed to be a flexible tail region, this position has been shown to tolerate various reporter proteins with little effect on function (22), even when truncated to remove several known phosphorylation sites for receptor regulation (30, 31). Conversely, insertions of a CFP moiety into the first intracellular loop resulted in nonsorting receptors that remained essentially trapped within biosynthetic organelles. This sorting defect effectively limited their exposure to applied ligand and thus prevented secretin binding and biological activity. The first loop was an interesting candidate for tag insertion in that modification of a nearby histidine residue at the cytosolic end of the second TM helix created a fully functional receptor with moderate constitutive activity (32). Placement of the CFP tag at either of two positions within the small, highly conserved loop yielded no significant differences in sorting, and neither position afforded the constitutive activity observed with the single histidine substitution. Each of the first-loop variants did, however, exhibit CFP, YFP, and FRET fluorescence signals that indicated at least some level of fusion protein maturation. Although the presence of fluorescent aggregates in some transfected cells suggested likely disruption in the folding and/or maturation of these receptors, it seems clear from the data presented here that the first intracellular loop may provide a turn region that is critical for the proper insertion of the first and second TM helices into the membrane or for the proper spacing and spatial relationships between these helices. Nonetheless, the severity of the functional defects of these variants prevented their use in meaningful FRET studies.

CFP insertions within the second intracellular loop generated a unique set of receptor variants that were functionally distinct from the nonsorting first-loop mutants. The former were found localized in the plasma membrane and were competent for secretin binding with wild-type affinities. Of note, the abilities of these second-loop variants to bind agonist did not correlate with their abilities to stimulate cAMP production, suggesting that agonist binding may have been unable to elicit the conformational changes needed for receptor activation. Similar conclusions were drawn from a PTH receptor mutant in which an endogenous histidine at the cytosolic end of helix three supported the formation of zinc-binding bridges to helix six that inhibited downstream signaling but not ligand binding (4). The second intracellular loop is an important determinant of the efficiency with which a receptor activates its cognate G protein(s) (33), although this characteristic has not been verified to great detail for class II receptors. Nonetheless, it is likely that the steric hindrance exerted by the second-loop CFP prevented SecR from interacting efficiently with its downstream effectors. Although this provides an interesting and unusual means for uncoupling ligand binding from receptor activation, these constructs again could not be used for meaningful FRET studies because of their functional defects.

Of all the receptor variants prepared for the present study, the third-loop variant receptor construct retained function that was most like wild type. Specifically, this receptor bound secretin saturably and with high affinity at the plasma membrane, and this binding was shown to elicit cAMP production as evidence of biological activity. Half-maximal cAMP responses required an effective concentration of secretin (EC50) that was approximately 100 times greater than that recorded for wild-type or carboxyl terminally tagged receptors. This result is consistent with the biological activities of third-loop variants of the {alpha}2A adrenergic and PTH receptors (19), indicating clearly that third-loop CFP insertions most likely obstruct, but do not eliminate, coupling to G protein subunits. Thus, the larger third intracellular loop seems to be a flexible linker that is more tolerant of modifications than other intracellular regions, making this loop the best candidate for insertions of bulky fluorescent reporter proteins.

FRET signals were recorded consistently from the third-loop dual reporter SecR construct, using both a standard spectrofluorometer and our spectral imaging workstation, but the latter provided several advantages for imaging FRET with increased sensitivity at the single-cell level. In particular, spectral imaging circumvented the limitations of standard epifluorescence microscopes that use bandpass filters with fixed cutoff parameters, thus allowing for better spectral separation of fluorescence signatures that could be registered to every pixel in the image (34, 35). In practice, these features allowed for more accurate removal of background autofluorescence and donor bleed-through, the latter of which is a significant factor for the CFP/YFP FRET pair (22, 30). Spectral imaging and linear unmixing have been reported previously (36, 37), and AOTF devices have been applied using static wavelengths (38, 39) and for unmixing multiple spectral signatures that did not include FRET (40). The combination of several of these elements in the current study is significant, because the microsecond switching capabilities of the AOTF instrument (38) make it theoretically possible to detect rapid changes in distances.

