Kevin R. LynchSn, and Joel LindenWl. From the Departments of $Internal Medicine, ...... Aspects (Siegel, G. L., ed) pp. 401-416, Raven Press, New York.
THE JOURNALOF BIOLOGIC~L CHEMISTRY
Vol. 269. No. 45, Issue ofNovember 11. pp. 27900-27906, 1994 Printed in U.S.A.
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
A, Adenosine Receptors TWO AMINO ACIDS ARE RESPONSIBLE FOR SPECIES DIFFERENCES IN LIGAND RECOGNITION* (Received for publication, July 12, 1994, and in revised form, September 8,1994)
Amy L. Tucker+§, AnnaS. RobevaS, Heidi E. Taylor*, Diane Holeton+, Matthew Bockner$, Kevin R. LynchSn, and Joel LindenWl From the Departments of $Internal Medicine, Cardiovascular Division, Wharmacology, and §Biochemistry, Health Sciences Center, University of Virginia, Charlottesville, Virginia22908
Species differences in ligand binding A, to adenosine The A, adenosine receptorhas been cloned from five species, receptors were localized to the seventh transmembrane dog, cow, rat, rabbit, and human (10-12). Despite > 90% recep(TM7) region based on the binding of [8-SH]cyclopentyltor identityat the aminoacid level, the pharmacology of aden1,3-dipropylxanthine and three other ligands to wild osine A, receptors differs markedly between species (15-19). type andsix bovinelcanine interspecies receptor chimeThere are differences, not only in the affinities for different ras expressed in COS-1 cells. Subsequent site-directed ligands, but in rank order potencies as well. Bovine and canine 270 receptors differ the most, with bovine A, adenosine receptors mutagenesisexperimentsidentifiedaminoacid (isoleucine/methionine, bovinelcanine) as being primarhaving higheraffinity forNG-substituted adenine analogs such ily responsible for species differences in the binding ofas the agonist R-PIA’ and the antagonist N-0861; bovine A, NB-adenine-substituted compounds, R-W-phenylisoproreceptors also have higher affinity forC-8-substituted xanthine and (S)-A@-endonorbornan-2-yl-Spyladenosine (R-PIA) antagonists such as CPX (11,15,20). Rat and human receptors methyladenine, and the C-8-substituted xanthine, are intermediate in their binding characteristics (15, 17, 18). [SHlcyclopentyl-1,3-dipropylxanthine.Thesedataare consistent with the hypothesis that NB theregion of ad- These binding properties are similar for native brain memenines and the C-®ion of xanthines bind to the samebrane receptors and for recombinant receptors expressed in region of the receptor. A second TM7 amino acid, 277 COS-1 cells.’ regions of the A, aden(serinelthreonine, bovinehanine), selectively influences The goal of this study was to identify the bindingof the ribose-substituted adenosine analog,osine receptors responsible for conferring speciesspecificity in binding. Our approach was to construct chimeric caninehovine 5’-N-ethylcarboxamidoadenosinetoavariableextent, depending on the nature of amino acid270. We hypoth- receptors to identify the domaids)of the receptor responsible esize that amino acid 270 of the A, receptor interacts for imparting species-dependent characteristics. This was folwith theW region of adenosine, while amino acid 277 is lowed by site-directed mutagenesis to target individual amino important, especially in the absence of an W substitu- acids. The results implicate two amino acids in TM7, 270 and tion, for interactions with a distinct nucleoside region, 277 (isoleucine/methionine (I/M) andserinehhreonine (SI”) possibly on the ribose. bovinekanine) as conferring species specificity in ligand binding. The contributionof these two amino acids varies depending on the nature of the ligand. Adenosine mediates physiological responses in virtually evEXPERIMENTALPROCEDURES ery organ system. In addition to its intracellular metabolic role, Materials-Restriction enzymes were obtained from Promega adenosine also acts extracellularly viacell surface receptorsin (HindIII, KpnZ, NcoI) and Life Technologies, Inc. (Spel, HpaI, SspI). the G protein-coupled superfamily, Receptor-mediated effects DNA Sequenase I1 kits were obtained from United States Biolabs. 