Human 5-HT3A -- Tyr 68

Species Original Mutated to Mutation
Human Tyr 68  

 

Rat Equivalent Tyr 73    
Mouse Equivalent Tyr 73 Ala

Ser

Y 73A

Y 73S

 

mTyr73Ala

Eleven residues (Tyr 50, Tyr 73, Tyr 88, Tyr 91, Tyr 94, Tyr 141, Tyr 143, Tyr 153, Tyr 167, Tyr 234, Tyr 240) were mutated individually for alanine, serine and some residues for phenylalanine. The ligands [3H]granisetron and 5-HT were used to examine the properities of ligand binding sites and function of the 5-HT3A receptor, respectively.

Mouse 5-HT3A receptors were expressed in Xenopus Oocytes (cRNA injection) and studied using two-electrode voltage-clamp technique. HEK293 cells were used in radioligand binding and calcium imaging assays to study the affinity of [3H]granisetron and the potency of 5-HT, respectively.

TheY73A and Y73S mutations did not significantly altered the affinity of the antagonist [3H]granisetron.

 

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT YES ++ 1.47 +/-0.42 2.39 +/- 0.23
Y50F NB - NR NR
Y50A NB -/+ NR NR
Y50S NB -/+ NR 1.59 +/- 0.18
Y73A/S YES NT NT NT
Y88A/S YES NT NT NT
Y91F YES NT 2.13 +/- 0.51 4.22 +/-0.25
Y91A NB - NR 13.7 +/-1.25*
Y91S NB + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NB ++ NR NT
Y143F YES NT NT NT
Y143A YES NT NR NT
Y143S YES NT >500* NT
Y153A YES NT 74.3 +/- 8.9* NT
Y153S NB ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NB ++ NR NT
Y234S NB + NR NT
Y240A/S YES NT NT NT

NT, Not tested; NB, No binding.

++, Presence of cell surface receptors revealed by immunocytochemistry

-/+, Reduced cell surface receptor expression compared to WT 5-HT3A

-, Absence of cell surface receptors

* significantly different from the WT 5-HT3A receptor

 

Price KL, Lummis SCR (2004). The role of Tyrosine residues in the extracellular domain of the 5-Hydroxytryptamine3 receptor.The Journal of Biological Chemistry 279(22):23294-23301

Twenty six residues (I71, Y73,W90, R92, N128, E129, Y143, Y153, T179, T181, S182,W183, L184, W195, V201, R202, S203, S206, I207, F226, I228, D229, I230, Y234, E236, K238) were replaced by either alanine or a residue with similar physicochemical properties to the native residue, to examine their roles in docking of [3H]granisetron into the 5-HT3A receptor binding site. Mouse 5-HT3A receptors were expressed in HEK293 cells and homology modeling, ligand-docking and radioligand binding were used.

The Y73A and Y73S mutations did not significantly altered the affinity of the antagonist [3H]granisetron. The table below lists conservative and non-conservative substitutions that either both, or individually affected [3H]granisetron binding.

Receptor [3H]granisetron binding (Kd)
WT 0.31+/-0.04
W90A*

NB

W90Y* 0.90 +/- 0.06
R92A* 1.80 +/- 0.40
R92K 1.00 +/- 0.30
E129A* NB
E129D* NB
Y153A* 2.36 +/- 0.53
Y153F 0.90 +/- 0.20
T179A* 3.20 +/- 0.10
T179S 0.38 +/- 0.20
S181A* 0.12 +/- 0.04
S181S 0.58 +/- 0.10
S182A* 1.00 +/- 0.20
S182T* 1.80 +/- 0.09
W183A*/Y* NB
L184A* 4.11 +/- 0.94
L184I* 0.71 +/- 0.05
W195A* 5.08 +/- 0.88
W195Y* 8.70 +/- 2.40
S203A* 0.08 +/-0.02
S203T 0.26 +/- 0.11
S206A* 1.67 +/- 0.27
S206T* 4.40 +/- 0.49
I228A* 1.40 +/- 0.30
I228N 0.30 +/- 0.05
D229A* 3.80 +/- 0.26
D229E* 0.11 +/- 0.03
I230A 0.30 +/- 0.10
I230N* 1.70 +/- 0.40
Y234A* NB
Y234F 1.30 +/- 0.36

NB, No binding

* significantly different from the WT 5-HT3A receptor

Thompson AJ, Price KL, Reeves DC, Chan SL, Chau PL, Lummis SC(2005). Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding.The Journal of Biological Chemistry 280(21):20476-82

                                                               

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mTyr73Ser

Eleven residues (Tyr 50, Tyr 73, Tyr 88, Tyr 91, Tyr 94, Tyr 141, Tyr 143, Tyr 153, Tyr 167, Tyr 234, Tyr 240) were mutated individually for alanine, serine and some residues for phenylalanine. The ligands [3H]granisetron and 5-HT were used to examine the properities of ligand binding sites and function of the 5-HT3A receptor, respectively.

