Complex chimeras to map ligand binding sites of GPCRs

K.L. Gearing1,2, A. Barnes1, J. Barnett1,3, A. Brown4, D. Cousens4, S. Dowell1, A. Green4, K. Patel5, P. Thomas6, F. Volpe7 and F. Marshall1,8

Departments of 1Gene Expression and Protein Biochemistry, 4Systems Research, 5Screening Sciences, 6Computational Analytical and Structural Sciences and 7Bioinformatics, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK 3Present address: University of the West of England, Bristol, UK 8Present address: Department of Pharmacology, University of Cambridge, Cambridge, UK

2 To whom correspondence should be addressed. e-mail: katy.l.gearing{at}gsk.com


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices. In this study, receptor–ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors. Regions of P2Y1, P2Y2 and BLT1 predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor. Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay. Binding of ligand to a P2Y2/BLT1 chimera confirmed that the ligand binding determinants of BLT1 are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.

Keywords: chimera/expression/GPCR/leukotriene/purine


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The recent publication of the crystal structure of rhodopsin has provided the first view of a G-protein coupled receptor (GPCR) at the molecular level (Palczewski et al., 2000Go). This is a significant breakthrough in the understanding of how this class of proteins functions. However, there are major differences between rhodopsin and ligand activated receptors and the details of how liganded receptors work at the molecular level are unlikely to become clear until further structures have been solved. In the meantime, much of the understanding of how GPCRs bind their cognate ligands has come from targeted mutagenesis studies. Here we describe the use of complex receptor chimeras that have been engineered within the transmembrane (TM) helices to map ligand binding sites.

The P2Y family of receptors was chosen as the subject of this study. There are seven cloned human receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12 and P2Y13) which mediate the cell’s response to extracellular nucleotides. Their principal physiological agonists are ADP (P2Y1, P2Y12, P2Y13), UTP/ATP (P2Y2), UTP (P2Y4), UDP (P2Y6) and ATP (P2Y11) (Parr et al., 1994Go; Communi et al., 1995Go, 1996a,b, Go1997, Go2001; Nguyen et al., 1995Go; Nicholas et al., 1996Go; Schachter et al., 1996Go; Leon et al., 1997Go; Hollopeter et al., 2001Go). Closely related to the P2Y receptors are the UDP-glucose receptor (Chambers et al., 2000Go) and the leukotriene receptors, BLT1 and BLT2, which have unrelated non-nucleotide ligands (Yokomizo et al., 1997Go, Go2000; Kamohara et al., 2000Go). In addition, there are a number of orphan receptors which have sequence homology to the known purinergic receptors, e.g. P2Y5, P2Y9 and P2Y10 (Janssens et al., 1997Go; Li et al., 1997Go).

Several key residues for ligand-induced activation in P2Y1 have been identified and molecular models have been built to explain where ligands might bind to this receptor (Jiang et al., 1997Go; Moro et al., 1998Go, 1999; Hoffmann et al., 1999Go; Jacobson et al., 1999Go; Jacobson, 2001Go). A limited set of mutants of P2Y2 has also been described (Erb et al., 1995Go). Based on these studies, the P2Y receptor ligands are thought to activate by binding to a site buried in the transmembrane region of the protein. This ligand-binding pocket is thought to be composed of several regions of the protein residing on different transmembrane helices. In addition, there is evidence that the extracellular loops play a role in ligand recognition (Hoffmann et al., 1999Go; Moro et al., 1999Go).

Evolutionary trees of GPCRs based on sequence homology have been used to predict potential ligands for orphan receptors. However, GPCRs are complex proteins and whilst they share a basic architecture, they have the ability to recognize and transduce structurally diverse messages to a variety of intracellular signalling pathways. These complexities are reflected in the three-dimensional structure and in their primary amino acid sequences. Ligand predictions based on full-length sequence homologies do not always prove to be correct. For example, sequence alignment of GPCRs shows that the amino acid sequences of the nucleotide binding receptors such as P2Y1 are closely related to each other. A receptor identified originally as GPR16 or P2Y7 and subsequently identified as the leukotriene receptor BLT1 is closely related to P2Y1 at the level of its full-length sequence. BLT1 was originally reported as a potential nucleotide receptor based on homology with P2Y receptors but was subsequently found to be unresponsive to nucleotide ligands (Herold et al., 1997Go). Furthering our understanding of which residues and sequence motifs are important for binding distinct ligands and separating these from the background responsible for other aspects of the receptor’s function may help refine predictions of ligand class. In this study it was proposed that it might be possible to define experimentally a set of non-contiguous regions that together comprise the ligand-binding site of a receptor.


