User:Anat Levit/Sandbox 1: Difference between revisions

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Almost all GPCRs are regulated by [http://en.wikipedia.org/wiki/Phosphorylation phosphorylation ] and this is a key process in determining the signaling properties of these receptors. Receptors are multiply phosphorylated at sites that can occur throughout the intracellular regions of the receptor. It is well established that GPCR phosphorylation is a complex process involving a range of different protein kinases able to phosphorylate the same receptor at different sites and that this results in differential signaling outcomes, which can be tailored in a tissue specific manner to regulate biological processes.

Sequence analysis of the PKR subtypes revealed that the majority (63%) of putative phosphor-acceptor residues (Ser, Thr and Tyr) are fully conserved between the subtypes, and the rest are either removed in one of the proteins (23%), i.e., the homologues position does not contain a phosphor-acceptor residue, or is changed to another (11%), for example, a switch from Ser to Thr.

We ~~hypnotized~~ that differential signaling of the PKR subtypes may result from phosphorylation of homologus residues by different kinases due to presence of phosphovariants in positions surrounding the phosphor-acceptor residue, and not due to change/elimination of this residue between the subtypes.

We hypothized that differential signaling of the PKR subtypes may result from phosphorylation of homologus residues by different kinases due to presence of phosphovariants in positions surrounding the phosphor-acceptor residue, and not due to change/elimination of this residue between the subtypes.

To this aim, we performed prediction of the putative phosphorylation sites in human PROKR1 and PROKR2 using the GPS, PPSP, NetPhos and NetPhosK webservers. Only sites predicted to be phosphorylated by all methods were considered as putative phosphorylation sites.

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To determine the location of a potential TM binding cavity for each receptor, we structurally aligned and superimposed each of the models onto the structures of bovine Rhodopsin ([[1l9h]], [[1f88]]), human β2-Adrenergic receptor ([[2rh1]]) and human A2-Adenosine receptor ([[3eml]]). The Prokineticin receptors binding sites were determined based on homology to the known binding site-composing residues of the templates.

To determine the location of a potential TM binding cavity for each receptor, we structurally aligned and superimposed each of the models onto the structures of bovine Rhodopsin ([[1l9h]], [[1f88]]), human β2-Adrenergic receptor ([[2rh1]]) and human A2A-Adenosine receptor ([[3eml]]). The Prokineticin receptors binding sites were determined based on homology to the known binding site-composing residues of the templates.

The TM cavity of PKR1 and PKR2 is ~~quite a narrow cleft,~~ similar to the epinephrine binding site of β2-Adrenergic receptor. It is a narrow and deep cleft that is largely concealed from solvent, which may enable ligand interaction with both walls. Based on the comparison to 2RH1, we can see that the residues lining the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_2rh1_based_residues/2'>PROKR1</scene> binding site are hydrophobic, which may contribute to potential affinity and polar, which can allow for strong directional constraints through electrostatic interactions.

The TM cavity of PKR1 and PKR2 is similar to the epinephrine binding site of β2-Adrenergic receptor. It is a narrow and deep cleft that is largely concealed from solvent, which may enable ligand interaction with both walls (via van der Waals contacts). Based on the comparison to 2RH1, we can see that the residues lining the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_2rh1_based_residues/4'>PROKR1</scene> binding site are hydrophobic, which may contribute to potential affinity and polar, which can allow for strong directional constraints through electrostatic interactions.

The <scene name='User:Anat_Levit/Sandbox_1/Pkr2_2rh1_residues/5' target='PROKR2'>PROKR2</scene> predicted binding site is almost identical to the PROKR1 site, except for an addition of Ala322 in PROKR2, which is not present in PROKR1, and Glu240 in PROKR1, which is not present in PROKR2.

The ligand binding pocket of bovine Rhodopsin is similar in position to the β2-Adrenergic receptor binding site, and also involves TMs II, III, V and VI. The position does not vary considerably with alternate ligands or between different subtypes of different species. The retinal binding pocket relies mainly on hydrophobic interactions in addition to a covalent linkage with TM VII. In both, <scene name='User:Anat_Levit/Sandbox_1/Pkr1_1l9h_based_residues/3' target='PROKR1'>PROKR1</scene> and

<scene name='User:Anat_Levit/Sandbox_1/Pkr2_1l9h_residues/1' target='PROKR2'>PROKR2</scene>, Trp288 is part of the binding pocket. This position is homologues to Trp265 (6.48) of Rhodopsin which interacts with retinal (a tryptophan is in general conserved at this position in class A receptors and is thought to be involved in receptor signaling).