33P are numerous and include inhibition of neurotransmitter reused in sequencing was obtained from Amersham Corp. L3HICPX was from DuPont-NEN. N-0861 was a g&t from Whitby Research, Inc. Rlease in the central nervous system (1);negativeinotropic, chronotropic, and dromotropic effects in the heart (2); dilata- PIA, NECA, and b&erreagents were purchased from Sigma. Mutagenthe Altered Sites Kit from Promega. Media for tion of arterial smoothmuscle, including coronary arteries (3); esis was performed using bacterial culturewere from Life Technologies, Inc. Reagents used in the effects on immunologic functions (4), and mast cell degranula- electrophoresis of sequencingreactionswerefromKodakIBI.The tion (5).Four subtypesof adenosine receptors have beencloned canine A, adenosine receptorcDNA (RDC7) was a gift from G. Vassart, to date, A,, ha, 4band A,, each from several species (6-10) Brussels, Belgium.Thebovine A, adenosine receptor was cloned as (reviewed in Refs. 11 and 12). Additional subtypes may also described previously (20). Construction of Chimera-Canine adenosine A, cDNA in Bluescript receptor subtypes stimulate adenylyl exist. The 4, and was digestedwithKpnI andHindIII (37 “C, 2 h) andthe released insert cyclase upon activation (11). Additionally, the recombinant42b subcloned into the mutagenesis vectorPALTERwhichhadbeen direceptor activates a C1- conductance in Xenopus oocytes (13). A, gested with the same two restriction enzymes. The bovineA, receptor and A, receptors are inhibitory to adenylylcyclase and couple to pBOV13 in Bluescript was digested with SpeI (37 “C, 2 h) to remove a phospholipase C (5,14). large portion of the noncoding regionthat includes a unique HpaI site. The plasmid was religated and the insert subcloned into PALTER using KpnI andXbaI sites (37 “C, 2 h). Oligonucleotides complementaryto the * This work was supported by National Institutes of Health Grant noncoding strand were designed to introduce unique, silent restriction R01-HL37942. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be R-h@-phenylisopropyladenosine; The abbreviations used are: R-PIA, hereby marked “aduertisenent”in accordance with 18 U.S.C. Section N-0861,(S)-h@-endonorbornan-2-yl-9-methyladenine; CPX, 8-cyclopent1734 solely toindicate this fact. I(To whom correspondence should be addressed: Box 158, Dept. Car- yl-1,3-dipropylxanthine;NECA, 5’-N-ethylcarboxamidoadenosine;TM, diology, Health Sciences Center, Universityof Virginia, Charlottesville, transmembrane. A. Tucker, unpublished results. VA 22908. Tel.: 804-924-5600 Fax: 804-982-3183.
hb
27900
27901
A , Adenosine Receptor Chimeras and Mutants TABLEI Summary of restriction enzymes and DNA fragments used for constructing chimeric bovinelcanine A, adenosine receptors The triple letter code on the leR denotes the chimera (refer to Fig. 3). Enzymes listed are those used to excise DNA fragments from wild type receptor cDNAs in PALTER following introduction of silent restriction sites. Fragments sized from each receptor are noted for each chimeric receptor. Chimera name Enzymes RDC7 fragment size Bovl3 fragment size DBB BDB DDB BDD DBD BBD a
Kpnl, Hpal Hpal, Ncol Kpnl, Ncol Kpnl, Hpal Hpal, Ne01 Kpnl, Ncol
(dog)
(bovine)
472 bpa 108 bp 580 bp 6.4 kbp 6.8 kbp 6.3 kbp
6.9 kbpb 7.3 kbp 6.8 kbp 498 bp 108 bp 606 bp
bp, base pairs.
* kbp, kilobase pairs.