    Methods
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 Abstract
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 Methods
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 Discussion
 References
 
Construction of GPCR expression plasmids

Chimeric receptors were assembled from splice overlap extension of short DNA fragments produced by PCR using standard techniques. Splice junctions are depicted in the amino acid sequences in Figure 2. Full-length and chimeric receptors were cloned in the yeast expression vector p426GPD (Mumberg et al., 1995Go).



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Fig. 2. Amino acid sequences of wild-type and and chimeric receptors.

 
BLT1 and the chimera BLT1/Y2 were modified by addition of an HA tag at the N-terminus (MYPYDVPDYARILK) and were cloned in pcDNA3.

Accession numbers for receptor sequences are u42029 (P2Y1) and u41070 (BLT1). The sequence of P2Y2 is that of Brown et al. (Brown et al., 2000Go).

Transient transfections

For transient transfections, CHO-K1 cells were were seeded in either black, clear-bottomed 96-well culture plates for FLIPR assays at 25 000 cells per well or 75 cm2 culture flasks, to be used for membrane preparation, at 50% confluency prior to transfection. For transfection of plates, 10 µg of DNA were mixed with 80 µl of Lipofectamine in 2.0 ml of Opti-MEM (Life Technologies) and incubated at room temperature for 30 min prior to the addition of 8 ml of Opti-MEM. Then 100 µl of transfection mixture were added to each well. The same mixture was used for transfection of each 75 cm2 flask. Cells were exposed to the Lipofectamine–DNA mixture for 5 h and 2 ml of 20% (v/v) newborn calf serum in DMEM F12 HAM were then added. FLIPR assays or membrane preparations were performed 48 h post-transfection.

P2 membrane preparation

All procedures were carried out at 4°C. Cell pellets were resuspended in 10 mM Tris–HCl, 0.1 mM EDTA, pH 7.4 (buffer A) followed by homogenization for 20 s with an Ultra Turrax and passed five times through a 25-gauge needle. Cell lysates were centrifuged at 1000 g for 10 min to pellet the nuclei and unbroken cells and P2 particulate fractions were recovered by centrifugation at 16 000 g for 30 min. These fractions were resuspended in buffer A and stored at –80°C. Protein concentrations were determined using the bicinchoninic acid (BCA) procedure (Smith et al., 1985Go) using BSA as a standard.

Transient expression of epitope-tagged proteins was visualized with specific mouse 12CA5 antibodies following immunoblotting of P2 membrane fractions.

FLIPR assays

Cells were incubated with 4 µM FLUO-3AM at 37°C in 5% CO2 for 90 min and then washed once in Tyrodes buffer (145 mM NaCl, 10 mM glucose, 2.5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, pH 7.4) containing 2.5 mM probenicid.

Basal fluorescence (11 000–15 000 FIU) was determined prior to drug additions.

Cell fluorescence was monitored ({lambda}ex = 488 nm, {lambda}em = 540 nm) using the FLIPR following exposure to agonist.

Antagonists were added to cells 4 min before addition of agonist.

For each response the peak increase above basal levels in fluorescence was calculated and iteratively curve-fitted using the ALLFIT model. Results are expressed as mean ± s.e.m. from three separate transfection experiments each performed in duplicate.

Yeast strains and assays of ß-galactosidase activity

Yeast strain MMY11 was co-transformed with Gpa1p ‘transplant’ chimeras (Brown et al., 2000Go) and receptor expression plasmids. Assays of FUS1-lacZ induction were performed 24 h after addition of ligands as described previously (Olesnicky et al., 1999Go).

