Dr. Thomas Frielle
Associate Professor of Biochemistry
Identifying a Human, Cell Membrane cAMP Receptor: Survival of an organism requires that all its cells sense their environment and respond to each other if they are to function cooperatively. Each cell senses numerous extracellular signaling molecules from other cells to which it must respond. Due to differences in chemical structure, each signaling molecule binds to only its own particular receptor protein on a cell's outer surface. Binding a particular signaling molecule by its receptor causes an intracellular response to each molecule, translating the extracellular signal into the intracellular response. The human receptors we study, the GPCR family, use neurotransmitters and hormones as their signaling molecules. We are currently attempting to identify a receptor whose signaling molecule and function are, as yet, unidentified.
G protein-coupled receptors (GPCRs) constitute one of the largest superfamilies in the human genome, encoding approximately 900 different receptor proteins. Localized on the cell membrane, each GPCR binds a specific extracellular signaling molecule or agonist, initiating an intracellular response requiring an accessory G protein. GPCRs are vital for cell-to-cell communication and recognition, suggested by their roles in sight, taste, olfaction, neurotransmission, hormonal regulation, blood clotting, cellular metabolism, inflammation and the immune responses. Supporting their significance, GPCRs and their signaling pathways are targeted by 50% of currently marketed pharmaceuticals, including Cymbalta® for depression, Hydrocodone®for pain relief, Prozac® for anxiety, Advair® for chronic, obstructive pulmonary disease, Sinemet® for Parkinson’s Disease, Lopressor® for heart disease, Sumatriptan® for migraines and Zantac® for acid reflux. Approximately $50 billion in pharmaceutical sales revenue results from drugs that target these receptors.
The genes of approximately 900 human GPCRs have been cloned using DNA-based molecular biological techniques; however, only 300 have been pharmacologically identified according to their specific activating agonist. The remaining 600 cloned GPCRs whose agonists and functions remain unknown are termed “orphan receptors.” The orphan GPCR of interest, GP133, was cloned through the Human Genome Project. Due to structural similarities, it was classified to the adhesion subfamily of GPCRs. Since they are believed to engage in cell-to-cell recognition, adhesion GPCRs appear to serve roles in the immune system, cell growth, cellular development and human cancer progression making them a focal point of pharmaceutical research.
All identified human GPCRs have corresponding homologs in other species, including simple eukaryotic (non-bacterial) organisms. As a consequence, identifying human homologs by comparison to the previously identified GPCRs of simpler organisms is possible due to their homology throughout the phylogenetic tree.
When compared to the several thousand GPCRs from all species, GP133 shows significant amino acid sequence similarity to four related GPCRs from the cellular slime mold Dictyostelium discoideum. GP133 and the D.discoideum GPCRs show a 14% amino acid identity and an overall 45% amino acid similarity compared to GP133’s amino acid sequence. D. discoideum GPCRs bind the natural agonist 3’,5’-cyclic adenosine monophosphate (cAMP) in vivo. The activation of D. discoideum GPCRs by extracellular cAMP initiates chemotaxis, the independent movement of cells toward a chemical attractant. Chemotaxis promotes the aggregation of single cells, initiating the development of a multicellular organism from the single cells. During chemotaxis, receptor activation by cAMP initiates an intracellular signaling pathway that stimulates the synthesis of intracellular cAMP. Activation of D. discoideum GPCRs by extracellular cAMP suggests that cAMP may play a role in cellular adhesion, leading to a developmental pathway resulting in a multicellular organism.
Experiments are ongoing to further investigate the proposed function of GP133 as a human cAMP receptor. Using cultured cells expressing GP 133, research students have recently collected preliminary data supporting the possibility that cAMP is the natural agonist of GP133. Using the D. discoideum GPCRs as a paradigm, the likelihood exists that GP133 is a cAMP receptor, possessing functions similar to the D. discoideum GPCRs. The structural and functional similarities to the D. discoideum GPCRs and the potential role of D. discoideum GPCRs in cell adhesion suggests that GP133 may also bind extracellular cAMP and stimulate a similar intracellular pathway, therefore playing a similar role in human cells.
