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Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research (M.W.B., J.v.O., P.P.W.v.d.H., C.C.B., A.P.IJ.) and Division of Molecular Genetics, Leiden Institute of Chemistry (H.d.D., J.B.), Leiden University, Leiden, the Netherlands.
Received March 3, 2003; accepted December 3, 2003
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Abstract |
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; Venter et al., 2001). Information on these proteins is collected in a target-specific database, the GPCRDB (www.gpcr.org).
Although all four adenosine receptors are activated by adenosine and several adenosine analogs, the adenosine A2B receptor has a markedly lower affinity for these agonists compared with the other three subtypes (Fredholm et al., 2001). Despite the synthesis and biological testing of a very large number of adenosine analogs, no selective, high-affinity adenosine A2B receptor agonists were obtained (De Zwart et al., 1998, 1999a). The prototypic, albeit nonselective, agonist for this receptor is 5'-N-ethylcarboxamidoadenosine (NECA). In contrast, several high-affinity antagonists have been developed for this receptor (Kim et al., 1998, De Zwart et al., 1999b). The most selective and potent antagonist currently available is MRS1754 (Kim et al., 2000).
Previously, we applied information from the GPCRDB to select amino acids of the human adenosine A2B receptor for site-directed mutagenesis (Beukers et al., 2000). The aim of that study was to gain more insight in the interaction of agonists with the human adenosine A2B receptor. Our bioinformatics approach yielded five amino acids that we mutated into the corresponding amino acids of the closely related, high-affinity, human adenosine A2A receptor. One of the mutated adenosine A2B receptors, the N273Y mutant, had 61-fold improved affinity for an adenosine analog, 2-(1-hexynyl) adenosine.
In the current study, we developed a random mutagenesis approach to identify amino acids of the human adenosine A2B receptor involved in receptor activation. We combined this random mutagenesis approach with a robust screening assay in yeast to identify amino acids involved in constitutive activation of the receptor as well as amino acids that increase the susceptibility of the receptor for agonists. An engineered pheromone signaling pathway in yeast allowed us to select mutations of interest based on growth. cAMP measurements in stably transfected CHO cells were carried out to verify the ability of the mutant receptors to activate a mammalian second messenger system.
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Materials and Methods |
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) was transferred to the pDT-PGK plasmid kindly provided by Dr. S. J. Dowell from GSK (Stevenage, UK). This plasmid was used to transfect yeast cells. The mutagenic PCR that we developed to introduce random mutations worked with DNA fragments up to 400 bp in length. To obtain such fragments, we introduced a silent mutation in the human adenosine A2B gene, resulting in a KpnI site at the border between the second intracellular loop and the fourth transmembrane domain, as shown in Fig. 1, a and b. The primer used to introduce this silent mutation was 5'-CTT GCT CGG GTA CCC GGT ACC AAA CTT TTA TAC C3'; the resulting KpnI site is underlined. A second endogenous BglII site located at the border between the fifth transmembrane domain and the third intracellular loop was exploited as well. Through restriction with EcoRI and KpnI, we obtained the ATG-KpnI fragment; through restriction with BglII and HindIII, we obtained the BglII-stop fragment (see Fig. 1b). Both fragments were subjected to random mutagenesis.
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Random Mutagenesis. The method we used to introduce random mutations by PCR was adapted from Fromant et al. (1995). The mutagenic PCR contained an excess of one of the four nucleotides and an altered ratio of [MgCl2] and [MnCl2]. The mutagenic reaction contained 10 ng of template DNA, 0.1 µM concentrations of each primer, 0.2 mM concentrations of dNTPs as well as 3.4 mM concentrations of the dNTP in excess, 0.5 mM MnCl2, 4.7 mM MgCl2, and 0.5 units of Super Taq polymerase in 100 µl of buffer consisting of 10 mM Tris-HCl, pH 9.0, 50 mM KCl, and 0.1% Triton X-100. The number of cycles was set to 10. The ATG-KpnI and the BglII-stop fragments were mutagenized separately with mutagenic PCR. For the ATG-KpnI fragment, the 5'-CAT GAA TTC CAT ATG CTG CTG CAG ACA CAG GA-3' primer and the previously mentioned primer used to introduce the silent KpnI site were used. To obtain the BglII-stop fragment, the primers 5'-CAT TAA GAT CTT CCT GGT GGC CTG CAG GCA GCT TC-3' and 5'-GAT AAG CTT CTA GAT CAT AGG CCC ACA CCG AGA G-3' were used (the restriction site for BglII is underlined).
The mutagenic PCR products were submitted to agarose gel electrophoresis and the gel bands containing the mutated fragments were isolated from the gel and purified. Subsequently, the mutated fragments were amplified further with 10 cycles of a regular PCR with the same primer sets.
