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dc.contributor.advisor Ruan, Ke-He
dc.creator Chillar, Annirudha
dc.date.accessioned 2012-09-28T12:35:51Z
dc.date.available 2012-09-28T12:35:51Z
dc.date.created 2010-08
dc.date.issued 2012-09-28
dc.date.submitted August 2010
dc.identifier.uri http://hdl.handle.net/10657/ETD-UH-2010-08-48
dc.description.abstract Prostaglandin E1 (PGE1) and E2 (PGE2) are ligands for the prostaglandin E2 receptor (EP) family, which consists of four subtype receptors, designated as EP1, EP2, EP3 and EP4. Interestingly, PGE2 mediates inflammation whereas PGE1 acts as an anti-inflammatory factor. However, the molecular basis of their opposite actions on the same set of EP receptors is poorly understood. To study the ligand recognition differences, a potential high throughput mutagenesis and constrained peptide was used. A peptide constrained to a conformation of the second-extracellular loop of human prostaglandin-E2 (PGE2) receptor subtype 3 (hEP3) was synthesized. The contacts between the peptide residues at S211 and R214, and PGE2 were first identified by NMR spectroscopy. The results were used as a guide for site-directed mutagenesis of the hEP3 protein. The S211L and R214L mutants expressed in HEK293 cells lost binding to [3H]PGE2. This study found that the non-conserved S211 and R214 of the hEP3 are involved in PGE2 recognition. The mutant S211L was able to give a calcium signal with PGE1, but not with PGE2. This implied that the corresponding residues in other subtype receptors could be important in distinguishing the different configurations of PGE2 and PGE1 ligand recognition sites. Direct transfection of point mutants in the EP1 receptor extracellular loop (using PCR products) was evaluated in HEK293 cells. Twenty-four EP1 extracellular loop mutants (alanine scan) were generated using phosporylated primers and ligase. The PCR product was directly transfected into HEK293 cells and the [3H]-PGE2 binding and PGE1 and PGE2 calcium signaling assay evaluated. Three mutants, A104G, P105A and P184A, showed reduced [3H]PGE2 binding, but could not differentiate between PGE1 and PGE2 in the calcium signaling assay. However, we propose that this novel high throughput mutagenesis approach using direct PCR product transfection can be integrated into a high throughput screening machine in the future. The PGE1 and PGE2 binding affinity on the four human recombinant EPs expressed in the live HEK293 as stable cell lines was determined by [3H]PGE2 binding. The PGE1 and PGE2 signaling on the four EPs was determined by the calcium (Ca2+) and cyclic AMP signaling. The Kd for [3H]PGE2 was calculated using saturation kinetic experiments. The IC50 of PGE2 and PGE1 were calculated from [3H]PGE2 displacement experiments using cold PGE2 and PGE1, respectively. PGE2 showed higher affinity or preference for EP3 and EP4 as compared to that of EP1 and EP2. PGE1 also showed a higher Ca2+ signal in EP1 as compared to that of PGE2. There was a two-log concentration difference between PGE2 and PGE1 for generation of Ca2+ signal in EP4. There was no difference in cAMP accumulation with PGE1 and PGE2. Leukotriene C4/D4/E2 levels were higher in the EP1 stable cell line upon stimulation with PGE2, but not PGE1. An anti-inflammatory molecule, 20 hydroxy lipoxin B4, peak was observed using mass spectroscopy with PGE1 and not PGE2. We also used a newly engineered hybrid enzyme (COX-2-10aa-mPGES-1) linking COX-2 and mPGES-1 together thus adopting the full biological activity of COX-2 and mPGES-1 in directly converting AA to PGE2. This enzyme was genetically introduced into HEK293 cells. These cells expressing the COX-2-10aa-mPGES-1 were producing higher level of PGE2 using endogenous AA as confirmed by LC/MS analysis. A new mouse model of cancer was developed by subcutaneous injection of these cells into Balb/c/nu/nu mice. A 100% (8 out 8) occurrence rate of cancer mass was detected in these cells. In contrast, 30% occurrence of cancer mass were determined for the groups of the cells co-expressing the individual COX-2 and mPGES-1. The presence of EP1 and EP2 stable cell line growth around these tumor masses confirmed their involvement in cancer. In conclusion, the Ca2+ signal indicated that the EP1 is likely the dominant and ligand-differentiating receptor in terms of signaling in tissues that co-express the EPs (cancer cells). PGE2 is likely to cause inflammation through leukotrienes and PGE1 is likely to be anti-inflammatory due to its ability to produce Lipoxin B4. High throughput mutagenesis for producing multiple-point mutations using direct PCR product transfection is a promising new method for the future. The experiments on nude mice indicated that the sole coupling of COX-2 to mPGES-1 is a powerful cancer-advancing factor, which implies that the coupling of COX-2 to mPGES-1 is a promising target for anti-cancer drug development. EP1 and EP2 receptors were identified as the likely receptors, to induce cancer. This study provides a molecular basis to understand the biological functions of PGE1 and PGE2 through their binding and signaling properties.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subject PGE1
dc.subject PGE2
dc.subject COX-2
dc.subject mPGES-1
dc.subject EP receptors
dc.title Binding and Signaling Differences between Prostaglandin E1 and E2 Mediated by Prostaglandin E Subtype Receptors
dc.date.updated 2012-09-28T12:35:56Z
dc.identifier.slug 10657/ETD-UH-2010-08-48
dc.type.material text *
dc.type.genre thesis *
thesis.degree.name Pharmacology (Ph.D.)
thesis.degree.level Doctoral
thesis.degree.discipline Pharmacology
thesis.degree.grantor University of Houston
thesis.degree.department Pharmacological and Pharmaceutical Sciences
dc.contributor.committeeMember Chow, Diana
dc.contributor.committeeMember Gao, Xiaolian
dc.contributor.committeeMember Williams, Louis
dc.contributor.committeeMember Das, Joydip

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