Agonists were dissolved in water [ADP, UTP, 2-methylthio-ADP (2MeSADP)] or EtOH (LTB4, 20-OH-LTB4) and antagonists in DMSO. Experiments with adenosine 3'-phosphate 5'-phosphosulphate (A3P5PS) were carried out at a final DMSO concentration of 1% and ticolubant or GW288327x at 0.1%. Results were expressed relative to the maximum response to control agonist.

[3H]LTB4 filter binding assays

For competition experiments P2 membranes were incubated with ~0.2 nM [3H]LTB4 in 10 mM HEPES, 20 mM MgCl2 pH 7.4 in the presence or absence of competitor compound for 60 min at room temperature. P2 membrane protein fractions (10 µg/well) were used for all competition studies in a total volume of 200 µl; 1 µM LTB4 was used to define non-specific binding. Assays were terminated by vacuum filtration (using a Tomtek 96-plate harvester) over GF/C filters, pre-soaked in assay buffer and the filters were washed twice with 1 ml of ice-cold buffer. Bound radioactivity [corrected counts per minute (ccpm)] was counted by solid scintillation spectrometry using a Microbeta Counter (Wallac). Specific binding (ccpm) was determined and curves were fitted to the specific binding data using the ALLFIT model. Results are expressed as means ± s.e.m. of three separate transfection experiments.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Chimeric receptor design

Despite their overall similarity, differences in sequence between the leukotriene and nucleotide receptors would be expected to be found on faces of the transmembrane helices oriented towards the putative ligand binding pockets of these receptors. Upon alignment of partial non-contiguous sequences encompassing the extracellular regions of the TM helices, the nucleotide and leukotriene receptors fall into clusters that reflect the family of ligand that will bind. This is illustrated in the comparison of P2Y1, P2Y2 and BLT1 shown in Figure 1. The most striking differences are found in TM7.



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Fig. 1. Alignment of receptor partial TM sequences using the Clustal W program. Black shading, residues present in all three sequences; grey shading, residues conserved in two out of three. Asterisks indicate significant differences between purinergic and leukotriene receptors.

 
Using this analysis as a starting point, chimeric receptors were designed in which the extracellular loops and tops of the transmembrane regions of one receptor were transposed on to the intracellular loops and bottoms of the transmembrane segments of a second, related receptor. A molecular model of P2Y1 (Moro et al., 1998Go) was used to aid their design in order to minimize the perturbation of resulting proteins in the TM bundle. Three chimeras were generated: P2Y1/Y2, P2Y2/Y1 and BLT1/Y2. The positions of splice junctions used to generate them are shown in Figure 2.

Ligand-induced receptor activity in yeast

Wild-type and chimeric receptors were transformed into yeast strain MMY11 together with a series of G-protein chimeras in which the five C-terminal amino acids of Gpa1p were exchanged with residues from mammalian G{alpha} subtypes. These Gpa1-G{alpha} transplant chimeras have previously been reported to allow coupling of mammalian GPCRs, including P2Y1 and P2Y2, to the pheromone response pathway in yeast (Brown et al., 2000Go). Expression of this series of G-proteins allowed coupling of the receptors to a pathway which results in the growth of yeast in the absence of histidine and the production of ß-galactosidase.

The resulting yeast strains were tested with known ligands to determine whether the chimeras were functional and, as predicted from the model, retained the ligand binding ability of the receptor from which the superior part of the protein was derived. The results are shown in Figure 3.



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Fig. 3. Dose–response curves of receptors. Data presented are the means of three colonies. (a) Comparison of ATP and UTP responses of P2Y2 and P2Y2/Y1. (b) Comparison of ADP and 2MeSADP responses of P2Y1 and P2Y1/Y2. (c) Antagonism of 2MeSADP responses of P2Y1 and P2Y1/Y2. (d) Comparison of LTB4 and 20-OH-LTB4 responses of BLT1 and BLT1/Y2. (e) Antagonism of LTB4 responses of BLT1/Y2.