American Chemical Society Poster Presentations (Shippensburg University students indicated in bold)
- Frieben, E.E. and Frielle, T., Characterization of a human orphan G-protein coupled receptor, GP133, 2016.
- Miller, T.C. and Frielle, Characterization of a human orphan G-protein coupled receptor, GP133, 2015.
- Miller, T.C., Frieben, E.E., Frielle, T., Richardson, J.N. Quantitative analysis of glucose and kinetic study of glucose oxidase for use in an introductory quantitative analysis laboratory, 2015.
- Heck, C.J.S. and Frielle, T. Cellular localization of a human, cell membrane cyclic AMP (cAMP) receptor, 2014.
- Lougheed, C.S. and Frielle T. Determination of the relationship between human G-protein coupled receptor GP-133 expression and intracellular 3',5'-cyclic adenosine monophosphate (cAMP) production, 2014.
- Sidone, C.L. and Frielle, T., Expression and identification of human G-protein coupled receptor GP133, 2013.
- Gotthold, J.M. and Frielle, T. Ethanol determination by forensic blood alcohol gas chromatographic analyses, 2013.
- Frielle, T., Chemistry Experience: A course instructing incoming students in necessary skills and responsibilities, 2012.
- DiSalvo, D. S. and Frielle, T., Cell surface expression and characterization of a human orphan G protein-coupled receptor, 2012.
- DiSalvo, D. S. and Frielle, T., Cloning and identification of a human, cell-surface cAMP receptor, 2011.
- Moore, K. E. and Frielle, T., Protection from oxidation-induced cell death by the antioxidant glutathione, 2010.
- Prettner, C.T., Frielle, T., McCann, R.L., and Richardson, R.N., Attenuated total reflectance sensing of ethanol in blood, 2010.
- Bender, C., DiSalvo, D. S., Frielle, T. and Zaleski, C.M. Reactions of transferrin with cysteinylglycine., 2010.
- Mason, M.E. and Frielle, T., Roles of g-glutamyltranspeptidase in the cellular response to reactive oxygen species, 2009.
- Crum, S.L. and Frielle, T., Dual roles of g-glutamyltranspeptidase in the protective cellular response to reactive oxygen species, 2008
- Zbegner, S. and Frielle, T., Role of g-glutamyltranspeptidase in the protective cellular response to reactive oxygen species, 2008
- Frielle, T., Moore, K.E., Mason, M.E. Crum, S.L. and Zbegner, S. Protective role of g-glutamyltranspeptidase in the cellular response to reactive oxygen species. J. Undergrad. Chem. Res., 12: 68-71, 2013.
- Ellis, C.E. and Frielle, T. Characterization of two human b1-adrenergic receptor transcripts: Cloning and alterations in the failing heart. Biochem. Biophys. Res. Commun. 258: 552-558, 1999.
- Yue, J., Hartsough, M.T., Frey, R.S., Frielle, T. and Mulder, K.M. Cloning and expression of a rat Smad1: Regulation by TGF beta and modulation by the Ras/MEK pathway. J. Cellular Physiol. 178: 387-396, 1998.
- Evanko, D. S., Ellis, C.E., Venkatachalam, V. and Frielle, T. Preliminary analysis of the transcriptional regulation of the human b1-adrenergic receptor gene. Biochem. Biophys. Res. Commun. 244: 395-402, 1998.
- Penn, R.B., Frielle, T. . and Benovic, J.L. Comparison of R-, S-, and RS-albuterol interaction with human b1- and b2-adrenergic receptors. Clin Rev. Allergy Immunol. 14: 37-45. 1996.
- Saito, M, Frielle, T., Benovic, J.L. and Ledeen, R.W. Modulation by GM1 ganglioside of b1-adrenergic receptor-induced cyclic AMP formation in Sf9 cells. Biochem. Biophys. Acta. 1267: 1-5,1995.