Transformation in Yeast. The mutated fragments were ligated in the pDT-PGK plasmid containing the wt human adenosine A2B receptor with the KpnI site after restriction with the appropriate restriction enzymes (EcoRI/KpnI or BglII/HindIII). To obtain mutant libraries, the plasmids were transformed to XL-10 gold Escherichia coli bacteria via electroporation.
DNA from these bacteria was isolated and used to transform yeast according to the method described by Gietz et al. (1995). Dr. S. J. Dowell kindly provided the yeast Saccharomyces cerevisiae strain we used. This strain is derived from the MMY11 strain (Olesnicky et al., 1999) and was further adapted to communicate with mammalian GPCRs through the introduction of a chimeric G protein (Brown et al., 2000). The genotype of this strain is: MATahis3 leu2 trp1 ura3 can1 gpa1
::G
i3 far1::ura3 sst2::ura3 Fus1::FUS1-HIS3 LEU2::FUS1-lacZ ste2::G418R. To measure signaling of GPCRs, the pheromone signaling pathway of this strain was coupled via the FUS1 promotor to HIS3, a gene encoding the key enzyme in histidine production, imidazole glycerol-phosphate dehydrase.
Growth Selection. The growth selection plates contained minimal agar medium to which tryptophan and adenine were added. The lack of leucine allowed selection for the strain, whereas uracil omission was used to select for the plasmid. To select for the presence of human adenosine A2B receptors, histidine was omitted and 7 mM 3-AT was added to the medium. 3-AT is a competitive inhibitor of imidazole glycerol-phosphate dehydrase and is used to suppress the basal growth that occurs in histidine-deficient medium. This optimal concentration of 3-AT was achieved through careful titration to suppress growth. In some experiments, we added 1.3 IU/ml adenosine deaminase to the agar plates, to address a potential role of endogenous adenosine (Price et al., 1996).
The initial screen was carried out in the presence of 4 nM NECA, a concentration at which the wt human adenosine A2B receptor barely grows. Plasmids from yeast colonies that did grow under these conditions were isolated and used in a second round of transformation. A few cells of the resulting yeast colonies were precultured for 16 h in minimal yeast nitrogen base without amino acids supplemented with tryptophan, adenine, and leucine. The cell suspensions thus obtained were adjusted to a concentration of 400,000 cells/ml and droplets of 1.5 µl were spotted on growth selection plates containing 7 mM 3-AT and either 0, 4, 40, or 400 nM NECA. Growth was quantified with Quantity One imaging software from Bio-Rad (Hercules, CA). Growth was corrected for local background on the plate and expressed as a ratio with respect to the basal growth of the wt receptor, in the absence of NECA.
Stable Transfection of CHO Cells. To transfect CHO cells with the wt and the mutated human adenosine A2B receptor, the DNA encoding these receptors was transferred from the yeast pDT-PGK to the mammalian pcDNA3 vector. CHO cells were cotransfected with peYFP-N1, a plasmid encoding enhanced yellow fluorescent protein, to identify transfected cells. peYFP-N1 was obtained from BD Biosciences Clontech (Etten Leur, The Netherlands).
The day before transfection, cells were seeded at 50,000 cells per well in a 24-well plate. For each well, 2.3 µl of N-(2,3-dioleoyloxy-1-propyl)trimethylammonium methyl sulfate (1 mg/ml) was carefully added to 0.7 µg of each of the DNAs in a total volume of 50 µl of DMEM without serum. This mixture was left at room temperature for 20 min to form liposomes. Cells were washed twice with DMEM without serum; next, 50 µl of DMEM without serum was added followed by the transfection mix. Cells were left for 2 h at 37°C, 5% CO2. Subsequently, the cells were washed with phosphate-buffered saline and treated with 5% dimethyl sulfoxide in phosphate-buffered saline for 3 min. The dimethyl sulfoxide was removed, and 500 µl of a 1:1 mixture of DMEM and Ham's F12 medium, containing 10% newborn calf serum, 2 mM glutamax, 50 IU/ml penicillin, and 50 µg/ml streptomycin, was added to each well. After 48 h, the cells were harvested and the medium was replaced with fresh medium containing 0.8 mg/ml G418 to select for cells that had taken up the plasmid. The cells were transferred from the 24-well plate to a 5-ml Petri dish, and the medium was replaced every other day. After 10 days, individual colonies were selected and transferred to separate wells in a 24-well plate. Clones with sufficiently high expression levels to perform cAMP measurements were used in the cAMP assays.