 
When co-expressed with Gpa1-G{alpha}q, the P2Y2/Y1 chimera was activated by UTP and ATP (EC50 0.7 and 2.8 µM, respectively), as was the wild-type P2Y2 (EC50 3.5 and 10 µM). This chimera therefore gained the ability to respond to UTP relative to the parental P2Y1, which was not activated by UTP (data not shown).

When tested in the same yeast strain, the P2Y1/Y2 chimera was activated by ADP and 2MeSADP and was antagonized by the selective P2Y1 antagonist A3P5PS (Boyer et al., 1996Go). In addition, P2Y1/Y2 lost the wild-type P2Y2 receptor’s ability to respond to UTP (data not shown). The maximum response to ADP or 2MeSADP for P2Y1/Y2 was ~3-fold lower than that of the wild-type P2Y1 when co-expressed with Gpa1-G{alpha}q. EC50 values observed for P2Y1/Y2 were 8.8 µM (ADP) and 1.7 µM (2MeSADP) compared with P2Y1 EC50 values of 2.3 µM (ADP) and 0.3 µM (2MeSADP).

BLT1/Y2 in MMY11 expressing Gpa1-G{alpha}q was activated by LTB4 (EC50 5.5 nM) and 20-OH-LTB4 (EC50 21 nM) but was inactive in the presence of UTP. Since the wild-type BLT1 was not functional in MMY11 expressing Gpa1-G{alpha}q (see below), this receptor was tested in yeast expressing a Gpa1-G{alpha}i1 chimera. EC50 values of 46 nM (LTB4) and 130 nM (20-OH-LTB4) were observed for BLT1 in this strain. The full-length BLT1 receptor from which BLT1/Y2 was derived was responsive to LTB4 and 20-OH-LTB4 but not to UTP or ATP. In contrast, P2Y2 was responsive to UTP and ATP but not LTB4 or 20-OH-LTB4. Furthermore, the BLT1/Y2 chimera was antagonized both by Ticolubant, a selective competitive antagonist of BLT1 (Daines et al., 1996Go), and GW288327x (CP105696) (Griffiths et al., 1995Go), a non-competitive antagonist of the wild-type receptor. Similar results were obtained when the BLT1/Y2 chimera was tested in yeast expressing a Gpa1-G{alpha}i1 chimera (data not shown).

Further constructs were designed in which either one, two or three of the P2Y1 extracellular segments were exchanged for P2Y2 sequences (Figure 4), rather than exchanging all four segments together (as in the P2Y2/Y1 chimera described above). These intermediate chimeric receptors were all unresponsive to ADP, ATP and UTP.



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Fig. 4. Additional chimeric P2Y1/Y2 receptors which did not respond to nucleotide ligands when expressed in yeast.

 
G{alpha} specificity of chimeric receptors

Using the yeast assay we were also able to test whether the G-protein coupling characteristics of these receptors have been separated from their ligand binding properties in this system.

Each of the wild-type and chimeric receptors were transformed into yeast together with a series of G-protein constructs expressing wild-type yeast Gpa1p or chimeras of Gpa1p with G{alpha}16, G{alpha}o, G{alpha}s, G{alpha}14, G{alpha}q, G{alpha}i1 or G{alpha}i3 (Table I). Wild-type P2Y2 was found to couple to some degree through all of these. P2Y1 only showed ligand-induced activity with four of the Gpa1p chimeras: Gpa1-G{alpha}q, Gpa1-G{alpha}14, Gpa1-G{alpha}i1 and Gpa1-G{alpha}i3. BLT1 was only active when co-expressed with G{alpha}i1 or G{alpha}i3 chimeras. The coupling profiles of P2Y1/Y2, P2Y2/Y1 and BLT1/Y2 were compared qualitatively with those of the full-length receptors from which they were derived (Table I). In contrast to the limited coupling specificity of unmodified BLT1, BLT1/P2Y2 had a broad coupling to all the G-protein chimeras, similar to P2Y2. The P2Y2/Y1 chimera coupled only to Gpa1-G{alpha}q, Gpa1-G{alpha}14, Gpa1-G{alpha}i1 and Gpa1-G{alpha}i3, as seen for P2Y1. P2Y1/Y2 coupling was also only seen with these four chimeric G-proteins.


