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A Retrospective of Recombinant P2Y Receptor Subtypes and Their Pharmacology
Archives of Biochemistry and Biophysics Vol. 397, No. 1, January 1, pp. 131–136, 2002 doi:10.1006/abbi.2001.2616, available online at http://www.idealibrary.com on
RESEARCH REPORT A Retrospective of Recombinant P2Y Receptor Subtypes and Their Pharmacology Katrin Sak* and Tania E. Webb† ,1 ¨ likooli 18, Tartu 50090, Estonia; and †Cell Signaling Laboratory, *Hematology–Oncology Clinic, Tartu University, U Department of Biological Sciences, De Montfort University at Leicester, The Hawthorn Building, The Gateway, Leicester LE1 9BH, United Kingdom
Received September 18, 2001; published online December 4, 2001
Since the first cloning of P2Y receptor sequences in 1993 it has become apparent that this family of Gprotein-coupled receptors is omnipresent. At least 25 individual sequences entered in the GenBank sequence database encode P2Y receptors from a variety of species ranging from the little skate Raja erinacea to man. In man, six receptor subtypes have been cloned and found to be functionally active (P2Y 1, P2Y 2, P2Y 4, P2Y 6, P2Y 11, and P2Y 12). In this article a review of the P2Y receptor subtypes is presented considering both their sequences and the pharmacological profiles of the encoded receptors expressed in heterologous expression systems. © 2001 Elsevier Science
Drury and Szent-Gyo¨rgyi published the first report of an extracellular action of adenine compounds in 1929. The work of Burnstock and colleagues has been fundamental in the classification of the receptors for extracellular purine nucleosides and both extracellular purine and pyrimidine nucleotides (1, 2), leading to clearly recognized subdivisions: P1, at which adenosine is the principal natural ligand, and P2, which are selective for nucleotides. Later the P2 receptors were divided into P2X receptors (ATP-gated cation channels) and P2Y receptors (G-protein-coupled seven-transmembrane receptors activated by purine or pyrimidine nucleotides) (3). Today there is a considerable literature on the wide-ranging cellular effects of extracellular nucleotides, mediating several physiologically essential processes, which places them as ubiquitous signaling molecules (4 – 8). This article focuses on the P2Y receptors. The first DNA sequences for members of this receptor were published in 1993 (9, 10) and enabled the isolation of related sequences by
1 To whom correspondence should be addressed. Fax: 44 116 257 7286. E-mail: [email protected].
0003-9861/01 $35.00 © 2001 Elsevier Science All rights reserved.
homology cloning strategies. A number of P2Y receptor subtypes have been cloned and designated P2Y 1 to P2Y n, in which the subscript allocation was based on the chronology of cloning (11). Among them six subtypes (P2Y 1, P2Y 2, P2Y 4, P2Y 6, P2Y 11, and P2Y 12) have been cloned from man and have been demonstrated to be functionally active. Sequence variations as well as differences in ligand activities between species homologues of P2Y receptor subtypes have raised several queries on the appropriateness of the present classification (7, 11). In this review we present a survey of the amino acid sequence identity shared between members of this receptor family as well as the activity of several natural and synthetic compounds at these recombinant receptors. HOW MANY P2Y RECEPTORS ARE THERE? To date at least 25 individual sequences present in the GenBank database encode functional P2Y receptors from a range of species. A summary of the percentage amino acid sequence identities of these receptors carried out by using the ClustalW service (http://www2.ebi.ac.uk/clustalw/; EMBL, European Bioinformatics Institute) is presented in Table I and a phylogenetic analysis of these sequences is presented in Fig. 1. It can be seen that an identity level of above 80% is highly indicative of species homologues (i.e., sequences belong to a receptor subtype). At the same time rather significant variations in amino acid sequences (up to 85%) can be observed comparing sequences belonging to different receptor subtypes. There are a number of reports in the literature which indicate that there are other subtypes yet to be cloned (12–14), which may or may not include a P2YApnA receptor (15). OVERVIEW OF LIGAND ACTIVITIES AT P2Y RECEPTORS Of the human recombinant receptors, P2Y 1, P2Y 2, P2Y 4, and P2Y 6 are coupled to phospholipid turnover and intracellular calcium mobilization (16). The P2Y 11 receptor activates both phospholipase C and adenylyl cyclase (17), while the 131
Human P2Y 1 Cloned from brain (27), erythroleukemia cells (42), prostate and ovary (43), placenta (44) Bovine P2Y 1 Cloned from brain (30), endothelium (29) Rat P2Y 1 Cloned from insulinoma cells (45) Mouse P2Y 1 Cloned from insulinoma cells (45) Chicken P2y 1 Cloned from brain (9) Turkey P2y 1 Cloned from brain (27) Human P2Y 2 Cloned from airway epithelial CF/T43 cells (46) Canine P2Y 2 Cloned from MDCK-D1 renal epithelial cells (47) Rat P2Y 2 Cloned from microvascular coronary endothelial cells (48), alveolar type II cells (49), a pituitary (50), aorta (51) Mouse P2Y 2 Cloned from NG108-15 neuroblastoma cells (10), mammary tumor cells (52) b Chicken P2y 3 Cloned from brain (53) Turkey P2y 3 Cloned from genomic DNA (54) Human P2Y 4 Cloned from placenta (55), chromosome X (56), c placenta genomic library (57) Rat P2Y 4 Cloned from heart (39), brain (40) Mouse P2Y 4 Cloned from gallbladder epithelial cells (41) Human P2Y 6 Cloned from placenta (58), activated T-cells (59) Rat P2Y 6 Cloned from aortic smooth muscle cells (60)
1
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
Receptor
No.