cAMP Assay. cAMP experiments were performed as described previously (Beukers et al., 2000). In short, cells were seeded at 300,000 cells/well in a 24-well plate 24 h before cAMP generation. Cells were washed twice with 50 mM HEPES-buffered DMEM, pH 7.4. Cells were incubated for 30 min at 37°C in 300 µl of HEPES-buffered DMEM, pH 7.4, containing 50 µM rolipram and 50 µM cilostamide to prevent breakdown of cAMP as well as 1 IU/ml adenosine deaminase. Subsequently, either DMEM (basal), 10 µM forskolin (maximal stimulation), or varying concentrations of NECA were added in a volume of 100 µl. After 15-min incubation at 37°C, the medium was removed and 200 µl of ice-cold 0.1 N HCl was added. The samples were stored at -20°C. cAMP production was measured with a radioligand binding assay based on the interaction of cAMP with protein kinase A as described previously (Beukers et al., 2000).
Bioinformatics. The G protein-coupled receptor database, GPCRDB, is a target-specific database that contains, among others, the amino acid sequences of the GPCRs (http://www.gpcr.org; Horn et al., 1998). In addition, computationally derived data are available, such as amino acid sequence alignments. The adenosine receptors belong to the class A or rhodopsin-like family of GPCRS. The alignment of these class A receptors is based on the presence of highly conserved amino acids in each of the seven transmembrane domains and is visualized in so-called snake plots (see Fig. 1). Moreover, mutation data of the GPCRs are available in tinyGRAP (http://www.grap.fagmed.uit.no/GRAP/homepage.html; Beukers et al., 1999), a subdatabase of the GPCRDB, that can be accessed via the snake plots. Amino acid residues for which mutation data are available are colored white. Next to snake plots for individual receptors, the GPCRDB also contains snake plots for receptor families. In this study, we used the snake plot for class A receptors and investigated whether point mutation data were available in other class A receptors for the amino acids that were involved in constitutive activity of the human adenosine A2B receptor.
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; Parnot et al., 2002). Mutations in the ATG-KpnI as well as the BglII-stop fragment were introduced at random with mutagenic PCR with either excess dTTP, dCTP, or dGTP. The mutations were in accordance with the nucleotide excess present. A mutagenic PCR in the presence of an excess dATP resulted in early termination of the PCR reaction. This phenomenon might be caused by excessive build-in of dATP leading to extensive misalignment of the DNA template in the active site of DNA polymerase. An average of 4000 mutants was screened for every construct.
Screening in Yeast. To screen for receptor activation, we used a yeast assay based on growth. Human adenosine A2B receptors have been demonstrated to couple to the endogenous signal transduction pathway of yeast (Brown et al., 2000). After transformation of the yeast strain with the wt human adenosine A2B receptor, we determined the sensitivity of the strain for the agonist NECA. As shown in Fig. 2, growth of the yeast strain is NECA-dependent.
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Growth is a very suitable selection criterion, because only mutants of interest show up on the selection plate. The selection plate contained 4 nM NECA, a concentration at which only very little growth of yeast strains expressing the wild-type receptor occurs. Strains showing increased growth were picked and their plasmids were isolated and used to transform another yeast cell. These cells were then transferred to plates containing NECA at concentrations of 0, 4, 40, and 400 nM. As a control, the wt human adenosine A2B receptor was transferred in triplicate to these plates. The concentration-response curve of growth of the wt receptor is shown in Fig. 3. The EC50 value of NECA to stimulate growth was 97 ± 36 nM for the wt receptor.
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Constitutively Active and Gain of Function Mutations. From our combined random mutagenesis and screening method, we selected 14 mutated receptors. Growth of these receptors in the absence of NECA was quantified and related to the growth of the wt human adenosine A2B receptor (Fig. 4). As can be seen in this graph, all mutated receptors are constitutively active although the degree of constitutive activity varies. Growth of the wt receptor is the mean of the three transformations as presented in Fig. 3. Because not all the dose-response curves show full saturation, we will comment only on those curves that show a shift in NECA responsiveness of 
4-fold. Three point-mutated receptors were obtained twice independently: V54L (a and b), F84L (a and b), and S91G (a and b). The constitutive activity of each of the copies of the mutants was quite similar demonstrating the reproducibility of the assay.
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After constitutive activity in the absence of NECA, we determined the growth concentration-response curves of these mutants for NECA. Again, the two copies of the V54L, F84L, and S91G mutants behaved similarly (see Fig. 5). The EC50 values of V54La and V54Lb were 14 and 17 nM, respectively; of F84La and F84Lb, 44 and 24 nM, respectively; and of S91Ga and S91Gb, 56 and 66 nM, respectively (Table 1). Thus, these three mutations result not only in constitutive activity but also in gain of function with respect to NECA.
