Human 5-HT3A -- Tyr 229

Species Original Mutated to Mutation
Human Tyr 229  

 

Rat Equivalent Tyr 234    
Mouse Equivalent Tyr 234

Phe

Ala

Ser

Y234F

Y234A

Y234S

mTyr234Ala

In this study amino acids from E225 to Y234 of a murine 5-HT3ASR were individually mutated to alanine. Conservative mutations were made if the initial alanine mutation showed no binding and/or function. The mutated receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies.

The Y234A mutation abolished binding to [3H]granisetron. The EC50 could not be determined as the receptor was not functional.

Suryanarayanan A, Joshi PR, Bikádi Z, Mani M, Kulkarni TR, Gaines C, Schulte MK. (2005) The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide. Biochemistry 44(25):9140-9.

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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.

The Y234F mutation did not significantly altered the affinity of the antagonist [3H]granisetron. No specific binding of [3H]granisetron was observed with Y234A and Y234S mutants.

 

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT 0.32 +/- 0.035 ++ 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 NO - NR 13.7 +/-1.25*
Y91S NO + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NO ++ 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 NO ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NO ++ NR NT
Y234S NO + NR NT
Y240A/S YES NT NT NT

NT, Not tested

++, 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

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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) thought to be within 5A of the 5-HT binding site were substituted with either alanine or a residue with somewhat similar physicochemical properties to the wildtype residue. The effects of these substitutions were investigated by measuring the binding of [3H]granisetron. The results were used to map the granisetron binding site within an homology model of the mouse 5-HT3A receptor based on the crystal structure of the AChBP. Mouse 5-HT3A receptors were expressed in HEK293 cells for radioligand binding.

In addition to [3H]granisetron binding, immunofluorescence was used to examine whether non-binding mutant receptors reached the cell surface. This approach demonstrated that W90A, E129A, E129D, W183A, W183Y and Y234A mutant receptors were expressed at the cell membrane even though there was no [3H]granisetron binding. The table below lists those mutations that significantly affected [3H]granisetron binding.

The Y234A mutant 5-HT3A receptor does not bind to [3H]granisteron. The wild type receptor bound [3H]granisteron with a Kd = 0.31 +/- 0.04 nM. The Y234F (Kd = 1.30 +/- 0.36 nM) mutant bound [3H]granisetron with a reduced affinity. Phenylalanine, like tyrosine contains an aromatic ring which is presumably required for the correct structure of the antagonist binding site.

 

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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mTyr234Phe

In this study amino acids from E225 to Y234 of a murine 5-HT3ASR were individually mutated to alanine. Conservative mutations were made if the initial alanine mutation showed no binding and/or function. The mutated receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies.

The Y234F mutation caused an 11-fold reduction in the Kd value for [3H]granisetron, and no significant changes to Bmax. There was a 185-fold reduction in the Ki value for 5-HT, with a 2.7-fold reduction in the EC50 value and no significant change in Imax. There was a 125-fold reduction in the Ki value for mCPBG, with a 7.3-fold reduction in the EC50 value and no significant change in Imax.

Suryanarayanan A, Joshi PR, Bikádi Z, Mani M, Kulkarni TR, Gaines C, Schulte MK. (2005) The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide. Biochemistry 44(25):9140-9.

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back to 5-HT3A

 

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.

The Y234F mutation did not significantly altered the affinity of the antagonist [3H]granisetron. No specific binding of [3H]granisetron was observed with Y234A and Y234S mutants.

 

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT 0.32 +/- 0.035 ++ 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 NO - NR 13.7 +/-1.25*
Y91S NO + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NO ++ 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 NO ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NO ++ NR NT
Y234S NO + NR NT
Y240A/S YES NT NT NT

NT, Not tested

++, 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

back to top
back to 5-HT3A

 

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) thought to be within 5A of the 5-HT binding site were substituted with either alanine or a residue with somewhat similar physicochemical properties to the wildtype residue. The effects of these substitutions were investigated by measuring the binding of [3H]granisetron. The results were used to map the granisetron binding site within an homology model of the mouse 5-HT3A receptor based on the crystal structure of the AChBP. Mouse 5-HT3A receptors were expressed in HEK293 cells for radioligand binding.

In addition to [3H]granisetron binding, immunofluorescence was used to examine whether non-binding mutant receptors reached the cell surface. This approach demonstrated that W90A, E129A, E129D, W183A, W183Y and Y234A mutant receptors were expressed at the cell membrane even though there was no [3H]granisetron binding. The table below lists those mutations that significantly affected [3H]granisetron binding.

The Y234A mutant 5-HT3A receptor does not bind to [3H]granisteron. The wild type receptor bound [3H]granisteron with a Kd = 0.31 +/- 0.04 nM. The Y234F (Kd = 1.30 +/- 0.36 nM) mutant bound [3H]granisetron with a reduced affinity. Phenylalanine, like tyrosine contains an aromatic ring which is presumably required for the correct structure of the antagonist binding site.

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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mTyr234Ser

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.

The Y234F mutation did not significantly altered the affinity of the antagonist [3H]granisetron. No specific binding of [3H]granisetron was observed with Y234A and Y234S mutants.

 

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT 0.32 +/- 0.035 ++ 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 NO - NR 13.7 +/-1.25*
Y91S NO + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NO ++ 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 NO ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NO ++ NR NT
Y234S NO + NR NT
Y240A/S YES NT NT NT

NT, Not tested

++, 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

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