328
328
361
361
365
328
328
373
374
373
377
362
362
373
373
373
373
Amino acid number
100
h P2Y 1
100
96
b P2Y 1
100
94
95
r P2Y 1
100
97
93
94
m P2Y 1
TABLE I
100
84
85
85
85
ch P2y 1
100
99
85
85
85
85
t P2y 1
100
34
34
30
31
31
31
h P2Y 2
100
87
33
33
30
30
31
31
ca P2Y 2
100
83
87
35
35
31
32
33
33
r P2Y 2
100
97
83
89
35
35
31
31
32
33
m P2Y 2
100
38
38
39
39
36
36
36
36
35
36
ch P2y 3
100
97
38
38
39
39
36
36
36
36
35
36
t P2y 3
100
38
38
50
48
49
51
34
34
33
33
33
34
h P2Y 4
100
83
38
38
48
48
49
49
34
34
32
33
33
34
r P2Y 4
Percentage
100
94
81
39
39
47
49
47
48
34
34
32
33
33
34
m P2Y 4
Amino Acid Similarity of P2Y Receptor Sequences
100
39
40
38
60
60
36
37
38
37
34
34
34
34
34
33
h P2Y 6
100
87
40
41
39
60
60
39
39
40
40
34
34
32
33
33
32
r P2Y 6
96
86
39
39
39
59
59
39
39
39
39
34
34
32
32
32
32
m P2Y 6
37
38
50
48
49
36
35
43
44
44
44
38
38
35
36
36
36
x P2y 8
23
23
23
24
23
21
21
25
24
24
25
27
27
24
25
25
26
h P2Y 11
21
22
23
24
25
21
20
20
20
17
20
25
25
24
23
23
25
ca P2Y 11
19
20
23
22
21
29
20
20
21
18
20
20
20
20
20
19
19
h P2Y 12
19
19
23
23
22
18
19
21
22
20
20
20
20
20
20
20
20
r P2Y 12
38
39
54
55
54
40
40
47
45
44
45
39
39
34
34
35
34
t P2y
35
35
33
33
33
36
36
34
34
34
35
65
65
62
62
62
61
s P2y
132 SAK AND WEBB
357
374
343
342
370
371
25
24
23
22
21
20
Note. Similarities more than 80% are indicated in bold and similarities between 52 and 80% in italic. a Cys 132 is substituted by Ser, Ser 143 by Arg, Ala 166 by Thr, and Ala 188 by Asp in the rat P2Y 2 sequence cloned from rat alveolar type II cells (49), comparing with other rat P2Y 2 receptors. b Glu 17 is substituted by Asp, Ser 120 by Arg, Thr 125 by Asn, Val 196 by Met, Asp 355 by Asn, Lys 369 by Pro, Asp 370 by Tyr, and Ile 371 by Val in the mouse P2Y 2 sequence cloned from mammary tumor cells (52), comparing with other mouse P2Y 2 receptors. c Leu 86 is substituted by Val, Asn 178 by Thr, and Ser 234 by Ala in the human P2Y 4 sequence cloned from human chromosome X (56), comparing with other human P2Y 4 receptor sequences.