were repeated two to six times. Specific binding was fit to a single site binding model using Marquardt's nonlinear least squaresinterpolation (24). For graphical comparison of changes in the affinity of r3H1CPX among receptor chimera and mutants,plots wereconstructed of B /Bmm versus B/B,Jfree, where free refers t o [free [3HlCPXl in nanomoles. The slopes of these plots are proportional to the ligand affinity (l/Kd, n"') and are independent of differences in expression (BmJ that occur with COS cell transfection. To calculate the KI of competing drugs, [3H]CPXwas added to tubes at about 0.5 x its K, for a given receptor, and competing ligand was added over a range of concentrations to tubes containing 25 pg of protein in a final volume of 100 pl. The experiments were incubated at 21 "C for 2 h. The IC, values were calculated using a three parameter logistic equation: B = Bo - (Bo - Ns)[IV(IC, + [I]). KI values were precisely calculated from IC,,, B,, and the concentration of r3H1CPX and its K,, as described by Linden (25). RESULTS
To identify regions of the adenosine A, receptor responsible
for conferring differences in ligand binding between bovineand canine adenosine A, receptors, caninehovine chimeric receptors were constructed by ligating the appropriate fragments summarized in Table I. Each chimera consists of three segments and is named according to the species from whicheach segment was derived. The wild type bovine and canine receptors are referred to as BBB and DDD, respectively. Chimera are named by three-letter codes indicating the species of the corresponding segment of the receptors. Eight receptors were characterized in this study (two wild type and six chimeras). Splice sites for the chimeras occur at Leu-140 in TM4 and Ser-176 in TM5 and flank the second exofacialloop containing a hypervariable region (Fig. 1). With the exception of the BDD chimeric receptor (Bm, 200 fmoVmg of protein), all of the receptors, including wild type, chimeric, and mutant(see below), showed good expression (>1pmoVmg of protein); however, because of the variability in B,, among the different receptors, the Scatchard plots are modified such that they are normalized to Bmu. The slopes of these modified Scatchard plots are proportional to ligand affinity. Pharmacologic characterization of the expressed receptors was done using four ligands. The two antagonists chosen were from different classes of compounds, 5"ATGCCCAGGATCCTCATGTACATCGCAATATTCCTCACCCACG- L3HlCPX is a C-8-substituted xanthine, while N-0861 contains GCAACTCGGCC-3' (1270M,T277S). For the canine mutations 5'-AA- an h@-substituted adenine ring, but does not have a ribose at position of the adenine as would adenosine. The ribose is GCCCAGCATCCTCATCTACATCGCAATATTCCTCACGCACGGC-3' the (M2701), 5'-ATCGCCATCTTCCTCTCCCATGGCAACTCGGCCATG-3' required for agonist activity (26).The two agonists chosen were (T277S), and 5'-AAGCCCAGCATCCTCATCTACATCGCAATATTCCT-R-PIA, an NG-substituted ligand, and NECA, a ribose 5'-subCTCCCACGGCAACTCGGCC-3' (M270I,T277S)wereused. The mu- stituted ligand. tated receptors were subcloned into the CLDNlOB expression vector Wild type bovine (BBB)A, adenosine receptors bind L3H1CPX using KpnI and HindIII for the canine mutants and HindIII alone for with a 20-foldhigher affinity than canine (DDD)receptors (Fig. the bovine mutants. DNA Sequencing-The nucleotide sequences of all chimeras and mu- 2A, Table 11). The NG-substituted ligands, R-PIA and N-0861 tants were verified by DNA sequencing on both strands with a Seque- also bind to bovine receptors with 54- and 26-fold a higher nase I1 kit (United States Biolabs). affinity, respectively; conversely,NECA binds to the bovine reMembrane Preparation and Radioligand Binding-Forty-eight h af- ceptor with a 20-fold lesser affinity than the canine receptor ter transfection, using the DEAE-dextran method (21), COS-1 cells were washed in phosphate-buffered saline and homogenized in 10 vol- (Fig. 2 B , Table 11). Similar patterns of relative binding affiniumes of buffer A (10 rm NaHEPES, pH 7.4, 10 mM EDTA, 1mM ben- ties of these ligands have been noted using brain membranes. Ligand Binding to A, Receptor Species