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As was already shown in Fig. 4, all mutated receptors demonstrate constitutive activity. The most constitutively active mutant, 20- to 22-fold over wt, contained three mutations: N36S, T42A, and T66A. A reliable EC50 value of NECA could not be determined for this mutant because of the high level of constitutive activity. The point mutant T42A itself was also constitutively active at 12-fold. In addition, the T42A mutant had a 4.9-fold decreased EC50 value for NECA. A double mutation at both T42A and V54A had no additional effect on the EC50 value of NECA but did increase the constitutive activity somewhat (15- versus 12-fold). These effects on constitutive activity and the EC50 value of NECA are also depicted in Fig. 6a. A point mutation at N36D had little effect on constitutive activity and resulted in a slight gain of function (Fig. 6b). The double mutant N36S V87A was slightly constitutively active and the EC50 value of NECA for this mutant was decreased 3.2-fold (Fig. 6b). Interestingly, the triple mutant N36S T42A T66A obtained twice was highly constitutively active (Fig. 6b).
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Another interesting position in the human adenosine A2B receptor was F84 (see Fig. 6c). This residue was mutated in three different receptor mutants. On two occasions, this mutation was a single-point mutation. As stated, the F84La and F84Lb mutants were slightly constitutively active (3.3- and 2.1-fold, respectively) and the EC50 value of NECA was decreased moderately (2.2- and 4.0-fold, respectively). Mutation of the same residue to serine (F84S) resulted in more constitutive activity (7.1-fold) and a stronger decrease of the EC50 value of NECA (6.5-fold). A double mutant that was found twice and contained both F84L and S95G was more constitutively active than the single F84L mutant and had an 18- to 19-fold lower EC50 value for NECA. A mutant with five mutations, A18T A23V C83Y A106V R112S, was considerably constitutively active, 12-fold compared with wt, and its EC50 value for NECA was 2.8-fold lower compared with wt.
The above-mentioned mutations were generated in the ATG-KpnI fragment of the human adenosine A2B receptor. The mutated receptors mentioned below resulted from at random mutagenesis of the BglII-stop fragment of the receptor (see Fig. 6d). Interestingly, this fragment did not yield any single-point mutations.
Mutation of four residues, I242T K269R V284A H302Q, resulted in a mutant with a 12-fold increased constitutive activity that in addition had a 9.8-fold lower EC50 value for NECA. Mutation of three residues in the C-terminal tail of the receptor, Y299C F301C L310P, resulted in a mutant with moderate constitutive activity (6.1-fold) as well as a moderate decrease in EC50 (4.6-fold).
In contrast to all the other mutants generated, the Q214L I230N V240M V250M N254Y T257S K269stop mutant no longer responded to stimulation with NECA. Despite the fact that this mutant effectively misses the seventh transmembrane domain and the C-terminal tail, it was highly constitutively active (18-fold) compared with the wt receptor.
In Table 1, an overview is presented of the mutated receptors that were selected and studied in this report. Their fold constitutive activity versus the wt human adenosine A2B receptor as well as their EC50 value for NECA are shown.
To address a potential role of endogenous adenosine we selected four representative mutants, V54L; A18T A23V C83Y A106V R112S; Q214L, I230N, V240M, V250M, N254Y T257S K269stop; and F84L, S95G, and tested their growth in the presence and absence of 1.3 IU/ml adenosine deaminase (Table 2). Adenosine deaminase had no effect on the growth of these mutants. To investigate whether an effect of endogenous adenosine might be masked by the presence of 3-AT, we also tested the growth of these four mutants in the absence of 3-AT. Again, adenosine deaminase had no effect. Hence, these data demonstrate that the growth in the presence of 3-AT is caused by the constitutive activity of the receptor and is not caused by binding of endogenous adenosine to the receptor.
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cAMP Measurements in CHO Cells. To verify whether the mutant human adenosine A2B receptors were able to activate a second-messenger system of mammalian cells, DNA encoding the point mutants T42A, F84S, the double mutant containing F84L and S95G, and the wt human adenosine A2B receptor was transferred to pcDNA3 to allow transfection of CHO cells. Unfortunately, human embryonic kidney 293 and COS-7 cells, two other mammalian cell systems, express endogenous adenosine A2B receptors. Because the expression levels of transiently transfected cells were too low, stable transfections were carried out. The various clones were tested for their ability to generate cAMP upon activation with varying concentrations of NECA. For the wt adenosine A2B receptor and the T42A mutant receptor, cAMP levels were sufficiently high to allow analysis. The EC50 values of NECA for cAMP stimulation were 1.3 ± 0.4 µM and 0.15 ± 0.04 µM for the wt and the T42A mutant receptor, respectively (see Fig. 7). Thus, the responsiveness of the T42A receptor for NECA was increased 8.7-fold. Basal cAMP production was slightly higher (2.3-fold) for the T42A receptor compared with the wt receptor.