38
22
16
100
22 24
100
100
23 24 85 100
100
21
23 20 17
68 100
25 25 537 19
18
Mouse P2Y 6 Cloned from gallbladder epithelial cells (41) Xenopus P2y 8 Cloned from neurula cDNA library (61) Human P2Y 11 Cloned from placenta (17) Canine P2Y 11 Cloned from MDCK-D1 renal epithelial cells (21) Human P2Y 12 Cloned from platelets (18) Rat P2Y 12 Cloned from platelets (18) Turkey P2y Cloned from blood cDNA library (62) Skate P2y Cloned from hepatocytes (63)
328
100
21
18
18
38
55
26 15
20 23 100
35 23 37
39
P2Y RECEPTOR SUBTYPES AND THEIR PHARMACOLOGY
133
FIG. 1. A phylogenetic analysis of cloned P2Y receptors using the Clustal algorithm is shown. The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
P2Y 12 receptor is coupled to the inhibition of adenylyl cyclase (18, 19). P2Y receptors can be characterized as purinoceptors, pyrimidinoceptors, and receptors with mixed selectivity. These receptors differentiate also between nucleoside di- and triphosphates. However, analysis of the activity data of several natural and synthetic compounds at P2Y receptors does not mirror the grouping that can be made from amino acid sequences analysis. Most of the published activity data have been collected into the “P2Y Receptor Ligand Database” [http://bioorg.chem.ut.ee/p2y/; Institute of Chemical Physics, Tartu University; (20)]. The activity constants of the most frequently used ligands, measured in mammalian cells transfected with the recombinant receptors, are presented in Table II. The divergence of amino acid sequence due to species differences within a receptor subtype, commonly up to 20% (Table I, Fig. 1), may influence the affinity of ligands and thus change the ligand activity order in the case of species homologues (21). Therefore, the mean activity constants of nucleotides and their analogues may differ within one subtype. Indeed, data have been published which suggest a contradictory activity type (agonist versus antagonist) for some ligands at species homologues within a P2Y receptor subtype (Table II). As mentioned above, this has been a consequence of species differences in some cases but is complicated further by the following observations. It has been demonstrated that the activity of ligands measured in some experimental assays depends also on the number of functionally active receptors, a factor especially important in the recombinant receptor systems (22). Moreover, action of ectoenzymes, catalyzing the conversion of nucleotides at the
134
SAK AND WEBB TABLE II
Mean Activity Constants of Nucleotides and Their Analogues at P2Y Receptors No.
Receptor
ATP
2MeSATP
2ClATP
ATP␥S
ADP
2MeSADP
ADPS
UTP
UDP
1
Human P2Y 1
5.64 ⴞ 0.06 1 6.11 ⫾ 0.07 1 7.80 ⫾ 0.10 1 — — — 5.97 ⫾ 0.45 2 5.64 ⫾ 0.06 1 — —
6.88 ⫾ 0.15 9
8.29 ⫾ 0.13 13
6.34 ⫾ 0.28 4
N.A. a
—
Bovine P2Y 1 Rat P2Y 1 Mouse P2Y 1 Chicken P2y 1 Turkey P2y 1 Human P2Y 2 Canine P2Y 2 Rat P2Y 2
5.24 ⴞ 0.03 1 7.53 ⫾ 0.16 9 7.40 ⫾ 0.00 2 7.52 ⫾ 0.00 2 — 8.00 ⫾ 0.00 2 7.52 ⫾ 0.02 3 N.A. — N.A.
—
2 3 4 5 6 7 8 9
5.07 ⴞ 0.23 2 5.84 ⫾ 0.18 5 5.77 ⫾ 0.54 3 6.85 ⫾ 0.08 1 — 6.10 ⫾ 0.60 4 5.68 ⫾ 0.20 3 6.75 ⫾ 0.12 6 — 6.50 ⫾ 0.20 2
6.50 ⫾ 0.10 1 — — — 5.96 ⫾ 0.41 2 6.08 ⫾ 0.16 3 — —
8.30 ⫾ 0.10 1 9.24 ⫾ 0.04 1 — — 7.77 ⫾ 0.18 2 — — —
— — — ⬃6.82 1 6.74 ⫾ 0.11 2 — — —
N.A. — — N.A. N.A. 6.91 ⫾ 0.29 6 — 6.10 ⫾ 0.19 5
— — — — — N.A. — 4.80 1
10
Mouse P2Y 2
5.99 ⫾ 0.06 5
N.A.
—
5.04 ⫾ 0.06 2
—
—
6.20 ⫾ 0.12 5
N.A. (64) or partial agonist (65)
11 12 13
Chicken P2y 3 Turkey P2y 3 Human P2Y 4
— 5.04 ⫾ 0.11 1 6.15 1
5.09 ⫾ 0.20 1 — N.A.
— — —
4.89 ⫾ 0.23 1 — N.A.
— 6.55 ⫾ 0.15 1 —
— 5.75 ⫾ 0.09 1 —
5.77 ⫾ 0.18 1 6.05 ⫾ 0.16 2 6.12 ⫾ 0.11 9
6.92 ⫾ 0.18 1 7.79 ⫾ 0.17 2 N.A.