Chimeras-Reprezamidine). The membranes were washed twiceby centrifugation a t 20,000 x g for 30 min in 10 volumes of buffer B (10 rm NaHEPES, pH sentative experiments showing equilibrium binding of L3H]CPX 7.4, 1 rm EDTA, 1 mM benzamidine), and the final pellet was resus- to the six bovinelcanine chimericreceptors are shown in Fig. 3 pended at a membrane protein concentration of 1 mg/ml in buffer B and data from multiple experiments are summarized in Table supplemented with 10%(w/v) sucrose and frozen in aliquots at -20 "C. Protein concentrations were determined using fluorescarnine (22) with 11. Allof the receptor constructs ending in bovine sequence, bovine serum albumin standards. It is notable that treating membranes containing TMs 5-7, bind L3H1CPXwith relatively high affinity, with 10 m~ EDTA and conducting subsequent radioligand binding as- with KD values between 0.56 to 1.05 m, while all of those says in the absence of added divalent cation results in the absence of ending in canine sequence have lower affinity, with KD values GTP sensitive high affinity agonist binding (23).These conditions were between 11and 15 m. A similar pattern is seen in competition chosen to avoidthe complication of two af€inity states for agonists that binding assays with the other ligands. Table I1 shows the K, are found in the presence of divalent cations. Thus, under the conditions values of R-PIA, N-0861,and NECA for wildtype and chimeric used in the study, only a single low affinity agonist binding site is detected. For equilibrium binding assays six concentrations of [3H]CPX receptors. The binding affinities of these ligands depend prewere used in triplicate in tubes containing membranes with 25 pg of dominantly on the carboxyl third of the chimeric receptors. membrane protein a t 21 "C for2-3 h in a volume of 100p1. Experiments This point is illustratedgraphically in Fig. 4. Thus, a consistent sites into TM regions 4 and 5 , which flank the second exofacial loop. The oligonucleotides 5'-TCCTTCGTGGTGGGGTTAACGCCCATGTTCGG3' and 5'-TCC'ITCGTGGTGGGGTTAACCCCGCTGTTCGG-3' were used as primers to introduce silent HpaI sites into the fourth transmembrane region of the bovine and canine clones, respectively. NcoI sites were placed into TM5 of both receptors using 5"TCGAGAAGGTCATCTCCATGGAGTACATGG-3'. The mutated cDNAs were used to construct six bovine-canine adenosine A, receptor chimeras. pALT-RDC7 and pALT-BOV13S were digested with restriction endonucleases as defined in Table I and the resulting DNA fragments purified on a low melt agarose gel. The recovered DNA fragments were ligated together in the combinations depicted in Table I. The resulting chimera were subcloned into the expression vector CLDNlOB (a gift from M. Reff, Smith KlineBecham Laboratories) using KpnI and HindIII for DBB, DDB, DBD, and HindIII alone for BDD, BBD, and BDB. The CLDNlOB vector contains a cytomegalovirus early promoter and an SV40 ori. Site-directed Mutagenesis-Mutations were introduced into RDC7 and Bovl3 cDNAs subcloned into PALTER. Oligonucleotides used to introduce aminoacid changes were engineered to contain a silent, unique restriction site (NcoI forthose changing amino acid 277,Ssp1 for 270 alone and double 270/277 mutants) for rapid identification of mutated cDNA by restriction digestion. The oligonucleotides used to mutate thebovine adenosine receptor cDNA were 5"ATGCCGAGGACCTCATGTACATCGCAATATTCCTCTCACACGGC-3' (1270M),5"TACATCGCCATCTTCCTAACCCATGGCAAACTCGGCCATG-3' (T277S), and
27902
A , Adenosine Receptor Mutants Chimeras and
FIG.1. Diagrammatic representation of the structure of the A, adenosine receptor of the dogand cow. The canine rrcrptorforms t h r hackhone of the diagram with each amino acid represented by its single-letter code. Whrrc the hovinr sequence difTrrs. its amino acid srqurncr is designated with black circles. The putative transmembrane regions are embedded in the grey banner rrpresenting the cell mcmhmnr.. Spl~cr sites for chimera, Leu-140and Ser-176, are indicated with bold circles. The initial methionineis orirntatrd extracrllularl?; thr trrminnl aspartatr. intracellularly. The inset denotes the amino acidsin TM7 targeted for mutagenesis.