Mouse 5-HT3A receptors were expressed in Xenopus Oocytes (cRNA injection) and studied using two-electrode voltage-clamp technique. HEK293 cells were used in radioligand binding and calcium imaging assays to study the affinity of [3H]granisetron and the potency of 5-HT, respectively.

TheY73A and Y73S mutations did not significantly altered the affinity of the antagonist [3H]granisetron.

 

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT YES ++ 1.47 +/-0.42 2.39 +/- 0.23
Y50F NB - NR NR
Y50A NB -/+ NR NR
Y50S NB -/+ NR 1.59 +/- 0.18
Y73A/S YES NT NT NT
Y88A/S YES NT NT NT
Y91F YES NT 2.13 +/- 0.51 4.22 +/-0.25
Y91A NB - NR 13.7 +/-1.25*
Y91S NB + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NB ++ NR NT
Y143F YES NT NT NT
Y143A YES NT NR NT
Y143S YES NT >500* NT
Y153A YES NT 74.3 +/- 8.9* NT
Y153S NB ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NB ++ NR NT
Y234S NB + NR NT
Y240A/S YES NT NT NT

NT, Not tested; NB, Not binding

++, Presence of cell surface receptors revealed by immunocytochemistry

-/+, Reduced cell surface receptor expression compared to WT 5-HT3A

-, Absence of cell surface receptors

* significantly different from the WT 5-HT3A receptor

Price KL, Lummis SCR (2004). The role of Tyrosine residues in the extracellular domain of the 5-Hydroxytryptamine3 receptor.The Journal of Biological Chemistry 279(22):23294-23301

Twenty six residues (I71, Y73,W90, R92, N128, E129, Y143, Y153, T179, T181, S182,W183, L184, W195, V201, R202, S203, S206, I207, F226, I228, D229, I230, Y234, E236, K238) were replaced by either alanine or a residue with similar physicochemical properties to the native residue, to examine their roles in docking of [3H]granisetron into the 5-HT3A receptor binding site. Mouse 5-HT3A receptors were expressed in HEK293 cells and homology modeling, ligand-docking and radioligand binding were used.

The Y73A and Y73S mutations did not significantly altered the affinity of the antagonist [3H]granisetron. The table below lists conservative and non-conservative substitutions that either both, or individually affected [3H]granisetron binding.

Receptor [3H]granisetron binding (Kd)
WT 0.31+/-0.04
W90A*

NB

W90Y* 0.90 +/- 0.06
R92A* 1.80 +/- 0.40
R92K 1.00 +/- 0.30
E129A* NB
E129D* NB
Y153A* 2.36 +/- 0.53
Y153F 0.90 +/- 0.20
T179A* 3.20 +/- 0.10
T179S 0.38 +/- 0.20
S181A* 0.12 +/- 0.04
S181S 0.58 +/- 0.10
S182A* 1.00 +/- 0.20
S182T* 1.80 +/- 0.09
W183A*/Y* NB
L184A* 4.11 +/- 0.94
L184I* 0.71 +/- 0.05
W195A* 5.08 +/- 0.88
W195Y* 8.70 +/- 2.40
S203A* 0.08 +/-0.02
S203T 0.26 +/- 0.11
S206A* 1.67 +/- 0.27
S206T* 4.40 +/- 0.49
I228A* 1.40 +/- 0.30
I228N 0.30 +/- 0.05
D229A* 3.80 +/- 0.26
D229E* 0.11 +/- 0.03
I230A 0.30 +/- 0.10
I230N* 1.70 +/- 0.40
Y234A* NB
Y234F 1.30 +/- 0.36

NB, No binding

* significantly different from the WT 5-HT3A receptor

Thompson AJ, Price KL, Reeves DC, Chan SL, Chau PL, Lummis SC(2005). Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding.The Journal of Biological Chemistry 280(21):20476-82

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