The ligand binding pocket of A2A-adenosine receptor assumes a very different location to that of Rhodopsin and β2-Adrenergic receptor. The interface is shifted to TMs VI and VII and there is also extensive interaction with ECL2. However, there is still minor contribution from TMs II, III and V, which is seen in our structural superposition of the

<scene name='User:Anat_Levit/Sandbox_1/Pkr1_3eml_based_residues/1' target='PROKR1'>PROKR1</scene> and <scene name='User:Anat_Levit/Sandbox_1/Pkr2_1l9h_residues/2' target='PROKR2'>PROKR2</scene> models.

Based on this analysis, we defined the core residues which line the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_consensus/2' target='PROKR1'>PROKR1</scene> and <scene name='User:Anat_Levit/Sandbox_1/Pkr2_consensus/2' target='PROKR2'>PROKR2</scene> binding pocket (the selected residues appear in at least two of the superpositions described). The pockets of the two receptors are almost identical, except for the additional Tyr140 and Glu319 residues in PROKR2. Tyr140 is known to be mutated in Kallmann syndrome. The p.Y140X nonsense mutation probably results in a PROKR2 with complete loss of function through the generation of an aberrant transcript that can be unstable or encodes for a truncated protein, lacking the carboxyl terminal domain.

===Conclusion===

The high conservation of the ligand binding pocket of the prokineticin receptors may explain the very similar affinity of the receptors to their cognate ligands.

This has also been observed in other subfamilies of GPCRs (such as dopamine, serotonin, histamine and the adrenergic receptors) and may probably explain the difficulty in obtaining potent subtype-selective compounds in pharmaceutical discovery programs.

For more information about our lab, please visit us at [http://departments.agri.huji.ac.il/biochemfoodsci722/teachers/niv_masha/index.htm HUJI].