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). The pheromone signaling pathway of the MATa mating type yeast uses the Ste2p receptor, a G protein-coupled receptor. Upon activation of this receptor, a mitogen-activated protein kinase cascade is activated through the endogenous Gpa1p G protein, resulting in the induction of mating genes such as FUS1. The engineered yeast strain lacks the endogenous Ste2p receptor and expresses a humanized G protein instead of the Gpa1p G protein. Moreover, in this engineered yeast strain, the HIS3 gene encoding an enzyme crucial for the production of the essential amino acid histidine is placed behind the FUS1 promoter. Finally, a human GPCR, in our case the adenosine A2B receptor or one of its mutants, is expressed in these cells. As a result, in the absence of histidine, the only yeast cells that can grow are those in which the human adenosine A2B receptor or one of its mutants is active.
The strength of the screening assay is best illustrated by the fact that we obtained 14 highly interesting receptor mutants. Quantification of the amount of growth turned out to be very reproducible, as illustrated by the three transformations of the wt receptor (see Fig. 3) as well as by the reproducibility of the data obtained for all the mutants that we found twice independently (see Figs. 5 and 6, a–c). Moreover, the EC50 value for NECA we obtained for the wt receptor, 97 ± 36 nM, agreed very well with the EC50 value of 87 ± 8 nM reported in the literature (Brown et al., 2000). Not all the dose-response curves showed full saturation. Therefore, we confined our comments to shifts in responsiveness to NECA of 4-fold. In addition, the four NECA concentrations allow for a rough estimate of the EC50 values only.
To determine whether the yeast assay was predictive of the functional properties of these mutant receptors in mammalian cells, cAMP measurements were performed on stably transfected CHO cells. Three mutants were selected for this purpose, T42A, F84S, and a double mutant F84L S95G. Only for the T42A mutant and the wt receptor were clones obtained with high enough expression levels to perform cAMP studies. The screening assay in yeast proved predictive of the increased NECA responsiveness. In the yeast assay, NECA had a 4.9-fold lower EC50 for the T42A receptor than for the wt receptor, which was mimicked in the CHO cells, in which the EC50 for cAMP production was decreased 8.7-fold compared with the wt receptor (Fig. 7). Basal cAMP production in the CHO cells was slightly higher (2.3-fold) for the T42A receptor compared with the wt receptor. In a previous study, we introduced point mutations in the human adenosine A2B receptor based on the criterion that the equivalent amino acid was different but conserved among the other adenosine receptor subtypes (Beukers et al., 2000). In our current study, none of the mutant receptors fulfilled this criterion. In fact, the mutated amino acids that yielded a 4-fold increased potency for NECA were either conserved among all four adenosine receptors (T42) or were present in at most two of the three other human adenosine receptors (see Table 3). The latter amino acids were V54, T66, F84, and S95.
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Unfortunately, no experimentally determined three dimensional structure is available for the adenosine A2B receptor. To get some indication of the location of the four amino acids mentioned above, we used the receptor model for the most closely related human adenosine A2A receptor. This model is present in the protein database (PDB code 1mmh; http://www.rcsb.org/pdb) and has NECA as a ligand in it. We evaluated the location of the four mutated amino acids with respect to the homologous amino acids of the human adenosine A2A receptor. Although we are aware of the limitations of the receptor models, we were struck by the fact that all four amino acids turned out to be located far away from NECA (9 Å).
Several studies have been published in which the equivalent amino acid of the human adenosine A2B receptor was mutated in other adenosine receptors. For example, mutation of Ser90 to Ala of the human adenosine A2A receptor, the equivalent of S91G in this study, resulted in a 4.7-fold increase in affinity for NECA (Jiang et al., 1996). Pro86 in the human adenosine A1 receptor is the equivalent of Phe84. Mutation of Pro86 to Phe resulted in a >10-fold decrease in the affinity of NECA (Rivkees et al., 1999). Apparently, the phenylalanine residue hampers the interaction with NECA in both receptors. Finally, the equivalent of Ser95 in the human adenosine A1 receptor was subjected to mutation. Mutation of this residue, Ser94 to Ala, resulted in a complete loss of affinity for NECA, whereas mutation to Thr resulted in a 2-fold decrease in NECA affinity (Barbhaiya et al., 1996).