The spectral imaging approach provided a highly sensitive means for detecting agonist-induced changes in the FRET signal in cells stably expressing the fully functional third-loop variant. An observed decrease in FRET suggested that the relative positions of the carboxyl terminus and third loop of SecR moved away from each other during binding of agonist but not antagonist. Analogous conclusions were derived from the decreased FRET observed with {alpha}2A adrenergic and PTH receptor dual reporter constructs (19, 20), in which a subsequent study also observed differential effects of specific agonist and antagonist ligands at the former (41). In those studies, it was argued for {alpha}2A adrenergic receptors that the possible movement of the third loop away from the carboxyl terminus correlated with predicted agonist-induced changes in the TM helices of class I GPCRs and that a common mechanism could exist for class I and II receptors. Our current results confirm and extend this experience with a second class II GPCR. Further experimental experience with additional receptors in both families will be important to truly understand how common this mechanism might be.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Molecular biology reagents for receptor mutagenesis were obtained from New England Biolabs (Beverly, MA), Stratagene (La Jolla, CA), Bio-Rad (Hercules, CA), Eppendorf (Hamburg, Germany), and Qiagen (Valencia, CA). Cell culture supplies and Lipofectamine were purchased from Invitrogen (San Diego, CA), serum supplements from HyClone (Logan, UT), and nonenzymatic cell dissociation solution from Sigma (St. Louis, MO). Formaldehyde and Vectashield were provided by Ted Pella (Redding, CA) and Vector Laboratories (Burlingame, CA), respectively. Binding and biological activity assays used [Tyr10]secretin and/or natural rat secretin peptides that were synthesized in our laboratory (42) and shown to be active at human SecRs expressed in intact cells (9). The Tyr10 residue was derivatized by oxidative radioiodination to form the [125I-Tyr10]secretin radioligand as described previously (42). Reagents not listed here were of the highest grade available, as appropriate for the given experiment.

Receptor Mutagenesis
Recombinant coding sequences were expressed constitutively from the cytomegalovirus promoter in the eukaryotic expression vector pcDNA3 (Invitrogen). All receptor mutations were incorporated into the plasmids pcDNA3/SecR and pcDNA3/SecR-YFP, which code, respectively, for native and carboxyl terminally tagged versions of human SecR (30, 43). Its predicted amino acid sequence was aligned with other closely related receptors using the CLUSTALW algorithm and, from these alignments, were determined the most highly nonconserved residues within the intracellular loop regions of the receptor that would be most tolerant of fluorescent protein insertions. These regions corresponded to first-loop positions 510 and 513, second-loop positions 732 and 735, and the third-loop position 993, in which the numbers refer to the nucleotide positions within the human SecR open reading frame after which was inserted the coding sequence for an enhanced CFP (numbering begins with the first nucleotide of the start codon). The latter was amplified from pECFP-N1 (Clontech, Mountain View, CA) with primers that added an in-frame NheI site in place of the start codon and an in-frame XbaI site that replaced the stop codon. The resulting products were ligated by TA cloning into pCRII (Invitrogen) to yield pCRII/NheI-CFP-XbaI. The NheI/XbaI fragment from this plasmid was ligated into NheI-digested versions of pcDNA3/SecR and pcDNA3/SecR-YFP that had been mutated in QuikChange (Stratagene) site-directed mutagenesis reactions with primers that inserted a unique NheI site at each of the nonconserved loop positions identified previously. These reactions yielded a set of CFP donor-only control receptors, as well as the corresponding CFP/YFP dual-tagged variants (Table 1Go). All DNA sequences were confirmed by both restriction digestion and automated dye-terminator cycle sequencing.

Cell Cultures and Transfections
African green monkey kidney (COS) cells were propagated in DMEM and transfected in diethylaminoethyl-dextran solutions as described previously (44). Transfected cells were lifted with trypsin, transferred to coverslips or 24-well plates as appropriate, and then incubated for an additional 24 to 48 h to allow transient gene expression. All transfections received a constant 3 µg of plasmid DNA per 10-cm Petri dish regardless of the downstream application. CHO cells obtained from the American Type Culture Collection (Manassas, VA) were grown in a Ham’s F-12 nutrient mixture containing 5% (vol/vol) Fetal Clone II serum. The cells were passaged twice per week on Corning (Acton, MA) tissue culture flasks and maintained in a humidified atmosphere of 5% (vol/vol) CO2 at 37 C. Stable transfections were accomplished with Lipofectamine and PLUS reagents according to the instructions of the manufacturer. The transfected, G418-resistant cells then were subjected to our standard limiting dilution procedure, followed by screening of the resulting clonal lines in secretin binding and/or fluorescence assays (see below). Those clones exhibiting binding and fluorescence attributes most like YFP-tagged wild-type receptors were characterized further as stably expressing dual reporter cell lines.

Confocal Microscopy
Subcellular localizations of the YFP-tagged receptor variants from each group of dual reporter constructs were evaluated in transfected COS cells seeded to 25-mm coverslips in six-well plates. The adherent cells were fixed immediately after removal from the growth incubator in freshly diluted 2% (wt/vol) formaldehyde in PBS for 15 min at room temperature. The fixed cells were washed in three 5-min exchanges of PBS (pH 7.4) and then mounted on microscope slides in Vectashield to prevent photobleaching. Receptors were localized within cells as described previously (30) using a Zeiss (Thornwood, NY) LSM 510 confocal microscope that was configured specifically for capturing YFP emissions. Digital images were background subtracted, adjusted for contrast when necessary, and then assembled into figures using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).