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Table I. Yeast strain MMY11 co-transformed with expression plasmids for receptor and G-proteins assayed for concentration-dependent activation of receptora
 
BLT1/Y2 activity in mammalian cells

CHO-K1 cells were transfected with BLT1/Y2 or BLT1 expression constructs. Measurement of calcium signalling in a FLIPR assay showed that the transiently expressed chimeric receptor responded to LTB4 with similar potency to that observed at the wild-type receptor although with reduced efficacy (Figure 5a and b). LTB4-mediated responses at the chimeric receptor were ~10–20% of those observed with BLT1. In addition, Ticolubant was shown to be an antagonist at both receptors in the FLIPR assay but had no effect on P2Y2 responses (data not shown).





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Fig. 5. (a, b) The effect of SB209247 on LTB4-mediated calcium responses determined in the FLIPR assay. Data points are plotted as mean ± s.e.m. responses in fluorescence intensity units (FIU) determined from three separate transfections each performed in duplicate. (c) Competition binding for LTB4 displacement of [3H]LTB4 binding to crude membrane fractions. Data are plotted as mean ± s.e.m. specific binding determined from three separate transfections. (d) Immunoblot of HA epitope-tagged BTL1/Y2 (A) and BLT1 (B) from membrane fractions. Expression of HA tagged protein was determined from three separate transfections and compared with Mock transfected cells (M).

 
[3H]LTB4 binding at the wild-type and chimeric receptors was compared in transiently transfected Chinese hamster ovary (CHO) cells (Figure 5c). Levels of specific binding were similar in all transfections and ligand was displaced by LTB4 with equal IC50s for both the chimeric and wild-type receptor.

Transiently expressed receptor was visualized by western blotting and detection using antibodies directed against the N-terminal HA tags. Protein of the correct size (~52 kDa) was observed as a doublet for both receptor constructs and was not seen in mock transfected cells (Figure 5d). However, the chimeric receptor appeared to be expressed at lower levels than the wild-type BLT1 in this system.


    Discussion
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 Abstract
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 Methods
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 References
 
The aim of this study was to improve our understanding of how receptors recognize their cognate ligands at the molecular level. Understanding how GPCRs recognize their ligands will aid the design of novel drugs and tools for studying receptor function. This information can be used to predict ligand binding motifs which may help to predict potential ligands for orphan receptors.

Using comparative sequence analysis combined with three-dimensional models of receptors to identify residues with the potential to interact with a ligand, chimeric receptors were designed in an attempt to separate ligand binding and G-protein coupling. The yeast system was chosen to study these chimeras because of the particular utility of its null receptor and G-protein background.

Functional assays of yeast strains expressing the chimeric receptors showed that they responded to agonists and were antagonized by compounds that acted on the receptor which made up the top half of the chimera. Yeasts expressing P2Y1/Y2 were responsive to ADP but were unresponsive to UTP, suggesting that the ligand binding characteristics of P2Y1 had been conferred on P2Y2 in this chimera. Similarly, the reciprocal chimera P2Y2/Y1 was responsive to UTP, suggesting that the ligand binding function of P2Y2 had been successfully conferred on the P2Y1 receptor in this construct.

Chimeric P2Y2/Y1 receptors that did not contain all four of the extracellular segments did not show any activity in the yeast assay. Possibly, ligand binding requires residues contained within all four of the segments encompassing the extracellular loops, the N-terminus and the tops of all seven helices. Alternatively, interactions involving all helices are required to coordinate a binding site, even though the ligand does not interact directly with each segment. Engineering these receptors within the helices may destabilize the resulting proteins so that they are not expressed or folded correctly unless all seven helices are engineered at the same time.

In addition to the effects of agonists, both P2Y1 and P2Y1/Y2 were antagonized by A3P5PS at similar concentrations, supporting the idea that the ligand binding specificity of P2Y1 had been conferred on this chimera.