finding for all of the ligands tested, whether they bind prefer- ligand examined in this study that hinds with higher affinity to entially to bovine or canine receptors, is that species dependent wild type canine than towild type bovine receptors. The hind5 and Table 111)suggests binding properties are conferred primarily by the carboxyl- ing of data to the eight receptors (Fig. an interesting interaction hetween amino acid 270 and 277 in terminal third of the receptors. the regulation of NECA binding. A change in amino acid 277 Ligand Binding to Zkansmemhrane 7 Mutants of Adenosine from T h r to Ser always reduces NECA binding affinity, hut the A , Receptors-Prior studies on G-protein linked receptors for ligands with low molecular masses have implicated the trans- reduction in affinityis small (1.4-1.9 fold when amino acid 270 is Met, and large (4.R10.9fold) when amino acid 270 is Ile. membrane regions as forming the ligand binding pocket (2729). There are two amino acid differences between bovine and Thus, the relatively low affinity of the bovine receptor can he g of canine adenosine A, receptors in putative TMs 5-7, amino acids attributed to both Ile-270 and Ser-277. and c h a n ~ n either 270 and 277, both in TM7. These were targeted for site-directed suhthese amino acids to the corresponding canine sequence mutagenesis.Mutantreceptorswereconstructedinwhich stantially increases NECA binding affinity. amino acids 270 and 277 were switched, individually or toDISCUSSlON gether, from bovine to canine orvice versa. Table 111 shows the K, (["HlCPX) andK, values (R-PIA, NECA, N-0861)of various Thisstudyidentifiesaminoacids in TM7responsihle for ligands for wild type and mutant receptors. Both amino acids confemng species selectivity in ligand binding to A, adenosine contribute to species differences in ligand binding to an extent receptors. The influence of these two amino acids on binding varies depending on the nature of the ligand. Adenine and that depends on the nature of the ligand. This point is illustrated graphically in Fig. 5.For the C-8-substituted xanthine, xanthine compounds substituted with aryl or cycloalkyl suhstituents at the W or the C-8 positions, respectively, hind prefCPX, and the W-substituted adenine compounds, R-PIA and N-0861, amino acid 270 is the major determinant of species erentially to bovine as opposed to canine receptors. primarily differences in receptor affinity. Of the eight receptors examined due to amino acid 270, isoleucine in the hovine receptor and (two wild type and six mutated), the four receptors containing methionine in the canine receptor. This is entirely consistent with the dataof Peet et al. (30)which suggests that theMi poIle in position 270 invariably bound these three ligands with of xanthines occupy the higher affinity than the four receptors with Met in position 270. sition of adenines and the C-8 position same position in the binding pocket of the A , adenosine receptor. For example, the affinities of ['HICPX for receptors with IleThis P I C - 8 model also is supported hy an analysisof steric and 270 are on average 5.9-fold higher than receptors with Met270. In the cases of R-PIA and N-0861 this factor is 7.3- and electrostatic properties of ligands (31 and hy binding studies 15-fold, respectively. By comparison, changing the amino acid with various ligands to membranes expressing recornhinant in position 277 (Sor T) produced small, directionally inconsis- dog, rat, and bovine A, adenosine receptors. The K'lC-8 orientent changes in the affinity of receptors for thePIC-8 ligands. tation appears also to hold for A, adenosine receptors (9, 321. The affinity ratio of I:'HICPX, R-PIA, and N-0861 for receptors NECA, which like adenosine is unsuhstituted in the N" position, binds preferentially to receptors that contain Thr-277 with Ser-277 versus Thr-277 is 2.13, 1.53, and 0.83, respec(canine) as opposed to Ser-277 (bovine). Changing amino acid tively. In sum these data indicate that the high relative affinity of WlC-8 ligands for bovine versus canine receptors is attrib- 277 from threonine to serine consistently reduces the affinity of receptors for NECA. It is striking that this effect is relatively utable primarily to amino acid 270, Ileversus Met. Amino acid 277 takes on greater significance in determining large if amino acid 270 is isoleucine rather than methionine. Consistent with these results is the recent finding that mutathe affinity of adenosine A, receptors for NECA, an agonist tion of threonine 277 in the human A, adenosine receptor to substituted on ribose, and not on adenine. This is the only
A , Adenosine Receptor Mutants Chimeras and
A
3000 2000 1000 0
8 0 3 6 9 1215 Free [3H1CPX (nM)
4
1.5-
0.5-
0.2
0
B
0.4
0.6 0.8 B/Bmax
1.2,
1
1
I
INECAl (log M)
1.2
.i
8
A
e
-10-9 -8 -7 -6 -5 -4 IN08611 (log M)
-3
FIG.2. Radioligand binding to transfectedCOS-1 cells. A, Top panels showspecific (circles) and nonspecific (squares) equilibrium binding of L3H1CPX to membranes of cells expressing bovine and canine A, adenosine receptors, respectively.Points are means f S.E., n = 3, the error bars are not visible becausethey are smaller than the size of the symbols. The bottom panel shows Scatchard transformations of these data and the structure of CPX. Nonspecific binding is defined by the addition of 1 CPX. Shown are representative curves from a series of three experiments. B , competition for L3H1CPXbinding and structures of other compounds that bind to A, adenosine receptors. Solid and open symbols depict binding to bovine and canine receptors, respectively. Points are means f S.E., n = 3. Shown are representative curves from a series of two t o six experiments. S.E. smaller than thesymbols are not shown.
