User:Anat Levit/Sandbox 1: Difference between revisions

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The prokineticin receptors have been found to be involved in various pathologies involving the cardiovascular, reproductive, endocrine and nervous systems. Notably, PROKR2 has been found to be <scene name='User:Anat_Levit/Sandbox_1/Pkr1_ks_mutations/1'>mutated in Kallmann syndrome</scene> with dilated cardiomyopathy, a hypogonadism caused by a deficiency of gonadotropin-releasing hormone (GnRH) (see Wikipedia:

[http://en.wikipedia.org/wiki/Kallmann_syndrome Kallmann syndrome]). Except for V331M and R357W which are Leu and Asn in PROKR1, respectively, all other residues mutated in PROKR2 are identical in PROKR1. Interestingly, two of the mutated residues, W178 (4.50) and P290 (6.50), are two of the most conserved residues in family A GPCRs (<scene name='User:Anat_Levit/Sandbox_1/~~Pkr_colored_by_homology~~/4'>restore initial scene</scene>).

[http://en.wikipedia.org/wiki/Kallmann_syndrome Kallmann syndrome]). Except for V331M and R357W ('''colored red''') which are Leu and Asn in PROKR1, respectively, all other residues mutated in PROKR2 are identical in PROKR1 ('''colored blue'''). Interestingly, two of the mutated residues, W178 (4.50) and P290 (6.50), are two of the most conserved residues in family A GPCRs (<scene name='User:Anat_Levit/Sandbox_1/Pkr1_consurf/1'>restore initial scene</scene>).

[[Image:ColorKey_ConSurf_NoYellow_NoGray.gif|right|200 px]]

Being highly homologues proteins, expressed in the same cell types and having similar nano-molar affinity to common ligands, it is of importance to understand the structural and functional differences between these receptors.

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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 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.

The phosphovariants can now be identified when the phosphorylation sites or interacting kinases were altered between subtype sequences.

The identified sites were classified according to one of the following types, defined by Ryu et al. 2009:

# In <scene name='User:Anat_Levit/Sandbox_1/Type_i/2'>type I</scene> the phosphorylation site is in the same location as the variation, and can either create a new phosphorylation site (Type I+) or eliminate an existing site (Type I-) ('''colored green''').

# In <scene name='User:Anat_Levit/Sandbox_1/Type_ii/2'>type II</scene> the variation is not in the same location as the phosphorylation site, and can either create a new phosphorylation site (Type II+) or eliminate an existing site (Type II-) ('''colored blue''').

# <scene name='User:Anat_Levit/Sandbox_1/Type_iii/1'>Type III</scene> are variations that change only the type of kinase involved, without affecting the phosphorylation site itself ('''colored red''').

<scene name='User:Anat_Levit/Sandbox_1/Type_i/3'>Identical</scene> phosphor-acceptor residues predicted to be phosphorylated by the same kinases are '''colored yellow''' (<scene name='User:Anat_Levit/Sandbox_1/All_phosphosites/1'>view all predictions</scene>).

As seen from the results, homologues residues in the receptors are predicted to be phosphorylated by different kinases, and some of the predicted phospho-sites are receptor unique.

By understanding the flexible nature of PK receptors phosphorylation it may be possible to develop agonists or allosteric modulators that promote a subset of phosphorylation events on the target GPCR and thereby restrict the action of the drug to a particular receptor mediated signaling response.

== Binding site analysis ==

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 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].

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 The prokineticin receptors have been found to be involved in various pathologies involving the cardiovascular, reproductive, endocrine and nervous systems. Notably, PROKR2 has been found to be <scene name='User:Anat_Levit/Sandbox_1/Pkr1_ks_mutations/1'>mutated in Kallmann syndrome</scene> with dilated cardiomyopathy, a hypogonadism caused by a deficiency of gonadotropin-releasing hormone (GnRH) (see Wikipedia:
-[http://en.wikipedia.org/wiki/Kallmann_syndrome Kallmann syndrome]). Except for V331M and R357W which are Leu and Asn in PROKR1, respectively, all other residues mutated in PROKR2 are identical in PROKR1. Interestingly, two of the mutated residues, W178 (4.50) and P290 (6.50), are two of the most conserved residues in family A GPCRs (<scene name='User:Anat_Levit/Sandbox_1/Pkr_colored_by_homology/4'>restore initial scene</scene>).
+[http://en.wikipedia.org/wiki/Kallmann_syndrome Kallmann syndrome]). Except for V331M and R357W (<font color='red'>'''colored red'''</font>) which are Leu and Asn in PROKR1, respectively, all other residues mutated in PROKR2 are identical in PROKR1 (<font color='blue'>'''colored blue'''</font>). Interestingly, two of the mutated residues, W178 (4.50) and P290 (6.50), are two of the most conserved residues in family A GPCRs (<scene name='User:Anat_Levit/Sandbox_1/Pkr1_consurf/1'>restore initial scene</scene>).
 [[Image:ColorKey_ConSurf_NoYellow_NoGray.gif|right|200 px]]
 Being highly homologues proteins, expressed in the same cell types and having similar nano-molar affinity to common ligands, it is of importance to understand the structural and functional differences between these receptors.
@@ Line 28: / Line 29: @@
+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 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.
+The phosphovariants can now be identified when the phosphorylation sites or interacting kinases were altered between subtype sequences.
+The identified sites were classified according to one of the following types, defined by Ryu et al. 2009:
+# In <scene name='User:Anat_Levit/Sandbox_1/Type_i/2'>type I</scene> the phosphorylation site is in the same location as the variation, and can either create a new phosphorylation site (Type I+) or eliminate an existing site (Type I-) (<font color='green'>'''colored green'''</font>).
+# In <scene name='User:Anat_Levit/Sandbox_1/Type_ii/2'>type II</scene> the variation is not in the same location as the phosphorylation site, and can either create a new phosphorylation site (Type II+) or eliminate an existing site (Type II-) (<font color='blue'>'''colored blue'''</font>).
+# <scene name='User:Anat_Levit/Sandbox_1/Type_iii/1'>Type III</scene> are variations that change only the type of kinase involved, without affecting the phosphorylation site itself (<font color='red'>'''colored red'''</font>).
+<scene name='User:Anat_Levit/Sandbox_1/Type_i/3'>Identical</scene> phosphor-acceptor residues predicted to be phosphorylated by the same kinases are <font color='yellow'>'''colored yellow'''</font> (<scene name='User:Anat_Levit/Sandbox_1/All_phosphosites/1'>view all predictions</scene>).
+As seen from the results, homologues residues in the receptors are predicted to be phosphorylated by different kinases, and some of the predicted phospho-sites are receptor unique.
+By understanding the flexible nature of PK receptors phosphorylation it may be possible to develop agonists or allosteric modulators that promote a subset of phosphorylation events on the target GPCR and thereby restrict the action of the drug to a particular receptor mediated signaling response.
 == Binding site analysis ==
+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' 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 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].