Characterization of Functional Regions of OGFr
Open Access
- Author:
- Kren, Nancy Porterfield
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 13, 2015
- Committee Members:
- Patricia McLaughlin, Dissertation Advisor/Co-Advisor
Michael Verderame, Committee Member
Ira Joseph Ropson, Committee Member
Lisa M Shantz, Committee Member
Samuel Shaomin Zhang, Committee Member - Keywords:
- OGFr
NES
Tandem Repeats - Abstract:
- The Opioid Growth Factor (OGF)-OGF receptor (OGFr) axis is present and tonically active in animal and human cancer cell lines, as well as human tumors. The OGF-OGFr pathway tonically mediates cell replication in cancer, with OGF serving as an autocrine-produced inhibitory pentapeptide. The inhibitory action of OGF on cell replication requires that OGF bind to OGFr, traffic into the nucleus to upregulate inhibitory kinases, p16 and p21 that act to stall the progression from G1-S phase of the cell cycle. Little is currently known about the functional regions required for this function. To begin to characterize the functional regions of OGFr, mutations in OGFr identified in cancer samples were characterized. Two mutations identified in cancer samples, S378I and R444H, were characterized with respect to their effects on localization of the receptor, as well as their ability to alter DNA synthesis. R444H demonstrated a significant decrease in the nuclear/cytoplasmic ratio while S378I showed no change. Both mutations demonstrated a loss of response to OGF and the long-acting opioid receptor antagonist naltrexone (NTX) in growth assays. R444H also demonstrated a loss of inhibition in DNA synthesis, while S378I showed no significant change compared to OGFr in the BrdU assay. These data demonstrate that cancer mutations such as R444H are capable of altering the inhibitory function of OGFr, while other mutations such as S378I may be capable of altering the response to the ligand OGF or the antagonist NTX. These data contribute to the understanding of the functionally required regions of OGFr and warrant further characterization of mutations identified in cancer samples.OGFr has three nuclear localization signals (NLS) that have previously been characterized. OGFr requires at least two of the three NLSs for entry into the nucleus and for inhibition of cell proliferation. When NLSs were mutated, OGFr was excluded from the nucleus and proliferation inhibition was lost. The nuclear export of OGFr is currently unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1 dependent nuclear export, suggesting OGFr is exported in a CRM1 dependent manner. One leucine rich sequence fitting the consensus sequence for nuclear export signals (NES) was identified. To better characterize this sequence, the associated leucines, L217 L220 L223 and L225, were mutated to alanine in isolation as well as in combinations. All of the mutations resulted in decreased nuclear accumulation. In support of decreased nuclear localization, subNES demonstrated a loss of inhibition. When the proposed NES was fused to EGFP, NES-EGFP, it responded to LMB, indicating this sequence is capable of functioning as an export signal in isolation. To investigate why the sequence functions differently in isolation than in the context of the full-length protein, the localization of subNES was evaluated in the presence of MG132, a potent inhibitor of proteosomal degradation, and LMB. Neither MG132 nor LMB affected the localization of subNES. Finally, the tandem repeat region of OGFr was evaluated to determine its contribution to OGFr localization. Six of the 7 tandem repeats found in the C-terminal end of OGFr were removed creating deltaTR. DeltaTR localized exclusively to the nucleus indicating that the tandem repeats may contribute to the localization of the receptor. Similar to the loss of inhibition seenwith subNES, deltaTR also demonstrated a significant loss of inhibition on DNA synthesis indicating that the tandem repeats are required for receptor function. These experiments demonstrate that OGFr contains one functional NES, L217 L220 L223 and L225, and that OGFr can be exported from the nucleus in a CRM1 dependent manner. However, OGFr may be exported by additional methods, as seen by altered localization when 6 of 7 tandem repeats were removed. The data presented in this thesis demonstrates that cancer mutations in OGFr are capable of altering the function of the receptor. Additionally, this work identifies on CRM1 dependent NES, L217 L220 L223 and L225, and demonstrates that the tandem repeats as well as the NES are required for DNA synthesis inhibition. The 7 tandem repeats are also demonstrated to contribute to the localization of the receptor. This thesis significantly contributes to the knowledge of the OGF-OGFr axis.