Date of Award

12-2011

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Erin G. Schuetz, PhD

Committee

Bernd Meibohm, PhD, FCP Alexander Sparreboom, PhD

Abstract

Background and Aim: MRP4/ABCC4 is an ABC transporter that can efflux the second-messenger, cAMP, from cells. MRP4 has a PDZ interacting motif at its carboxy terminal end through which it binds to scaffolding proteins NHERF1 and PDZK1. Previous studies have shown that PDZK1 serves as a scaffold physically coupling MRP4 with the cystic fibrosis transmembrane conductance regulator (CFTR). This protein complex functionally couples cAMP regulation of CFTR function with MRP4 cAMP transporter activity [Li, C., et al., Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia. Cell, 2007. 131(5): p. 940-51]. We hypothesized that the MRP4 PDZ domain can bind MRP4 to scaffolding proteins other than NHERF1 and PDZK1 and that those PDZ proteins serve to physically and functionally link MRP4 to other proteins involved in cAMP signaling. High expression of MRP4 has been observed in normal prostate and in human prostate cancer cell lines such as LNCaP cells [Cai, C., et al., Androgen induces expression of the multidrug resistance protein gene MRP4 in prostate cancer cells. Prostate Cancer Prostatic Dis, 2007. 10(1): p. 39-45]. In these cells, there are reports that the !2 adrenergic receptor (!2AR), a G protein-coupled receptor that ultimately signals through cAMP, is also highly expressed [Kasbohm, E.A., et al., Androgen receptor activation by G(s) signaling in prostate cancer cells. J Biol Chem, 2005. 280(12): p. 11583-9]. !2AR also has a PDZ interacting motif at its carboxy terminal end. We hypothesized that in these cells there is the possibility of interaction between MRP4 and !2AR through a shared PDZ protein, leading to physical and functional association of these proteins.

Methodology: We probed Panomics PDZ protein arrays with biotinylated MRP4 peptides consisting of MRP4 PDZ interacting motif, and identified potential PDZ partner proteins to which MRP4 peptide binds. We used LNCaP cells to quantify the mRNA expression of these putative PDZ partner proteins, as well as MRP4 and !2AR. We carried out pull down assays to test for physical association between the three proteins. We used a cAMP reporter assay to test whether activation of !2AR induced cAMP signaling and determine whether this signaling was modulated by MRP4 expression.

Results: Out of 93 PDZ domains, MRP4 showed interaction with 24 PDZ domains. The five candidate PDZ proteins chosen for further studies met the following criteria: (a) They were documented in the literature to interact functionally with GPCRs (G protein-coupled receptors) that upon stimulation couple with G" proteins and cause a rise in intracellular cAMP, and (b) They were expressed in prostate cells and co-localized with MRP4. Based on previous studies, only two PDZ domains, NHERF1 and MAGI3, were reported to bind with G"s, which is a subunit responsible for production of cAMP in response to activation of certain type of GPCRs. LNCaP cells have higher expression of MRP4 and !2AR (a G protein-coupled receptor that binds G"s subunit). Using pull down assays, we showed a physical association between MRP4 and !2AR. We also showed a functional association between !2AR and MRP4, because inhibition of MRP4 modulated !2AR-induced cAMP signaling in LNCaP cells.

Conclusion: MRP4 is physically and functionally associated with the !2AR in LNCaP cells. This association may be facilitated by a scaffolding protein, which may be MAGI3. This protein may be responsible for holding the macromolecular complex of two proteins, MRP4 and !2AR. This association may be important for regulating cAMP levels in LNCaP cells and affecting the downstream expression of certain genes that depend on the cAMP signal transduction.

DOI

10.21007/etd.cghs.2011.0252

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