Receptor Binding Assays
Transfected cells in 24-well plates were tested for the ability of expressed SecR variants to bind secretin agonists saturably and with high affinity (30). Cells were washed twice in Krebs-Ringers-HEPES (KRH) solution [in mM: 25 HEPES (pH 7.4), 104 NaCl, 5 KCl, 1 KH2PO4, 1.2 MgSO4, and 2 CaCl2] containing 0.2% (wt/vol) BSA and 0.01% (wt/vol) soybean trypsin inhibitor and then were treated with increasing amounts of unlabeled secretin (from 0 to 1 µM) for 1 h at room temperature in the presence of constant amounts of radiolabeled [125I-Tyr10]secretin (~20,000 cpm). Bound radioactivity collected from alkaline-lysed cells was quantified in a {gamma}-spectrometer for the generation of competitive displacement curves in nonlinear regression analyses performed with Prism 3.02 (GraphPad Software, San Diego, CA). LIGAND software (45) was used to calculate Ki values for those receptors at which 1 µM unlabeled secretin displaced at least 70% of the bound radioligand.

Receptor Activity (cAMP) Assays
Receptor-expressing cells in 24-well plates also were tested for their ability to stimulate the production of cAMP signaling molecules in response to agonist treatment (44). Briefly, intact PBS-washed cells were exposed to increasing amounts of secretin (from 0 to 1 µM) diluted in KRH containing 0.2% (wt/vol) BSA, 0.01% (wt/vol) soybean trypsin inhibitor, 0.1% (wt/vol) bacitracin, and 1 mM 3-isobutyl-1-methylxanthine for 30 min at 37 C. The cells then were acid lysed and neutralized as described previously (44). The resulting supernatants were applied to radioactivity-based (Diagnostic Products, Los Angeles, CA) or fluorescence-based (PerkinElmer, Wellesley, MA) competition binding assays according to the instructions of the manufacturer (the former were discontinued during the course of this study). Bound [3H]cAMP was recorded with a liquid scintillation counter; time-resolved FRET in LANCE (lanthide chelate excitation) assays was measured with an EnVision 2103 multilabel plate reader (PerkinElmer). In both cases, counts were compared with standard curves for quantification of cAMP production and analysis of saturable displacements with Prism software. Second messengers produced by the first- and second-loop receptor variants represented less than 10% of the maximal response elicited by wild-type receptors.

Fluorescence Anisotropy Measurements
Anisotropy measurements at room temperature were recorded using a Fluoromax 3 spectrofluorophotometer equipped with an automatic polarizer and a thermostatically regulated cuvette holder, as we described previously (17). The polarizer was aligned with excitation at 0° and emission at 55°. Fluorescence measurements were performed using cells expressing the third-loop dual reporter construct at a wavelength optimal for excitation of CFP. The anisotropy measurements were performed using constant wavelength analysis mode with 5 sec integration times at multiple emission wavelengths. Excitation wavelength was fixed at 435 nm, and emission wavelength was varied from 460 to 600 nm with a 10 nm interval.

Spectral Microscopy
Spectral information was collected microscopically from live COS or CHO cells grown on 42-mm coverslips in 6-cm Petri dishes. Transiently transfected COS cells were observed at steady state at 37 C after mounting coverslips in an open cultivation POC-R cell chamber (Zeiss) and then washing and incubating in warm KRH containing BSA and soybean trypsin inhibitor. Above-ambient temperatures were maintained with an aluminum Heating Insert P stage adaptor and Temp control 37-2 digital temperature regulator (Zeiss). Stably transfected CHO cells were observed similarly by mounting coverslips in a prechilled POC-R chamber and rinsing cells with chilled KRH. Below-ambient temperatures for ligand incubations were created conductively by cooling the cell chamber with dry ice that was placed on a prechilled stainless steel plate on top of the inactivated heatable stage adaptor. A temperature probe (Fluke, Everett, WA) was fed through one of the perfusion ports in the stage adaptor and was placed directly into the overlying KRH solution for real-time acquisition of sample temperature. Cold-acclimated cells were imaged as described below and then were treated for 30 min at 2–5 C with secretin or PG97-269 ligands that were added directly to the overlying solution from appropriate 10-fold dilutions in KRH.

