Life & Health Sciences
Project: Deorphanizing GPR88: Attempt at Ligand Validation in Vell-Based Assay Produces Distinct Responses in Common Host Cell Lines
Presenter: Hannah Nelson
Major: Biomedical Engineering
Classification: Senior
Faculty Mentor: Dr. Abby Parrill-Baker, Chemistry
Abstract: G protein-coupled receptors (GPCR) are a large class of cell surface receptors involved in generating intracellular signaling events. Upon ligand binding in a pocket at the extracellular end of the transmembrane region of the GPCR, there is a conformational change in the receptor, leading to G protein coupling. Receptors and heterotrimeric G proteins, composed of Gα and Gβγ subunits, function together to transmit signals via effectors of downstream pathways. The Gα subunits Gαs and Gαi/o modulate cyclic AMP (cAMP) through stimulation or inhibition of adenylyl cyclase while Gαq subunits activate phospholipase C and stimulate intracellular Ca2+release. Due to their roles in cellular signaling and function, an estimated 30-50% of prescription drugs target GPCR for therapeutic action. However, there exists a large group of GPCR, called “orphans,” for which there are no known endogenous ligands or defined functions. Specifically, the orphan GPCR, GPR88, has been linked with psychiatric disorders, but due to the lack of an endogenous agonist, the GPR88 signaling pathways are not well-defined. However, in vivo and in vitro studies with synthetic agonists suggest that GPR88 couples to Gαi to reduce intracellular cAMP. Efforts to identify endogenous ligands for GPR88 have resulted in studies classifying (1R,2R)-N-[(2S,3S)-2-amino-3-methylpentyl]-N-(4'-propyl[1,1'-biphenyl]-4-yl)-2-(2-pyridinyl)-cyclopropanecarboxamide (2-PCCA) as a synthetic GPR88 agonist. Additionally, our group has computationally predicted 3-iodothyronamine (ITA), among several other compounds, as potential endogenous ligands for GPR88.
In this work, we aim to experimentally validate 2-PCCA and ITA in cellular models as agonists for GPR88. The cell lines employed are human embryonic kidney 293-T (HEK293-T), RH-7777 from rat liver tissue, and COS-7 from monkey kidney tissue. GPR88 was transfected into the cells, and activation of the receptor was determined via Promega’s GloSensor cAMP assay, a reporter assay that monitors changes in intracellular cAMP concentrations. For Gαi pathways, the assay detects decreases in isoproterenol (ISO)-stimulated cAMP production using D-luciferin as an enzymatic substrate to induce a luminescent signal upon receptor activation. Dose-dependent responses to 2-PCCA were observed in transfected RH-7777 and HEK293-T cells; whereas, such responses to ITA are only present at higher drug concentrations. The IC50 values for 2-PCCA in transfected RH-7777 and HEK293-T cells are 24.3nM and 125nM, respectively, compared to a published 911nM IC50 in HEK293 cells. Neither 2-PCCA nor ITA produce dose responses in GPR88 transfected COS-7 cells; however, evidence of activity is seen in the controls. These results suggest that other candidate endogenous GPR88 agonists should be tested, as the responses seen in the RH-7777 and HEK293-T cells do not confirm ITA as an endogenous agonist for the receptor. Furthermore, the responses achieved were ultimately dependent on cell type, highlighting difficulties of cell-based assays, as true “null” immortal cell lines do not exist, and it is not often possible to find a cell line that endogenously expresses the GPCR of interest. Additionally, issues such as pathway crosstalk or receptor-protein promiscuity are subject to arise. Ultimately, further work must be done to determine a higher-affinity endogenous agonist than ITA for GPR88, and assays that address the issues mentioned above should be employed.
Project: Interaction of Sports Bra Support & Running Speed on Knee Joint Stiffness
Presenter: Emily Kaesberg
Major: Exercise, Sports & Movement Science
Classification: Senior
Faculty Mentor: Dr. Douglas Powell, Health Studies
Abstract: Running has many health benefits, but can also result in injuries with a high percentage of runners experiencing an injury annually. For females, certain values, such as the type of sports bra support, may negatively affect running biomechanics. Other research has determined that insufficient breast support alters trunk and upper extremity biomechanics during treadmill running. But limited research has focused on the interaction between sports bra support and speed on lower extremity biomechanics. Knee joint stiffness is being observed because it has been known to be closely related with lower extremity injury in runners.
Project: MAP3K4 Kinase Activity Dependent Control of Mouse Gonadal Sex Determination
Presenter: Amber Broadhurst
Major: Biology
Classification: Senior
Faculty Mentor: Dr. Amy Abell, Biological Sciences
Abstract: During sex determination, the bipotential gonads develop into either testes or ovaries. The deletion of the Map3k4 gene in XY mice results in sex reversal where ovaries are formed despite the presence of the Y chromosome. The Map3k4 gene encodes the protein Mitogen-activated protein kinase kinase kinase 4 (MAP3K4), which consists of a large N-terminus for protein-protein interaction and a C-terminal kinase domain. MAP3K4 phosphorylates and activates MAP2K3/6, which in turn phosphorylates and activates p38. This cascade is responsible for development, transcriptional regulation, and importantly sex determination. In addition to the kinase dependent function of MAP3K4, this protein is involved in many protein-protein interactions that are independent of MAP3K4 kinase activity. Deletion of the Map3k4 gene in mice leads to embryonic lethality making it a difficult topic to study. It is unknown if the sex reversal that occurs in XY mice homozygous for this MAP3K4 deletion is due to the loss of the MAP3K4 kinase dependent function or the loss of MAP3K4 kinase independent functions. Here, we use a mouse model with a point mutation in the active site lysine at position 1361 to arginine (K1361R), inactivating MAP3K4 kinase activity while still allowing protein-protein interactions to occur. The inactivation of MAP3K4 kinase activity was confirmed using Western Blotting. This K1361R mutation is represented as KI for Kinase Inactive. We found that adult mice homozygous for this mutation (Map3k4KI/KI) exhibit a 4:1 female-biased sex ratio. Interestingly, sixty percent of adult Map3k4KI/KI phenotypically female mice were positive for the Y chromosome measured using polymerase chain reaction (PCR). Measurement of the reproductive organs of the sex-reversed XY Map3k4KI/KI mice revealed a shorter anogenital distance (AGD) compared to XY Map3k4WT/WT that was comparable to XX Map3k4WT/WT mice. Sex reversed adult XY Map3k4KI/KI mice were also smaller in body size and weight relative to XX and XY Map3k4WT/WT mice. These XY Map3k4KI/KI mice with sex reversal also developed overtly female reproductive organs that were defective. The uterine horns in the sex-reversed XY Map3k4KI/KI were smaller than XX Map3k4WT/WT or the ovaries were absent. Together, these findings demonstrate that MAP3K4 kinase activity is required for gonadal sex reversal.
Project: MemphisEATS: Discussing the Impact of Mobile Applications on Health & Nutrition
Presenter: Ashlyn Redmond
Major: Health Studies
Classification: Senior
Faculty Mentor: Dr. Marie van der Merwe, Health Sciences
Abstract: Numerous Americans face the diagnoses of diabetes and obesity. Lacking basic knowledge about healthy eating fuels this problem. A mobile application has been developed to support these issues by providing healthy meals to families through meal delivery and tutorials. The survey group consisted of 20 individuals who came to the lab kitchen to taste-test the meals and record their responses though survey. Initially, the research showed that individuals are open to trying new foods and would benefit from a delivery service. Continued research will record the acceptance of meals and delivery while monitoring anthropometric measurements, relating to diabetes and obesity.