We compared our results not only with data on other adenosine receptors but also with literature data on constitutive activity in other class A receptors. To make this comparison we used data from the GPCRDB and its subdatabase tinyGRAP. Within the GPCRDB, a numbering system has been introduced to align more distantly related class A GPCRs. This numbering system is based on the presence of several highly conserved amino acids in each of the seven transmembrane domains (Ballesteros and Weinstein, 1995). These conserved residues are highlighted in the snake-like plot of the human adenosine A2B receptor (Fig. 1). In Table 4, the results of this literature search are presented. Indeed, many of the amino acids that showed up in our screen as being important for constitutive activity of the human adenosine A2B receptor were also involved in constitutive activity of other class A GPCRs. In addition, these data suggest that the amino acids Ile242 and Val284 might be responsible for the constitutive activity obtained in the quadruple mutant I242T, K269R, V284A, and H302Q. The amino acids Ala106, Arg112, Tyr299, Phe301, and Leu310 could not be taken into account because no reliable alignment can be made for these residues as a result of the absence of highly conserved amino acids in the intracellular domains in which they are located.
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However, mutations in the C terminus of class A GPCRs have frequently been shown to be involved in constitutive activity. For example, truncation of the C-terminal tail of the 5HT4 receptor (Claeysen et al., 1999), the prostaglandin E2 receptor EP4 subtype (Bastepe and Ashby, 1997), the TRHR receptor (Zaltsman et al., 2000), and the rat SSTR2 receptor (Schwartkop et al., 1999) resulted in constitutive activity. The Q214L, I230N, V240M, V250M, N254Y, T257S, K269 stop mutant is very interesting as well. A random mutagenesis study in yeast on the human C5a anaphylatoxin receptor revealed several truncated receptors that displayed constitutive activity. One of them, referred to as R92, resembled to a very large degree the mutant we obtained (Baranski et al., 1999). Next to various other amino acids that were mutated, the stop codon was present in the equivalent position; moreover, this mutated receptor contained mutations at the equivalent positions of Ile230, Val240, Val250, Asn254, and Thr257. These data provide support for our finding that a truncated receptor lacking both TM7 as well as the C terminus can be constitutively active. A recent report on the human adenosine A2A receptor showed that truncation of this receptor at position 311 results in a nonconstitutively active receptor, whereas the wt receptor, as well as a receptor truncated at amino acid 360, are constitutively active (Klinger et al., 2002).
As stated, many reports in the literature confirm our finding that truncation at the C terminus results in constitutive activation of GPCRs. Hence, this approach may be a way to introduce constitutive activity in orphan receptors to enable the search for inverse agonists for these receptors.
Interestingly, all selected mutated human adenosine A2B receptors turned out to be constitutively active despite the fact that our selection screen was based on increased growth in the presence of 4 nM NECA. Experiments in the presence of adenosine deaminase proved that the constitutive activity was not compromised by the presence of endogenous adenosine (Table 2).
Constitutive activity is generally thought to represent a shift in the population of receptors from R toward R* (Leff, 1995). Hence, the affinity and potency of agonists for constitutively active receptors are increased, compared with wt receptors. Indeed, constitutive activity of the mutant receptors was accompanied by an increased potency for NECA. This finding may suggest that the human adenosine A2B receptor itself is relatively silent, and consequently agonists will have a low affinity and potency for this receptor. In line with this hypothesis are the findings that 1) no agonists have been found to date with increased affinity for the receptor, and 2) the selected mutant receptors contained mutations in amino acids located far away from the putative binding site of NECA. The mutations apparently increase the activity of the receptor (causing constitutive activation), and are probably not improving the binding of the agonist NECA.
In our experiments, we have been unable to determine the receptor expression levels because they were too low to enable either radioligand binding or immunofluorescence studies (data not shown). Hence, we cannot completely rule out the possibility that our data may have been affected by differences in receptor expression levels (Lutz and Kenakin, 1999). Milligan et al. (2002) reported in their review article that constitutively active mutant receptors are generally expressed at lower levels than wt receptors. If this were also the case in our experiments, we might have underrather than overestimated the constitutive activity as well as the increased potency of the mutant receptors for NECA versus the wt receptor.
In conclusion, our random mutagenesis approach has yielded both highly constitutively active as well as strong gain of function mutant adenosine A2B receptors. The pharmacological analysis of the wt and T42A mutant receptors in mammalian CHO cells corroborated our findings in yeast. The latter assays, however, are rather cumbersome, particularly on adenosine A2B receptors. Evaluation of GPCRs with the screening assay in yeast overcomes such problems.