- Suzuki, T., Nguyen, C.Y., Nantel, F., Bonin, H., Valiquette, M., Frielle, T., Bouvier, M. Distinct regulation of b1- and b2-adrenergic receptors in Chinese hamster fibroblasts. Mol. Pharmacol. 41: 542-548, 1992.
- Yang-Feng, T.L., Xue, F., Zhong, W., Cotecchia, S., Frielle, T., Caron, M.G., Lefkowitz, R.J. Franke, U. Chromosomal organization of adrenergic receptor genes. Proc. Natl. Acad. Sci. USA, 87: 1516-1520, 1990.
- Dohlman, H.G., Caron, M.G., DeBlasi, A., Frielle, T., Lefkowitz, R.J. Role of extracellular disulfide-bonded cysteines in the ligand binding function of the b2-adrenergic receptor. Biochemistry 29: 2335-2342, 1990.
- Frielle, T., Daniel, K.W., Caron, M.G. and Lefkowitz, R.J. Structural basis of b-adrenergic receptor subtype specificity studied with chimeric b1/b2 adrenergic receptors. Proc. Natl. Acad. Sci. USA 85: 9494-9498, 1988.
- Frielle, T., Collins, S., Daniel, K.W., Caron, M.G., Lefkowitz, R.J., Kobilka, B.K. Cloning of the cDNA for the human b1-adrenergic receptor. Proc. Natl. Acad. Sci. USA 4: 7920-7924, 1987.
- Kobilka, B.K., Frielle, T., Collins, S., Yang-Feng, T., Kobilka, T.S., Francke, U., Lefkowitz, R.J., Caron, M.G. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature 329: 75-79, 1987.
- Kobilka, B.K., Frielle, T., Dohlman, H.G., Bolanowski, M.A., Dixon, R.A.F., Keller, P., Caron, M.G., Lefkowitz, R.J. Delineation of the intronless nature of the human and hamster b2-adrenergic receptor genes and their putative promoter regions. J. Biol. Chem. 262: 7321-7327, 1987.
- Kobilka, B.K., Dixon, R.A.F., Frielle, T., Dohlman, H.G., Bolanowski, M.A., Sigal, I.S., Yang-Feng, T.L., Franke, U., Caron, M.G., Lefkowitz, R.J. cDNA for the human b2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 84: 46-50, 1987.
- Dixon, R.A.F., Kobilka, B.K., Strader, D.J., Benovic, J.L., Dohlman, H.E., Frielle, T., Bolanowski, M.A., Bennett, C.D., Rands, E., Diehl, R.E., Mumford, R.A., Slater, E.E., Sigal, I.S., Caron, M.G., Lefkowitz, R.J., Strader, C.D. Cloning of the gene and cDNA for mammalian b-adrenergic receptor and homology with rhodopsin. Nature 321: 75-79, 1986
- Frielle, T., Curthoys, N.P. Characterization of the membrane binding domain of g-glutamyltranspeptidase by specific labeling techniques. Biochemistry 22: 5709-5714, 1983.
- Frielle, T., Brunner, J., Curthoys, N.P. Isolation of the hydrophobic membrane binding domain of rat renal g-glutamyltranspeptidase selectively labeled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine. J. Biol. Chem. 257: 14979-14982, 1982.
- Frielle, T., and Curthoys, N.P. Characterization of the amphipathic structure of g-glutamyl-transpeptidase. Biophys. J. 37: 193-195, 1982.
- Frielle, T., Crimaldi, A.A., Coffee, C.J. A continuous spectrophotometric assay for cyclic 3', 5'-nucleotide phosphodiesterase. Anal. Biochem. 97: 239-247, 1979.
- Niehaus, W.G. Frielle, T., Kingsley, E.A., Jr. Purification and characterization of a secondary alcohol dehydrogenase from a Pseudomonad. J. Bacteriol. 134: 177-183, 1978.