14
Rat P2Y 4
6.00 ⫾ 0.16 3
N.A. (40) or partial agonist (39) 5.68 ⫾ 0.06 1
—
Partial agonist 5.68 ⫾ 0.10 1
7.53 ⫾ 0.03 2 8.07 ⫾ 0.07 1 — 6.59 ⫾ 0.07 1 6.53 ⫾ 0.21 3 N.A. — Partial agonist 4.83 ⫾ 0.23 2 N.A. (10, 64) or partial agonist (65) 4.99 ⫾ 0.78 2 6.33 ⫾ 0.17 1 N.A. (55, 66) or partial agonist (38) N.A. (40) or partial agonist (38, 39) 5.95 ⫾ 0.23 2
—
—
6.17 ⫾ 0.32 3
15 16 17
Mouse P2Y 4 Human P2Y 6 Rat P2Y 6
6.36 ⫾ 0.13 1 N.A. N.A.
— 4.00 1 N.A.
— — —
— — —
N.A. 4.36 ⫾ 0.17 2 5.20 ⫾ 0.11 1
— — 5.75 ⫾ 0.30 1
— — 4.59 ⫾ 0.47 1
18
Mouse P2Y 6
N.A.
—
—
—
—
—
—
19 20
Xenopus P2y 8 Human P2Y 11
7.05 ⫾ 0.06 2 4.38 ⫾ 0.14 4
— 3.97 ⫾ 0.18 3
— —
— 5.17 ⫾ 0.30 2
— N.A.
— N.A.
21 22 23 24 25
Canine P2Y 11 Human P2Y 12 Rat P2Y 12 Turkey P2y Skate P2y
⬃4.22 1 — — 6.89 ⫾ 0.07 1 —
⬃6.00 1 — — N.A. —
— — — — —
— — — 4.95 ⫾ 0.33 1 —
⬃5.00 1 6.52 1 6.52 1 4.14 ⫾ 0.15 1 —
⬃6.00 1 9.05 1 9.05 1 N.A. —
— Partial agonist 4.15 ⫾ 0.39 2 5.65 ⫾ 0.35 2 — — N.A. —
6.59 ⫾ 0.14 1 5.22 1 N.A. (67) or full agonist (54, 68) 6.95 ⫾ 0.35 5 N.A. or weak agonist (41) — N.A. N.A. — — 6.99 ⫾ 0.08 1 —
N.A. (38, 40) or partial agonist (39) 5.38 ⫾ 0.01 1 N.A. 6.52 ⫾ 0.00 2 7.72 ⫾ 0.28 5
7.38 ⫾ 0.20 1 — N.A. N.A. N.A. N.A. 3.99 ⫾ 0.12 1 —
Note. The constants listed [agonistic pEC 50 ⫾ SE and antagonistic pK i ⫾ SE (in bold)] were calculated from P2Y Receptor Ligand Database as mean values of published data for transfected systems. Number of separate determination used for calculation is given as superscript. a N.A., no or very small agonistic activity up to 100 M ligand concentration; pEC 50 ⬍ 4.00.
cell surface (23), results in a measured response due to the applied compound, its conversion/breakdown products, or a mixture of both rather than solely the initial ligand. In this context the importance of the application of HPLC-purified ligands and the use of regenerating systems in the P2Y receptor assays are self-evident.
Thus, ATP can be either an antagonist (24 –26) or an agonist (27, 28) at the human P2Y 1 receptor and an agonist for the P2Y 1 receptors derived from the other species [bovine (29 –31), rat (32), chick (9, 33–35), and turkey (27, 36, 37)]. ATP is an antagonist at the human P2Y 4 receptor (38) but is an agonist at both rat and mouse P2Y 4 receptors
FIG. 2. Comparison of ligand activities at P2Y receptor-transfected cells measured by different assay procedures. Numbers within the graphs correspond to the numeration of P2Y receptors listed in Tables I and II (■—ATP, Œ—2MeSATP, —ATP␥S, F—ADP, }—2MeSADP, *—ADPS, 䊐—UTP, E—UDP).
P2Y RECEPTOR SUBTYPES AND THEIR PHARMACOLOGY
(38 – 41). Despite such complications, in general the scatter of the data presented in Table II is rather small, indicating that the errors caused by differences in assay procedures are generally not determinant, especially in the case of nonhydrolyzable compounds. Indeed, the comparison of activity data measured by inositol phosphate accumulation, intracellular calcium mobilization, electrophysiological recordings, and cyclic AMP formation revealed that there is no systematic influence of the assay procedures on the results (Figs. 2A–2C). However, there are still some ligands with an unclear activity type (Table II), in which cases some scattering can be seen in the comparison of results measured by different assay procedures, for example, the activity of UTP at rat P2Y 6 receptor in Fig. 2B. This emphasizes the need for special attention to be paid to ligand purity. CONCLUDING REMARKS Further studies with novel highly selective and nondegradable agonists and antagonists coupled with investigations into signal transduction pathways of this heterogeneous family of receptors are required to extend the existing classification system and most importantly correlate the recombinant receptors with their endogenous counterparts. ACKNOWLEDGMENTS K.S. is grateful to Professor Jaak Ja¨rv (Institute of Chemical Physics, Tartu University, Estonia) for helpful comments about the initial manuscript.