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Footnotes |
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Address correspondence to: Margot Beukers, Div. of Medicinal Chemistry, LACDR, Gorlaeus Laboratories, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, The Netherlands. E-mail: beukers{at}chem.leidenuniv.nl
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References |
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Baranski TJ, Herzmark P, Lichtarge O, Gerber BO, Trueheart J, Meng EC, Iiri T, Sheikh SP, and Bourne HR (1999) C5a receptor activation. Genetic identification of critical residues in four transmembrane helices. J Biol Chem 274: 15757-15765.
Barbhaiya H, McClain R, IJzerman A, and Rivkees SA (1996) Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding. Mol Pharmacol 50: 1635-1642.[Abstract]
Bastepe M and Ashby B (1997) The long cytoplasmic carboxyl terminus of the prostaglandin E2 receptor EP4 subtype is essential for agonist-induced desensitization. Mol Pharmacol 51: 343-349.
Beukers MW, Kristiansen K, IJzerman AP, and Edvardsen Ø (1999) TinyGRAP database: a bioinformatics tool to mine receptor mutant data. Trends Pharmacol Sci 20: 475-477.[CrossRef][Medline]
Beukers MW, den Dulk H, van Tilburg EW, Brouwer J, and IJzerman AP (2000) Why are A2B receptors low-affinity adenosine receptors? Mutation of Asn273 to Tyr increases affinity of human A2B receptor for 2-(1-hexynyl)adenosine. Mol Pharmacol 58: 349-356.
Brown AJ, Dyos SL, Whiteway MS, White JH, Watson MA, Marzioch M, Clare JJ, Cousens DJ, Paddon C, Plumpton C, et al. (2000) Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras. Yeast 16: 11-22.[CrossRef][Medline]
Claeysen S, Sebben M, Becamel C, Bockaert J, and Dumuis A (1999) Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain. Mol Pharmacol 55: 910-920.
Colson A-O, Perlman JH, Jinsi-Parimoo A, Nussenzveig DR, Osman R, and Gershengorn MC (1998) A hydrophobic cluster between transmembrane helices 5 and 6 constrains the thyrotropin-releasing hormone receptor in an inactive conformation. Mol Pharmacol 54: 968-978.
De Zwart M, Kourounakis A, Kooijman H, Spek AL, Link R, von Frijtag Drabbe Kunzel JK, and IJzerman AP (1999a) 5'-N-substituted carboxamidoadenosines as agonists for adenosine receptors. J Med Chem 42: 1384-1392.[CrossRef][Medline]
De Zwart M, Link R, von Frijtag Drabbe Kunzel JK, Cristalli G, Jacobson KA, Townsend-Nicholson A, and IJzerman AP (1998) A functional screening of adenosine analogues at the adenosine A2B receptor: a search for potent agonists. Nucleosides Nucleotides 17: 969-985.[Medline]
De Zwart M, Vollinga RC, Beukers MW, Sleegers DF, Von Frijtag Drabbe Künzel J, Groote de M, and IJzerman AP (1999b) Potent antagonists for the human adenosine A2B receptor. Derivatives of the triazolotriazine adenosine receptor antagonist ZM241385 with high affinity. Drug Dev Res 48: 95-103.[CrossRef]
Duprez L, Parma J, Van Sande J, Allgeier A, Leclere J, Schvartz C, Delisle MJ, Decoulx M, Orgiazzi J, Dumont J, et al. (1994) Germline mutations in the thyrotropin receptor gene cause non-autoimmune autosomal dominant hyperthyroidism. Nat Genet 7: 396-401.[CrossRef][Medline]
Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, and Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 53: 527-552.
Fromant M, Blanquet S, and Plateau P (1995) Direct random mutagenesis of genesized DNA fragments using polymerase chain reaction. Anal Biochem 224: 347-353.[CrossRef][Medline]
Gietz RD, Schiestl RH, Willems AR, and Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11: 355-360.[CrossRef][Medline]
Horn F, Weare J, Beukers MW, Hörsch S, Bairoch A, Chen W, Edvardsen Ø, Campagne F, and Vriend G (1998) GPCRDB: an information system for G protein-coupled receptors. Nucleic Acids Res 26: 275-279.
Hwa J, and Perez DM (1996) The unique nature of the serine interactions for 1-adrenergic receptor agonist binding and activation. J Biol Chem 271: 6322-6327.