27903 serine or alanine causes a relatively selective decrease in NECA binding affinity (33). Moreover, interaction of A, adenosine receptor ligands with amino acid 277 is predicted in the binding model of Dudley et al. (34). Our data indicate some interplay between amino acids in positions270 and 277 in determining affinities for ligands without bulky N6 or C-8 substitutions. One possibilityis that therelatively small isoleucine side chain promotes docking of aryl or cycloalkyl iWC-8 substituent, while the slightly larger methionine group repels these, but interacts favorably with the N6 region of unsubstituted agonists. In theabsence of the methionine at 270 (such as in the bovine receptor which has an isoleucine, or the human receptor containing a threonine), the amino acid at position 277, viainteractions with a different region of the ligand, has a larger influence on ligand affinity for 5' or C2-substituted ligands without N6 substitutions. A threonine at position 277 favors high affinity binding to such ligands. The rat adenosine A, receptor shows a ligand binding profile that is consistent with this theory; this receptor displays high affinity for N6- and C-8-substituted ligands and for ligands with 5'- or C2-substitutions. As would be predicted, it has anisoleucine at position 270 as does the bovine A, receptor, but a threonine at position 277 like the canine A, receptor. It is possible that mutations in receptor amino acids alter binding affinity either because ligands interact directly with mutated amino acids, or because the mutations change receptor conformation to indirectly influence binding to remote domains. We have attempted t o minimize the latterpossibility in this study by making conservative interspecies mutations that are not likely to produce changes in receptor structure. The mutations we have introduced apparently do not produce major changes in receptor structure or expression. Thus, we hypothesize that amino acid 270 is directly involved in the docking of the N6 portion of adenines and the C-8 portion of xanthines. However, in the absence of structural data it isnot possible to exclude the possibility of indirect effects of changing amino acids 270 and 277 on a remote ligand recognition domain. Our data support a model forligand binding in which the N6 or C-8 substituents of the ligand interact with a region of the receptor containing amino acid 270, whilea different region of the ligand, perhaps on the ribose, interacts with a hydroxyl on amino acid 277. Because the antagonists, CPX and N-0861, do not have ribose moieties and their binding is not significantly affected by the T/S mutations at position 277,the interaction of the ribose at this position is an attractive hypothesis. Either the 5' N-ethylcarboxyl substituent or some other portion of the ribose moiety of NECA may interact at position 277. Binding studies on mutant receptors using ligands substituted at other positions, such as 2-chloroadenosine, which has a chlorine at the C2 position on the adenine ring, will help t o clarify this. This study exploits unique species pharmacologyin order to better understandligand orientation in theadenosine A, receptor binding site(s). While there are computer-generated models for the A, binding domain, supportive structure-function analysis of adenosine receptors has been limited to date. Ijzerman et al. (35) have proposed interaction of histidines 251 and 278 with the N6 region of the adenine and ribose hydroxyl groups, respectively, in part based on early chemical modificationstudreceptors using the histidineies of the adenosine A, and ha selective agent diethylpyrocarbonate. These studies showed that alkylating histidine residues modified ligand binding characteristics (36-38). Examination of the sequences for the putative transmembrane amino acids of the adenosine A, receptor reveals two histidines, one in each of transmembranes 6 and 7. Data from mutational analyses of the two transmembrane histidines in the bovineh, receptor have been difficultto interpret.
27904
A , Adenosine Receptor Chimeras and Mutants
TBLE I1 Summary of binding affinities of the antagonists PHICPX and N-0861 and the agonists R-PIA and NECA for wild type and chimeric A, adenosine receptors Amnities are expressed as themeans * S.E.of two to six independent experiments performed in triplicate. Also indicated for eachligand tested is the fold change from the wild type canine affinity. R-PIA
CFX Receptor
DDD BDD DBD BBD BBB DBB BDB DDB
Change from DDD
Kd S.E. nM
-fold
11.05 f 0.46 11.56f 1.15 12.15f 2.80 15.0 * 1.69 0.56 * 0.08 0.89f 0.13 0.75* 0.15 1.05 * 0.18
1 0.96 0.91 0.74 19.63 12.4 14.7 10.5
N-0861
K, i S.E.