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RESEARCH REPORT A Retrospective of Recombinant P2Y Receptor Subtypes and Their Pharmacology Katrin Sak* and Tania E. Webb† ,1 ¨ likooli 18, Tartu 50090, Estonia; and †Cell Signaling Laboratory, *Hematology–Oncology Clinic, Tartu University, U Department of Biological Sciences, De Montfort University at Leicester, The Hawthorn Building, The Gateway, Leicester LE1 9BH, United Kingdom
Received September 18, 2001; published online December 4, 2001
Since the first cloning of P2Y receptor sequences in 1993 it has become apparent that this family of Gprotein-coupled receptors is omnipresent. At least 25 individual sequences entered in the GenBank sequence database encode P2Y receptors from a variety of species ranging from the little skate Raja erinacea to man. In man, six receptor subtypes have been cloned and found to be functionally active (P2Y 1, P2Y 2, P2Y 4, P2Y 6, P2Y 11, and P2Y 12). In this article a review of the P2Y receptor subtypes is presented considering both their sequences and the pharmacological profiles of the encoded receptors expressed in heterologous expression systems. © 2001 Elsevier Science
Drury and Szent-Gyo¨rgyi published the first report of an extracellular action of adenine compounds in 1929. The work of Burnstock and colleagues has been fundamental in the classification of the receptors for extracellular purine nucleosides and both extracellular purine and pyrimidine nucleotides (1, 2), leading to clearly recognized subdivisions: P1, at which adenosine is the principal natural ligand, and P2, which are selective for nucleotides. Later the P2 receptors were divided into P2X receptors (ATP-gated cation channels) and P2Y receptors (G-protein-coupled seven-transmembrane receptors activated by purine or pyrimidine nucleotides) (3). Today there is a considerable literature on the wide-ranging cellular effects of extracellular nucleotides, mediating several physiologically essential processes, which places them as ubiquitous signaling molecules (4 – 8). This article focuses on the P2Y receptors. The first DNA sequences for members of this receptor were published in 1993 (9, 10) and enabled the isolation of related sequences by
1 To whom correspondence should be addressed. Fax: 44 116 257 7286. E-mail: [email protected].
0003-9861/01 $35.00 © 2001 Elsevier Science All rights reserved.
homology cloning strategies. A number of P2Y receptor subtypes have been cloned and designated P2Y 1 to P2Y n, in which the subscript allocation was based on the chronology of cloning (11). Among them six subtypes (P2Y 1, P2Y 2, P2Y 4, P2Y 6, P2Y 11, and P2Y 12) have been cloned from man and have been demonstrated to be functionally active. Sequence variations as well as differences in ligand activities between species homologues of P2Y receptor subtypes have raised several queries on the appropriateness of the present classification (7, 11). In this review we present a survey of the amino acid sequence identity shared between members of this receptor family as well as the activity of several natural and synthetic compounds at these recombinant receptors. HOW MANY P2Y RECEPTORS ARE THERE? To date at least 25 individual sequences present in the GenBank database encode functional P2Y receptors from a range of species. A summary of the percentage amino acid sequence identities of these receptors carried out by using the ClustalW service (http://www2.ebi.ac.uk/clustalw/; EMBL, European Bioinformatics Institute) is presented in Table I and a phylogenetic analysis of these sequences is presented in Fig. 1. It can be seen that an identity level of above 80% is highly indicative of species homologues (i.e., sequences belong to a receptor subtype). At the same time rather significant variations in amino acid sequences (up to 85%) can be observed comparing sequences belonging to different receptor subtypes. There are a number of reports in the literature which indicate that there are other subtypes yet to be cloned (12–14), which may or may not include a P2YApnA receptor (15). OVERVIEW OF LIGAND ACTIVITIES AT P2Y RECEPTORS Of the human recombinant receptors, P2Y 1, P2Y 2, P2Y 4, and P2Y 6 are coupled to phospholipid turnover and intracellular calcium mobilization (16). The P2Y 11 receptor activates both phospholipase C and adenylyl cyclase (17), while the 131
Human P2Y 1 Cloned from brain (27), erythroleukemia cells (42), prostate and ovary (43), placenta (44) Bovine P2Y 1 Cloned from brain (30), endothelium (29) Rat P2Y 1 Cloned from insulinoma cells (45) Mouse P2Y 1 Cloned from insulinoma cells (45) Chicken P2y 1 Cloned from brain (9) Turkey P2y 1 Cloned from brain (27) Human P2Y 2 Cloned from airway epithelial CF/T43 cells (46) Canine P2Y 2 Cloned from MDCK-D1 renal epithelial cells (47) Rat P2Y 2 Cloned from microvascular coronary endothelial cells (48), alveolar type II cells (49), a pituitary (50), aorta (51) Mouse P2Y 2 Cloned from NG108-15 neuroblastoma cells (10), mammary tumor cells (52) b Chicken P2y 3 Cloned from brain (53) Turkey P2y 3 Cloned from genomic DNA (54) Human P2Y 4 Cloned from placenta (55), chromosome X (56), c placenta genomic library (57) Rat P2Y 4 Cloned from heart (39), brain (40) Mouse P2Y 4 Cloned from gallbladder epithelial cells (41) Human P2Y 6 Cloned from placenta (58), activated T-cells (59) Rat P2Y 6 Cloned from aortic smooth muscle cells (60)
1
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
Receptor
No.