Spectral microscopy was performed with a Zeiss Axiovert 200M epifluorescence microscope that was equipped with an AOTF imaging device custom made by Chromodynamics (Lakewood, NJ) (38). Samples were excited with 75-W xenon arc lamp (Zeiss) white light illumination that in the reflected light path was passed through a highly transmissive bandpass 400/45 nm excitation filter (Chroma Technology, Rockingham, VT). Although not optimal for CFP excitation, this filter was combined in a standard epifluorescence filter cube with 440 nm beamsplitter and long-pass 450 nm emission filters (Chroma Technology) for the collection of spectral information that included both CFP and YFP emissions. Signals greater than 450 nm were sent as a whole to the AOTF imaging device, which functioned as a dynamically tuned, incremental bandpass filter for secondary modification of fluorescence emissions. A Photometrics (Tucson, AZ) Cascade 128+ cooled CCD camera placed downstream of the AOTF imaging system was programmed to capture images in 5-nm increments from 460 to 600 nm without dynamic contrast adjustment. At these wavelengths, the AOTF device was capable of outputting very narrow bandwidths that increased evenly with the selected wavelength from approximately 1.5 to 3 nm, although this limited the overall throughput of the system to about 30% (including polarization losses) (38). This limitation was overcome in part by the multiplication gain capabilities of the Cascade camera. Digital 16-bit images were collected as 128 x 128 pixel frames with the camera operated in single-frame mode; exposure times ranged from 25 to 500 ms (no binning) at gain settings of 3024 to 4095 with 2- to 12-fold averaging. AOTF manipulations and image acquisitions were automated with QED InVivo 2.2.0 software (Media Cybernetics, Silver Spring, MD) that had been modified by the manufacturer to include modules for AOTF control. In CHO cell experiments, this software also was used in conjunction with an MCU 28 motorized stage control unit (Zeiss) to mark multiple points of interest on the same coverslip and thus allow for the collection of spectral data from the same cells before and after ligand treatments. Based on these conditions, each spectral dataset consisted of a {lambda}-stack of 29 separate TIFF images that, when combined in series, provided wavelength-specific intensity information for every pixel element of the micrograph.

FRET Calculations
FRET signals were derived from intensity-based spectral information according to Ecker et al. (34) by first selecting an appropriate ROI from a representative cell(s) in each dataset. Whole-cell ROIs for COS cells were selected in MetaMorph 6.3 (Molecular Devices, Sunnyvale, CA) by creating boundaries around thresholded fluorescence emissions; plasma membrane ROIs for CHO cells were selected in QED InVivo as 5 x 5 pixel squares that were positioned at the cell periphery over only those areas of the membrane that appeared to possess authentic CFP and/or YFP fluorescence. It is important to note that plasma membrane ROIs were selected from the same cells in each image series before and after ligand incubation so that comparisons could be determined on a cell-by-cell basis. Average fluorescence intensities within each ROI, including a separate noncellular ROI that represented nonspecific (background) levels, then were exported to an Excel (Microsoft, Seattle, WA) spreadsheet for every image in the spectral dataset. Because each image corresponded to a specific emission wavelength, these background-corrected intensities were normalized for the creation of spectral plots or were integrated over defined bandwidths for the calculation of FRET ratios as follows: [Em520–590 – (Em460–500 x Cf)]/Em460–500. The correction factor, Cf = Em520–590/Em460–500, defined the proportion of the acceptor signal that was attributable to donor bleedthrough and was calculated separately for each experiment from spectral datasets of cells expressing the appropriate CFP control construct that lacked the YFP acceptor. Donor-only controls in ligand experiments were treated and imaged identically to their corresponding dual reporter samples for the calculation of separate, treatment-specific correction factors. Note that correction factor is analogous to the donor bleedthrough coefficient for sensitized emission calculations; acceptor bleedthrough attributable to direct excitation of YFP with the 400/45 nm excitation filter was negligible (≤1% of maximal YFP emission). Spectral plots, bar graphs, and P values (unpaired Student’s t test) were generated in Prism 3.02.


   ACKNOWLEDGMENTS
 
We appreciate the excellent technical assistance of Renee Happs, Laura Bruins, and Delia Pinon. We also thank Elliot Wachman (Chromodynamics, Lakewood, NJ) and Ciprian Almonte (Media Cybernetics, Silver Spring, MD) for helpful discussions and suggestions regarding use and application of the AOTF system and QED InVivo software.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK46577 and the Fiterman Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 15, 2007

Abbreviations: AOTF, Acousto-optic tunable filter; CHO, Chinese hamster ovary; CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; GPCR, G protein-coupled receptors; Ki, dissociation constant; KRH, Krebs-Ringers-HEPES; ROI, region of interest; SecR, secretin receptor; TM, transmembrane; YFP, yellow fluorescent protein.

Received for publication January 31, 2007. Accepted for publication May 4, 2007.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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