Jiang Q, Van Rhee AM, Kim J, Yehle S, Wess J, and Jacobson KA (1996) Hydrophilic side chains in the third and seventh transmembrane helical domains of human A2A adenosine receptors are required for ligand recognition. Mol Pharmacol 50: 512-521.[Abstract]
Kim Y-C, de Zwart M, Chang L, Moro S, von Frijtag Drabbe Kunzel JK, Melman N, IJzerman AP, and Jacobson KA (1998) Derivatives of the triazoloquinazoline adenosine receptor antagonist CGS15943having high potency at human A2B receptors. J Med Chem 41: 2835-2845.[CrossRef][Medline]
Kim Y-C, Ji X-D, Melman N, Linden J, and Jacobson KA (2000) Anilide derivatives of an 8-phenylxanthine carboxylic congener are highly potent and selective antagonists at the human A2B receptors. J Med Chem 43: 1165-1172.[CrossRef][Medline]
Klinger M, Kuhn M, Just H, Stefan E, Palmer T, Freissmuth M, and Nanoff C (2002) Removal of the carboxy terminus of the A2A-adenosine receptor blunts constitutive activity: differential effect on cAMP accumulation and MAP kinase stimulation. Naunyn-Schmiedeberg's Arch Pharmacol 366: 287-298.[CrossRef][Medline]
Kosugi S, Van Dop C, Geffner ME, Rabl W, Carel JC, Chaussain JL, Mori T, Merendino JJ Jr, and Shenker A (1995) Characterization of heterogeneous mutations causing constitutive activation of the luteinizing hormone receptor in familial male precocious puberty. Hum Mol Genet 4: 183-188.
Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al. (2001) Initial sequencing and analysis of the human genome. Nature (Lond) 409: 860-921.[CrossRef][Medline]
Leff P (1995) The two-state model of receptor activation. Trends Pharmacol 16: 89-97.[CrossRef][Medline]
Lutz M and Kenakin T (1999) Quantitative Molecular Pharmacology and Informatics in Drug Discovery. John Wiley & Sons, Chichester, UK.
Milligan G, Stevens PA, Ramsay D, and McLean AJ (2002) Ligand rescue of constitutively active mutant receptors. Neurosignals 11: 29-33.[CrossRef][Medline]
Olesnicky NS, Brown AJ, Dowell SJ, and Casselton LA (1999) A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus. EMBO (Eur Mol Biol Organ) J 18: 2756-2763.[CrossRef][Medline]
Parnot C, Miserey-Lenkei S, Bardin S, Corvol P, and Clauser E (2002) Lessons from constitutively active mutants of G protein-coupled receptors. Trends Endocrinol Metab 3: 336-343.
Pauwels PJ and Wurch T (1998) Review: amino acid domains involved in constitutive activation of G-protein-coupled receptors. Mol Neurobiol 17: 109-135.[Medline]
Porcellini A, Ciullo I, Laviola L, Amabile G, Fenzi G, and Avvedimento VE (1994) Novel mutations of thyrotropin receptor gene in thyroid hyperfunctioning adenomas. Rapid identification by fine needle aspiration biopsy. J Clin Endocrinol Metab 79: 657-661.[Abstract]
Price LA, Strnad J, Pausch MH, and Hadcock JR (1996) Pharmacological characterization of the rat A2a adenosine receptor functionally coupled to the yeast pheromone response pathway. Mol Pharmacol 50: 829-837.[Abstract]
Rivkees SA, Barbhaiya H, and IJzerman AP (1999) Identification of the adenine binding site of the human A1 adenosine receptor. J Biol Chem 274: 3617-3621.
Rosenkilde MM, Kledal TN, Holst PJ, and Schwartz TW (2000) Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74. J Biol Chem 275: 26309-26315.
Schwartkop CP, Kreienkamp HJ, and Richter D (1999) Agonist-independent internalization and activity of a C-terminally truncated somatostatin receptor subtype 2 (delta349). J Neurochem 72: 1275-1282.[CrossRef][Medline]
Tonacchera M, Chiovato L, Pinchera A, Agretti P, Fiore E, Cetani F, Rocchi R, Viacava P, Miccoli P, and Vitti P (1998) Hyperfunctioning thyroid nodules in toxic multinodular goiter share activating thyrotropin receptor mutations with solitary toxic adenoma. J Clin Endocrinol Metab 83: 492-498.
Vage DI, Klungland H, Lu D, and Cone RD (1999) Molecular and pharmacological characterization of dominant black coat color in sheep. Mamm Genome 10: 39-43.[CrossRef][Medline]
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, et al. (2001) The sequence of the human genome. Science (Wash DC) 291: 1304-1351.
Zaltsman I, Grimberg H, Lupu-Meiri M, Lifschitz L, and Oron Y (2000) Rapid desensitization of the TRH receptor and persistent desensitization of its constitutively active mutant. Br J Pharmacol 130: 315-320.[CrossRef][Medline]
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) human adenosine A2B receptor versus both copies of V54L (
, 
), both copies of F84L (
, 
, 
). The intercept with the y-axis represents the growth expressed as the density ratio with respect to the basal growth of the wt receptor in the absence of NECA.