328
328
361
361
365
328
328
373
374
373
377
362
362
373
373
373
373
Amino acid number
100
h P2Y 1
100
96
b P2Y 1
100
94
95
r P2Y 1
100
97
93
94
m P2Y 1
TABLE I
100
84
85
85
85
ch P2y 1
100
99
85
85
85
85
t P2y 1
100
34
34
30
31
31
31
h P2Y 2
100
87
33
33
30
30
31
31
ca P2Y 2
100
83
87
35
35
31
32
33
33
r P2Y 2
100
97
83
89
35
35
31
31
32
33
m P2Y 2
100
38
38
39
39
36
36
36
36
35
36
ch P2y 3
100
97
38
38
39
39
36
36
36
36
35
36
t P2y 3
100
38
38
50
48
49
51
34
34
33
33
33
34
h P2Y 4
100
83
38
38
48
48
49
49
34
34
32
33
33
34
r P2Y 4
Percentage
100
94
81
39
39
47
49
47
48
34
34
32
33
33
34
m P2Y 4
Amino Acid Similarity of P2Y Receptor Sequences
100
39
40
38
60
60
36
37
38
37
34
34
34
34
34
33
h P2Y 6
100
87
40
41
39
60
60
39
39
40
40
34
34
32
33
33
32
r P2Y 6
96
86
39
39
39
59
59
39
39
39
39
34
34
32
32
32
32
m P2Y 6
37
38
50
48
49
36
35
43
44
44
44
38
38
35
36
36
36
x P2y 8
23
23
23
24
23
21
21
25
24
24
25
27
27
24
25
25
26
h P2Y 11
21
22
23
24
25
21
20
20
20
17
20
25
25
24
23
23
25
ca P2Y 11
19
20
23
22
21
29
20
20
21
18
20
20
20
20
20
19
19
h P2Y 12
19
19
23
23
22
18
19
21
22
20
20
20
20
20
20
20
20
r P2Y 12
38
39
54
55
54
40
40
47
45
44
45
39
39
34
34
35
34
t P2y
35
35
33
33
33
36
36
34
34
34
35
65
65
62
62
62
61
s P2y
132 SAK AND WEBB
357
374
343
342
370
371
25
24
23
22
21
20
Note. Similarities more than 80% are indicated in bold and similarities between 52 and 80% in italic. a Cys 132 is substituted by Ser, Ser 143 by Arg, Ala 166 by Thr, and Ala 188 by Asp in the rat P2Y 2 sequence cloned from rat alveolar type II cells (49), comparing with other rat P2Y 2 receptors. b Glu 17 is substituted by Asp, Ser 120 by Arg, Thr 125 by Asn, Val 196 by Met, Asp 355 by Asn, Lys 369 by Pro, Asp 370 by Tyr, and Ile 371 by Val in the mouse P2Y 2 sequence cloned from mammary tumor cells (52), comparing with other mouse P2Y 2 receptors. c Leu 86 is substituted by Val, Asn 178 by Thr, and Ser 234 by Ala in the human P2Y 4 sequence cloned from human chromosome X (56), comparing with other human P2Y 4 receptor sequences.
38
22
16
100
22 24
100
100
23 24 85 100
100
21
23 20 17
68 100
25 25 537 19
18
Mouse P2Y 6 Cloned from gallbladder epithelial cells (41) Xenopus P2y 8 Cloned from neurula cDNA library (61) Human P2Y 11 Cloned from placenta (17) Canine P2Y 11 Cloned from MDCK-D1 renal epithelial cells (21) Human P2Y 12 Cloned from platelets (18) Rat P2Y 12 Cloned from platelets (18) Turkey P2y Cloned from blood cDNA library (62) Skate P2y Cloned from hepatocytes (63)
328
100
21
18
18
38
55
26 15
20 23 100
35 23 37
39
P2Y RECEPTOR SUBTYPES AND THEIR PHARMACOLOGY
133
FIG. 1. A phylogenetic analysis of cloned P2Y receptors using the Clustal algorithm is shown. The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.
