TABLE 4

Analysis of the pharmacological effects and structural role of individual residues of the human P2Y₁ receptor, deduced from site-directed mutagenesis, molecular modeling, and homology to other GPCRs Where applicable, the data indicate the effects of single amino acid replacement on the activation of PLC by mutant human P2Y₁ receptors transiently expressed in COS-7 cells and results of molecular modeling of the docked complex of 2-MeSADP or ATP within the wild-type hP2Y₁ receptor (see Moro and Jacobson, 2002; Costanzi et al., 2004; and references therein).

Site	Mutation (if Performed)	Position^a	Putative Interactions, Pharmacological Role	Potency Loss (Ratio for 2-MeSADP)^a
TM1
Y58		1.39	H-bond donor to S314^b^,^c^,^d	N.A.
N69		1.50	H-bond donor to S317^c^,^d	N.A.
TM2
S87		2.40	H-bond acceptor from Y324^c^,^d	N.A.
N92		2.45	H-bond acceptor from W176^c^,^d	N.A.
D97		2.50	NA⁺ binding^c	N.A.
Y110		2.63	Agonist effect^e	N.A.
TM3
R128	A	3.29	Counterion to α-phosphate^d^,^f	>50,000
F131	A	3.32	Modulatory toward agonist, but not antagonist^f	8
H132	A	3.33	Modulatory^g	7
Y136	A	3.37	Modulatory^g	6
S138		3.39	H-bond acceptor from N316^c^,^d	N.A.
H148		3.49	H-bond acceptor from R255^c^,^d	N.A.
TM4
S172		4.46	H-bond acceptor from N92^c^,^d	N.A.
W176		4.50	H-bond donor to N92^c^,^d	N.A.
TM5
T221	A	5.42	Small effect^f	7
T222	A	5.43	Proximity to γ-phosphate^f	5
F226	A	5.47	Binding of antagonists^f	9
TM6
R255		6.30	H-bond donor to H148^c^,^d^h	N.A.
Y273	A	6.48	Receptor activation^d^,ⁱ	>3,000
	F	6.48	Receptor activation rescued^d	2
H277	A	6.52	Effect of agonist but not antagonist^f	45
K280	A	6.55	Counterion to β-phosphate of ADP; action of PPADS^d^,^e^,^j	810
TM7
Y306	A	7.35	Modulatory toward agonist effects only^d	5
	F	7.35	Modulatory^d^,^g	6
Q307	A	7.36	H-bond acceptor from exocyclic NH of ADP^d^,^f	210
R310	A	7.39	Counterion to α-phosphate^d^,^f	>50,000
	K	7.39	Rescue of agonist (partial) and antagonist (full) effects^f	190
S314	A	7.43	Activation, H-bond donor to adenine N1 or N3;^d^,^f H-bond acceptor from Y58^b	>50,000
	T	7.43	Rescue of agonist effect^f	5
N316		7.45	H-bond donor to S138^c^,^d	N.A.
S317	A	7.46	H-bond acceptor from N69; no pharmacological effect^d^,^f	0.7
D320		7.49	Receptor activation^c^,^k	N.A.
Y324		7.53	H-bond donor to S87^c^,^d	N.A.
Extracellular and cytoplasmic regions
C42	A	N-terminal	Disulfide with C296^f	22,000
T47		N-terminal	Meta-binding site^c^,^f	N.A.
C124	A	EL1	Disulfide with C202	>50,000
K125	A	EL1	No effect^f	3
L157		IL2	G protein interaction^c^,^l	N.A.
R195	A	EL2	No effect^f	2
K196	A	EL2	No effect^f	1
K198	A	EL2	Modulatory^f	8
Y203	A	EL2	Agonist effects^d^,^m	>3,000
	F	EL2	Rescue of agonist effects^d	5
D204	A	EL2	Meta-binding site^f	30
	N	EL2	Unable to rescue function^f	270
	E	EL2	Unable to rescue function^f	66
D208	A	EL2	No effect^f	2
E209	A	EL2	Meta-binding site; H-bonding to ribose^f	7800
	D	EL2	H-bonding rescued^f	1
	Q	EL2	H-bonding rescued^f	4
	R	EL2	H-bonding rescued^f	2
R212	A	EL2	Small effect^f	4
R285	A	EL3	Small effect^f	4
R287	A	EL3	Meta-binding site; H-bonding to E209^f	6700
	K	EL3	Charge rescue (partial) of agonist effects^f	34
	Q	EL3	Partial rescue^f	240
	E	EL3	Disrupts E209 interaction^f	>50,000
D289	A	EL3	No effect^f	2
C296	A	EL3	Disulfide with C42^f	2300
D300	A	EL3	Agonist effects^f	45
R301	A	EL3	No effect^f	1
D329		C-terminal	Beginning of H8^c^,^d^,ⁿ	N.A.
R333		C-terminal	Required for Gq coupling^o	N.A.
R334		C-terminal	Required for Gq coupling^o	N.A.
K341		C-terminal	End of H8^c^,^d^,ⁿ	N.A.

N.A. not applicable; EL extracellular loop; IL intracellular loop
↵ a Residue identifier of format X.YZ refers to the TM X and residue YZ with respect to a highly conserved amino acid in each TM numbered 50 (Ballesteros et al., 2001; Costanzi et al., 2004); potency ratio shown is EC₅₀ at mutant receptor divided by EC₅₀ at the wild-type; the EC₅₀ of 2-MeSADP at the wild-type human P2Y₁ receptors was reported as 2.2 ± 0.5^f or 3 ± 1^d nM
↵ b Applies only to ground state of receptor
↵ c Not mutated, prediction from modeling only
↵ d Costanzi et al. (2004)
↵ e Hoffmann et al. (2004)
↵ f Moro and Jacobson (2002) and references therein
↵ g Modulatory toward effects of agonist and nucleotide antagonist
↵ h Analogous to E(6.30) of the β₂ adrenergic receptor involved in an `ionic lock' with R(3.50) but no electrostatic interaction with V(3.50) of the P2Y₁ receptor is possible; for the P2Y₁ receptor, a similarly placed interaction is predicted between R(6.30) and H(3.49)
↵ i Mutation precludes only activation by agonist, but not binding of agonist or antagonist
↵ j Guo et al. (2002)
↵ k Analogous to the N of the NPxxxY motif involved in activation of thyroid-stimulating hormone (Claeysen et al., 2002) and other receptors
↵ l Moro et al. (1993)
↵ m Mutation precludes only activation and binding of agonist, but not binding of antagonist
↵ n An eighth helical region (cytoplasmic), by analogy to rhodopsin; a putative palmitoylation site (Cys) in this region is predicted for P2Y₄, P2Y₁₂, and P2Y₁₄, but not P2Y₁ receptors
↵ o Ding et al. (2005)