Change
PM
-fold 1
4.76f 0.27 8.582 0.45 1.57f 0.20 0.97f 0.13 0.09* 0.02 0.21* 0.01 0.07.+ 0.01 0.36* 0.04
0.55 3.03 4.91 52.9 22.7 68 13.2
K, f S.E. W
3.18* 0.72 1.93 2 0.7 1.96f: 1.01 2.972 0.45 0.12 2 0.03 0.30 2 0.01 0.08f 0.02 0.21f 0.10
NECA Change
-fold
1 1.65 1.62 1.07 26.5 10.6 39.8 15.1
K, i S.E.
Change
PM
-fold
2.18 2 1.72 3.63 2 1.83 1.892 0.75 0.48f 0.16 42.8 f 17.1 36.42 20.3 18.42 6.6 88.3 f 5.80
1 0.6 1.15 4.54 0.05 0.06 0.12 0.02
3.. DDB BDB DBB BED
DBD BDD
0.5
DDD
0
0.4
0.8
7.5 8.5
1.2
0
0:4
018
2,
DBD
-
I
0
2
*DDB
1
014
0:s
112
I
1:2
2,
B/Bmax FIG.3.Modified Scatchard transformations of [*HlCPX binding to wild type and chimericAI adenosine receptors.The solid and dashed lines show binding to wild type bovine and canine receptors, respectively. The dotted lines shown binding to the indicated chimeric receptors. Filled and open squares depict binding to receptor chimera ending with bovine and canine sequence, respectively. Each plot is representative of three experiments.
Mutation of the histidine residue in TM7 (His-278) to leucine dramatically decreased both agonist and antagonistbinding by 90% (39); however, this may have been due to poor receptor expression rather thanto the loss of a selective receptor-ligand interaction. Mutation of the histidine in TM6 effected no change in agonist affinity, but caused a 3.8-fold decrease in antagonist affinity. Again,there was a large (74%)decrease in receptor number. Interestingly, histidine 278 is adjacent to the threonine 277 mutated in this study. Given the dramatic decrease in receptor number and the nonspecific effects onligand binding seen with mutation of histidines 251 and 278, it is possible that these histidines are important for receptor processing or configuration of the ligand binding domain without
CPX
9.5 4.5 5.5
6.5 7.5 4.5 5.5 R-PIA
6.5 7.5
NO861
4
5
6
7
NECA
pKi or pKd FIG.4. Graphical comparison of binding constants of compounds for chimeric A, adenosine receptors.The composition of the chimeras is indicated by the three-lettercodes shownon the left (see Fig. 3). Solid and striped bars show binding to receptors ending with bovine and canine sequence, respectively. Average K, values are derived from competition for L3H1CPX binding and are listed in table 2. Error bars show the -log of the means f S.E.of binding constants.
directly interacting with ligands. In support of this, Ganitsen et al. (40) have shown that the degree of protonation of the histidine residues does not alter ligand affinity; this would be difficult to explain if this amino acid were directly involved in binding. Expression of high numbers of mutated receptors in this study, and ligand-selective effects of these mutations support roles for amino acids 270 and 277 in direct ligand interactions. Despite their different binding profiles, the amino acid sequences of the canine and bovine adenosine receptors are very similar over most of their length, especially in the transmembrane regions, which typically are thought to form the ligand binding domain. Exceptions t o this are seen among the glycoprotein hormone receptors, including the lutropin (LH), follitropin (FSH), and thyrotropin (TSH) receptors, which have large extracellular domains that bind ligands with high affinity (41,42). The A, adenosine receptors differ most between species in a hypervariable region of the second exofacial loop.We have speculated previously (20) that this region is variable either because it does not have an important role in receptor structure-function, so it accommodates frequent mutations, or that this region is responsible for species differencesin ligand binding. The results of this study support the former possibility. Previous studies of G protein-coupled receptors, most notably adrenergic and muscarinic receptors, using site-directed mutagenesis and intersubtype chimeric receptors have demonstrated the importance of transmembranes 5, 6, and 7 in agonist and antagonistbinding (43-47). In several receptors, TM7 has been shownto be important for antagonist binding (43,45, 46). For several G protein-coupled receptors, including the aZadrenergic, the 5-HT2,and the 5-HT,, receptors, marked pharmacologicaldifferences between homologuesfrom different
A, Adenosine Receptor Mutants Chimeras and
27905
TABLEI11 Summary of binding affinities of the antagonists PHICPX and N-0861and of the agonists R-PIA and NECA for wild type and mutant A , adenosine receptors All affinities are expressed as themean i: S.E. of two to six independent experiments performed in triplicate. Also indicated for eachligand tested is the -fold change from the wild type canine affinity. R-PIA Receptor
CPX
Amino acid 2701277
(DDD)
Dog Dog T277S Dog M270I Dog M2701, T277S (BBB) Bovine Bov 1270M Bov S277T Bov 1270M, S277T
M/T
MIS
m
US US MIS
m
M/T
N-0861 Change for DDD
Kd
nu 11.05 -c 0.46
4.72 i: 0.55 1.58 i: 0.22 0.82 i: 0.06 0.56 i: 0.08 2.76 i: 0.27 1.1-c 0.41 5.17 i: 0.13
KL
Change
K.