P2Y 12 receptor is coupled to the inhibition of adenylyl cyclase (18, 19). P2Y receptors can be characterized as purinoceptors, pyrimidinoceptors, and receptors with mixed selectivity. These receptors differentiate also between nucleoside di- and triphosphates. However, analysis of the activity data of several natural and synthetic compounds at P2Y receptors does not mirror the grouping that can be made from amino acid sequences analysis. Most of the published activity data have been collected into the “P2Y Receptor Ligand Database” [http://bioorg.chem.ut.ee/p2y/; Institute of Chemical Physics, Tartu University; (20)]. The activity constants of the most frequently used ligands, measured in mammalian cells transfected with the recombinant receptors, are presented in Table II. The divergence of amino acid sequence due to species differences within a receptor subtype, commonly up to 20% (Table I, Fig. 1), may influence the affinity of ligands and thus change the ligand activity order in the case of species homologues (21). Therefore, the mean activity constants of nucleotides and their analogues may differ within one subtype. Indeed, data have been published which suggest a contradictory activity type (agonist versus antagonist) for some ligands at species homologues within a P2Y receptor subtype (Table II). As mentioned above, this has been a consequence of species differences in some cases but is complicated further by the following observations. It has been demonstrated that the activity of ligands measured in some experimental assays depends also on the number of functionally active receptors, a factor especially important in the recombinant receptor systems (22). Moreover, action of ectoenzymes, catalyzing the conversion of nucleotides at the
134
SAK AND WEBB TABLE II
Mean Activity Constants of Nucleotides and Their Analogues at P2Y Receptors No.
Receptor
ATP
2MeSATP
2ClATP
ATP␥S
ADP
2MeSADP
ADPS
UTP
UDP
1
Human P2Y 1
5.64 ⴞ 0.06 1 6.11 ⫾ 0.07 1 7.80 ⫾ 0.10 1 — — — 5.97 ⫾ 0.45 2 5.64 ⫾ 0.06 1 — —
6.88 ⫾ 0.15 9
8.29 ⫾ 0.13 13
6.34 ⫾ 0.28 4
N.A. a
—
Bovine P2Y 1 Rat P2Y 1 Mouse P2Y 1 Chicken P2y 1 Turkey P2y 1 Human P2Y 2 Canine P2Y 2 Rat P2Y 2
5.24 ⴞ 0.03 1 7.53 ⫾ 0.16 9 7.40 ⫾ 0.00 2 7.52 ⫾ 0.00 2 — 8.00 ⫾ 0.00 2 7.52 ⫾ 0.02 3 N.A. — N.A.
—
2 3 4 5 6 7 8 9
5.07 ⴞ 0.23 2 5.84 ⫾ 0.18 5 5.77 ⫾ 0.54 3 6.85 ⫾ 0.08 1 — 6.10 ⫾ 0.60 4 5.68 ⫾ 0.20 3 6.75 ⫾ 0.12 6 — 6.50 ⫾ 0.20 2
6.50 ⫾ 0.10 1 — — — 5.96 ⫾ 0.41 2 6.08 ⫾ 0.16 3 — —
8.30 ⫾ 0.10 1 9.24 ⫾ 0.04 1 — — 7.77 ⫾ 0.18 2 — — —
— — — ⬃6.82 1 6.74 ⫾ 0.11 2 — — —
N.A. — — N.A. N.A. 6.91 ⫾ 0.29 6 — 6.10 ⫾ 0.19 5
— — — — — N.A. — 4.80 1
10
Mouse P2Y 2
5.99 ⫾ 0.06 5
N.A.
—
5.04 ⫾ 0.06 2
—
—
6.20 ⫾ 0.12 5
N.A. (64) or partial agonist (65)
11 12 13
Chicken P2y 3 Turkey P2y 3 Human P2Y 4
— 5.04 ⫾ 0.11 1 6.15 1
5.09 ⫾ 0.20 1 — N.A.
— — —
4.89 ⫾ 0.23 1 — N.A.
— 6.55 ⫾ 0.15 1 —
— 5.75 ⫾ 0.09 1 —
5.77 ⫾ 0.18 1 6.05 ⫾ 0.16 2 6.12 ⫾ 0.11 9
6.92 ⫾ 0.18 1 7.79 ⫾ 0.17 2 N.A.
14
Rat P2Y 4
6.00 ⫾ 0.16 3
N.A. (40) or partial agonist (39) 5.68 ⫾ 0.06 1
—
Partial agonist 5.68 ⫾ 0.10 1
7.53 ⫾ 0.03 2 8.07 ⫾ 0.07 1 — 6.59 ⫾ 0.07 1 6.53 ⫾ 0.21 3 N.A. — Partial agonist 4.83 ⫾ 0.23 2 N.A. (10, 64) or partial agonist (65) 4.99 ⫾ 0.78 2 6.33 ⫾ 0.17 1 N.A. (55, 66) or partial agonist (38) N.A. (40) or partial agonist (38, 39) 5.95 ⫾ 0.23 2
—
—
6.17 ⫾ 0.32 3
15 16 17
Mouse P2Y 4 Human P2Y 6 Rat P2Y 6
6.36 ⫾ 0.13 1 N.A. N.A.