@@ Line 31: / Line 31: @@
 Almost all GPCRs are regulated by [http://en.wikipedia.org/wiki/Phosphorylation phosphorylation ] and this is a key process in determining the signaling properties of these receptors. Receptors are multiply phosphorylated at sites that can occur throughout the intracellular regions of the receptor. It is well established that GPCR phosphorylation is a complex process involving a range of different protein kinases able to phosphorylate the same receptor at different sites and that this results in differential signaling outcomes, which can be tailored in a tissue specific manner to regulate biological processes.
 Sequence analysis of the PKR subtypes revealed that the majority (63%) of putative phosphor-acceptor residues (Ser, Thr and Tyr) are fully conserved between the subtypes, and the rest are either removed in one of the proteins (23%), i.e., the homologues position does not contain a phosphor-acceptor residue, or is changed to another (11%), for example, a switch from Ser to Thr.
-We hypnotized that differential signaling of the PKR subtypes may result from phosphorylation of homologus residues by different kinases due to presence of phosphovariants in positions surrounding the phosphor-acceptor residue, and not due to change/elimination of this residue between the subtypes.
+We hypothized that differential signaling of the PKR subtypes may result from phosphorylation of homologus residues by different kinases due to presence of phosphovariants in positions surrounding the phosphor-acceptor residue, and not due to change/elimination of this residue between the subtypes.
 To this aim, we performed prediction of the putative phosphorylation sites in human PROKR1 and PROKR2 using the GPS, PPSP, NetPhos and NetPhosK webservers. Only sites predicted to be phosphorylated by all methods were considered as putative phosphorylation sites.
@@ Line 51: / Line 51: @@
-To determine the location of a potential TM binding cavity for each receptor, we structurally aligned and superimposed each of the models onto the structures of bovine Rhodopsin ([[1l9h]], [[1f88]]), human β2-Adrenergic receptor ([[2rh1]]) and human A2-Adenosine receptor ([[3eml]]). The Prokineticin receptors binding sites were determined based on homology to the known binding site-composing residues of the templates.
+To determine the location of a potential TM binding cavity for each receptor, we structurally aligned and superimposed each of the models onto the structures of bovine Rhodopsin ([[1l9h]], [[1f88]]), human β2-Adrenergic receptor ([[2rh1]]) and human A2A-Adenosine receptor ([[3eml]]). The Prokineticin receptors binding sites were determined based on homology to the known binding site-composing residues of the templates.
-<applet load='PKR1_model1.pdb' size='300' frame='true' align='right' caption='Human PROKR1' SCENE='User:Anat_Levit/Sandbox_1/Initial_pkr1/1'/>
+<applet load='PKR1_model1.pdb' size='300' name='PROKR1' frame='true' align='right' caption='Human PROKR1' SCENE='User:Anat_Levit/Sandbox_1/Initial_pkr1/1'/>
-The TM cavity of PKR1 and PKR2 is quite a narrow cleft, similar to the epinephrine binding site of β2-Adrenergic receptor. It is a narrow and deep cleft that is largely concealed from solvent, which may enable ligand interaction with both walls. Based on the comparison to 2RH1, we can see that the residues lining the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_2rh1_based_residues/2'>PROKR1</scene> binding site are hydrophobic, which may contribute to potential affinity and polar, which can allow for strong directional constraints through electrostatic interactions.
+The TM cavity of PKR1 and PKR2 is similar to the epinephrine binding site of β2-Adrenergic receptor. It is a narrow and deep cleft that is largely concealed from solvent, which may enable ligand interaction with both walls (via van der Waals contacts). Based on the comparison to 2RH1, we can see that the residues lining the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_2rh1_based_residues/4'>PROKR1</scene> binding site are hydrophobic, which may contribute to potential affinity and polar, which can allow for strong directional constraints through electrostatic interactions.
+The <scene name='User:Anat_Levit/Sandbox_1/Pkr2_2rh1_residues/5' target='PROKR2'>PROKR2</scene> predicted binding site is almost identical to the PROKR1 site, except for an addition of Ala322 in PROKR2, which is not present in PROKR1, and Glu240 in PROKR1, which is not present in PROKR2.
+The ligand binding pocket of bovine Rhodopsin is similar in position to the β2-Adrenergic receptor binding site, and also involves TMs II, III, V and VI. The position does not vary considerably with alternate ligands or between different subtypes of different species. The retinal binding pocket relies mainly on hydrophobic interactions in addition to a covalent linkage with TM VII. In both, <scene name='User:Anat_Levit/Sandbox_1/Pkr1_1l9h_based_residues/3' target='PROKR1'>PROKR1</scene> and
+<scene name='User:Anat_Levit/Sandbox_1/Pkr2_1l9h_residues/1' target='PROKR2'>PROKR2</scene>, Trp288 is part of the binding pocket. This position is homologues to Trp265 (6.48) of Rhodopsin which interacts with retinal (a tryptophan is in general conserved at this position in class A receptors and is thought to be involved in receptor signaling).
+The ligand binding pocket of A2A-adenosine receptor assumes a very different location to that of Rhodopsin and β2-Adrenergic receptor. The interface is shifted to TMs VI and VII and there is also extensive interaction with ECL2. However, there is still minor contribution from TMs II, III and V, which is seen in our structural superposition of the
+<scene name='User:Anat_Levit/Sandbox_1/Pkr1_3eml_based_residues/1' target='PROKR1'>PROKR1</scene> and <scene name='User:Anat_Levit/Sandbox_1/Pkr2_1l9h_residues/2' target='PROKR2'>PROKR2</scene> models.
+Based on this analysis, we defined the core residues which line the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_consensus/2' target='PROKR1'>PROKR1</scene> and <scene name='User:Anat_Levit/Sandbox_1/Pkr2_consensus/2' target='PROKR2'>PROKR2</scene> binding pocket (the selected residues appear in at least two of the superpositions described). The pockets of the two receptors are almost identical, except for the additional Tyr140 and Glu319 residues in PROKR2. Tyr140 is known to be mutated in Kallmann syndrome. The p.Y140X nonsense mutation probably results in a PROKR2 with complete loss of function through the generation of an aberrant transcript that can be unstable or encodes for a truncated protein, lacking the carboxyl terminal domain.
+===Conclusion===
+The high conservation of the ligand binding pocket of the prokineticin receptors  may explain the very similar affinity of the receptors to their cognate ligands.
+This has also been observed in other subfamilies of GPCRs (such as dopamine, serotonin, histamine and the adrenergic receptors) and may probably explain the difficulty in obtaining potent subtype-selective compounds in pharmaceutical discovery programs.
+<applet load='PKR2_model1.pdb' size='300' frame='true' name='PROKR2' align='right' caption='Human PROKR2' SCENE='User:Anat_Levit/Sandbox_1/Pkr2_2rh1_residues/2'>
+For more information about our lab, please visit us at [http://departments.agri.huji.ac.il/biochemfoodsci722/teachers/niv_masha/index.htm HUJI].