Change
-fold
W
-fold
PM
1 2.34 7 13.5 19.6 4 10.1 2.13
4.76 2 0.27 3.02 f 0.03 0.20 f 0.08 0.72 f 0.01 0.09 f 0.02 0.91 f 0.67 0.44 * 0.29 1.84 2 1.56
1 1.58 23.8 6.61 52.9 5.23 10.8 2.58
3.18 2 0.72 4.97 i: 0.05 0.26 t 0.02 0.26 t 0.01 0.12 i: 0.03 0.61 i: 0.01 0.04 * 0.002 1.46 i: 1.28
W 1 2.18 i: 1.72 0.65 3.08 i: 1.51 12.2 6.56 i: 0.34 12.2 31.4 i: 1.50 26.5 42.8 i: 17.1 5.21 6.92 i: 0.28 79.5 3.9 i: 0.36 i: 0.87 2.18 3.75
oq
Dq=m Bm la7011 E m VU.U l t Bcdme ~m
sa7m
D q W7Ol Dog YII. TI8
7.5
8.5 CPX
9.5 5
6 7 R-PIA
5
6
NO86 1
7
NECA
4
5 6 NECA
7
pKi or pKd FIG.5. Graphical comparison of binding constants of compounds for binding to mutated A, adenosine receptors.Mutant receptors containing Met-270 and Ile-270 are depicted with solid bars and striped bars,respectively. Mutations are identified by wild type and mutated single letter codes placed before and after, respectively, the number of the mutated receptor amino acidb). Average K,values are derived from competition for [3H]CFXbinding and are listed in Table 111. Error bars show the -log of the means * S.E. of binding constants.
species can be attributed to single amino acid differences (4850). For the qadrenergic and the 5-HT2receptor, the critical amino acid is in TM5, but for 5-HT1, it is in TM7. Likewise, the results of this study of the A, adenosine receptor implicate TM7 as being largely responsible for interspecies variation in pharmacology. We have identified two amino acids responsible for conferring species selectivity for different ligands to adenosine A, receptors. Other amino acids in the TM regions would also be expected to be involved in ligand interaction. Only six amino acids differ between the bovine and canine adenosine A, receptors in the transmembrane domains, the most divergent TMs being 4 and 7. Among subtypes of adenosine receptors, TM2 and TM3 are most highly conserved and have been shownt o be important for ligand binding in the cationic amine receptors (44, 51). The lack of variability in these regions raises the possibility that these domains may be important in binding to invariant regions of agonist ligands, such as the 2' or 3' OH groups of the ribose moiety or unsubstituted portions of the adenine ring. These identical regions should not contribute to receptor subtype or species selectivity for ligands. Neither of the published models of adenosine A, receptor-ligand binding pocket (34,35) iscompletely consistent with the datafrom our mutational analysis of the adenosine A, receptor; however, both the models of Peet and of Ijzerman propose interactions between adenosine and amino acids in TM6 and TM7. Results from mutational analyses, including our own, should lead t o the development of more refined and accurate models for the adenosine receptor ligand binding domain. REFERENCES 1. Fredholm, B. B., and Dunwiddie, T.V. (1988) Den& Pharmacol. Sei. 9, 130134 2. Bellardinelli, L., Linden, J., and Berne, R. M. (1989)Prog. Cardiouasc. Dis. 32, 73-97
-fold
Ki
Change -fold
1 0.71 0.33 0.07 0.05 0.31 0.56 0.58
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