— 4.00 1 N.A.
— — —
— — —
N.A. 4.36 ⫾ 0.17 2 5.20 ⫾ 0.11 1
— — 5.75 ⫾ 0.30 1
— — 4.59 ⫾ 0.47 1
18
Mouse P2Y 6
N.A.
—
—
—
—
—
—
19 20
Xenopus P2y 8 Human P2Y 11
7.05 ⫾ 0.06 2 4.38 ⫾ 0.14 4
— 3.97 ⫾ 0.18 3
— —
— 5.17 ⫾ 0.30 2
— N.A.
— N.A.
21 22 23 24 25
Canine P2Y 11 Human P2Y 12 Rat P2Y 12 Turkey P2y Skate P2y
⬃4.22 1 — — 6.89 ⫾ 0.07 1 —
⬃6.00 1 — — N.A. —
— — — — —
— — — 4.95 ⫾ 0.33 1 —
⬃5.00 1 6.52 1 6.52 1 4.14 ⫾ 0.15 1 —
⬃6.00 1 9.05 1 9.05 1 N.A. —
— Partial agonist 4.15 ⫾ 0.39 2 5.65 ⫾ 0.35 2 — — N.A. —
6.59 ⫾ 0.14 1 5.22 1 N.A. (67) or full agonist (54, 68) 6.95 ⫾ 0.35 5 N.A. or weak agonist (41) — N.A. N.A. — — 6.99 ⫾ 0.08 1 —
N.A. (38, 40) or partial agonist (39) 5.38 ⫾ 0.01 1 N.A. 6.52 ⫾ 0.00 2 7.72 ⫾ 0.28 5
7.38 ⫾ 0.20 1 — N.A. N.A. N.A. N.A. 3.99 ⫾ 0.12 1 —
Note. The constants listed [agonistic pEC 50 ⫾ SE and antagonistic pK i ⫾ SE (in bold)] were calculated from P2Y Receptor Ligand Database as mean values of published data for transfected systems. Number of separate determination used for calculation is given as superscript. a N.A., no or very small agonistic activity up to 100 M ligand concentration; pEC 50 ⬍ 4.00.
cell surface (23), results in a measured response due to the applied compound, its conversion/breakdown products, or a mixture of both rather than solely the initial ligand. In this context the importance of the application of HPLC-purified ligands and the use of regenerating systems in the P2Y receptor assays are self-evident.
Thus, ATP can be either an antagonist (24 –26) or an agonist (27, 28) at the human P2Y 1 receptor and an agonist for the P2Y 1 receptors derived from the other species [bovine (29 –31), rat (32), chick (9, 33–35), and turkey (27, 36, 37)]. ATP is an antagonist at the human P2Y 4 receptor (38) but is an agonist at both rat and mouse P2Y 4 receptors
FIG. 2. Comparison of ligand activities at P2Y receptor-transfected cells measured by different assay procedures. Numbers within the graphs correspond to the numeration of P2Y receptors listed in Tables I and II (■—ATP, Œ—2MeSATP, —ATP␥S, F—ADP, }—2MeSADP, *—ADPS, 䊐—UTP, E—UDP).
P2Y RECEPTOR SUBTYPES AND THEIR PHARMACOLOGY
(38 – 41). Despite such complications, in general the scatter of the data presented in Table II is rather small, indicating that the errors caused by differences in assay procedures are generally not determinant, especially in the case of nonhydrolyzable compounds. Indeed, the comparison of activity data measured by inositol phosphate accumulation, intracellular calcium mobilization, electrophysiological recordings, and cyclic AMP formation revealed that there is no systematic influence of the assay procedures on the results (Figs. 2A–2C). However, there are still some ligands with an unclear activity type (Table II), in which cases some scattering can be seen in the comparison of results measured by different assay procedures, for example, the activity of UTP at rat P2Y 6 receptor in Fig. 2B. This emphasizes the need for special attention to be paid to ligand purity. CONCLUDING REMARKS Further studies with novel highly selective and nondegradable agonists and antagonists coupled with investigations into signal transduction pathways of this heterogeneous family of receptors are required to extend the existing classification system and most importantly correlate the recombinant receptors with their endogenous counterparts. ACKNOWLEDGMENTS K.S. is grateful to Professor Jaak Ja¨rv (Institute of Chemical Physics, Tartu University, Estonia) for helpful comments about the initial manuscript.
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