The BLT1/Y2 chimera behaved in a similar fashion to BLT1 in the yeast assay in that both responded to LTB4 and 20-OH-LTB4 and were antagonized by selective BLT1 receptor antagonists. These observations were confirmed in CHO cells.

The P2Y1/Y2 and P2Y2/Y1 chimera ligand responses are similar to, but not identical with, those obtained with the wild-type receptors in yeast (Figure 3). The data indicate that wild-type P2Y1 is better coupled than P2Y2 to the Gpa1-Gaq chimera, since P2Y2/Y1 was activated by 5-fold lower concentrations of UTP or ATP than wild-type P2Y2, whereas P2Y1/Y2 required higher concentrations of both ADP and 2MeSADP for activation than P2Y1. P2Y1/Y2 gave greatly reduced maximum levels of response compared with P2Y1, which may reflect a combination of weaker coupling, protein instability or differences in expression levels in this system. N-terminal epitope tagging of the wild-type receptors resulted in loss of function in yeast but not in mammalian cells (data not shown). It was therefore not possible to compare receptor expression levels in the different yeast strains, but levels of protein expression were compared in transiently transfected CHO cells for one pair of receptors.

Crude membrane preparations from transiently transfected CHO cells showed equivalent levels of LTB4 binding but higher levels of expression of the wild-type receptor by western blotting. This suggests that the ligand may only be labelling a subpopulation of expressed receptor.

The chimeric yeast-mammalian G{alpha} proteins used in the yeast assay can be considered as tools to explore the coupling characteristics of chimeric receptors. Interpreting the results for the receptors in this study is complicated since the wild-type receptors have overlapping coupling specificities and a quantitative comparison of expression levels of the receptors was not possible. However, generally the coupling specificity appears to reflect that of the receptor making up the intracellular portion of the chimera. BLT1/P2Y2 exhibited the broad coupling to all G{alpha} species as observed with wild-type P2Y2 in contrast to the restricted coupling of wild-type BLT1, suggesting that this chimera has the G-protein interacting properties of P2Y2. The failure of P2Y1/P2Y2 to couple to the whole panel of G-protein chimeras observed with wild-type P2Y2 could reflect the fact that assays for this chimeric receptor are not robust, indicative of low receptor expression. Native P2Y2 responses were greatest when G{alpha}14, G{alpha}q, G{alpha}i1 or G{alpha}i3 chimeras were co-expressed relative to responses obtained with the other mammalian G-protein transplant chimeras (data not shown), therefore a weakly expressed chimera would be seen to couple principally through these chimeras. The most promising evidence that coupling specificity is defined by the intracellular portion of the receptor comes from the P2Y2/P2Y1 chimera, which exhibited selective coupling to the Gpa1-G{alpha}q, -G{alpha}14, -G{alpha}i1 and -G{alpha}i3 chimeras as seen for wild-type P2Y1 and not the broad coupling of wild-type P2Y2. In this case poor expression seems unlikely to be the reason for the limited coupling specificity, since the chimeric receptor was better coupled than wild-type P2Y2 (see above) and gave a robust, sensitive response.

Through these experiments it has been demonstrated that G-protein-coupled receptors can be engineered within their helices to produce functional chimeras. GPCR chimeras have been engineered in many previous studies but these have usually been simpler in design involving substitution of a single region. Where multiple exchanges have been made these have been engineered outside of the transmembrane regions [for examples see the literature (McClintock and Lerner, 1997Go; Liu et al., 1999Go)]. Using this novel approach we have been able to define regions required for ligand-induced activation for P2Y1, P2Y2 and BLT1. These findings open up the possibility of producing functional chimeras containing the ligand binding domain of an unrelated receptor, for example an orphan GPCR, fused to a well coupled receptor. This technique could be useful for studying receptors that do not couple efficiently in a given system. Further site-directed mutagenesis studies and molecular modelling studies would be useful to explore and dissect the roles of individual extracellular loops and transmembrane helices in ligand binding and activation of leukotriene and purinergic receptors.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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Received April 20, 2002; revised March 31, 2003; accepted April 8, 2003.





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