- Frielle, T., Caron, MG. and Lefkowitz RJ. The mammalian beta-adrenergic receptors. Oxford Surveys on Eukaryotic Genes 6: 53-66, 1989.
- Frielle, T., Caron, M.G., Lefkowitz, R.J. Properties of the b1- and b2-adrenergic receptor subtypes revealed by molecular cloning. Clin. Chem. 35: 721-725, 1989.
- Frielle, T., Caron, M.G. and Lefkowitz, R.J. Human b1- and b2-adrenergic receptors: Structurally and functionally related receptors derived from distinct genes. Trends Neurosci. 11: 321-324, 1988.
- Caron, M.G., Kobilka, B.K., Frielle, T., Bolanowski, M.A., Benovic, J.L., Lefkowitz, R.J. Cloning of the cDNA and genes for the hamster and human beta2-adrenergic receptors. J. Receptor Res. 8: 7-21, 1988.
- Frielle, T., Kobilka, B.K., Dohlman, H., Caron, M.G., Lefkowitz, R.J. The b-adrenergic receptors and other receptors coupled to guanine nucleotide regulatory proteins. Federation Proceedings of APS Symposium, 1987 FASEB Meeting "Molecular Biology in Physiology", Raven Press, New York, 1987.
- Frielle, T., Tong, J., Curthoys, N.P. Changes in rat renal glutaminase activity studied in vivo and in primary cultures of proximal convoluted tubular cells. Contributions to Nephrology (A.C. Schoolwerth, ed.) S. Karger, 1985.
- Frielle, T., Curthoys, N.P. Specific labeling of the hydrophobic domain of rat renal g-glutamyltranspeptidase. Brush Border Membranes, (Ciba Foundation Symposium 95) pp. 73-91, Pitman Books Ltd., London, 1983.
- Curthoys, N.P., Frielle, T., Tsao, B., McIntyre, T.M., Hughey, R.P. Glutathione: Storage, Transport and Turnover (Y. Sakamoto, ed.). pp. 147-174, Japan Scientific Societies Press, Tokyo. 1983.
Current Research Students
- Emily E. Frieben: Accepted as a Ph.D. candidate in Pharmacology, Penn State College of Medicine
- Aaron M. Jefferys: Junior
- Colby E. Ott: Junior
Past Research Students
- Carley J.S. Heck: Ph.D. candidate in Biochemistry, Johns Hopkins University
- Caleb S. Lougheed: D.O. candidate, Pennsylvania College of Osteopathic Medicine
- Dale S. DiSalvo: M.D., Penn State College of Medicine
- Roxanne Jones: Laboratory technician, U.S. Army Medical Research Institute of Infectious Diseases
- Jane-Marie Gotthold: Clinical Trial Associate, Chiltern
- Katelyn E. Moore: High School chemistry teacher, Loudon County, VA
- Charles T. Prettner: Analyst, Pennsylvania Department of Environmental Protection
- Mark E. Mason: Ph.D. in Biochemistry, University of Pennsylvania
- Stacy L. Crum: M.S. in Biochemistry, University of Virginia, technical representative, Sigma-Aldrich-Suppelco
- Susan Zbegner: Research and Development Chemist, Fuchs Lubricants
After graduating from Bucknell University with a Chemistry degree, I received M.S. and Ph.D. degrees in Biochemistry from VA Tech and the University of Pittsburgh School of Medicine. I then completed a Post-doctoral Fellowship in Molecular Pharmacology at Duke University in the Howard Hughes Medical Institute. I directed basic research in Molecular Pharmacology and taught Ph.D. and M.D. students at Thomas Jefferson University and Penn State College of Medicine. Between then and coming to Shippensburg in 2006, I taught high school chemistry and biology and also wrote experiment-based elementary and middle school science curriculum.
I’m perpetually amazed by the chemical and biological world around us. When I'm not teaching or doing research at Ship, I still enjoy reading about and being involved in science. I often visit elementary and middle schools for science fairs and science outreach activities. I'm also the Co-Director of our summer Chemistry Camp for elementary students. It's incredible seeing the kids' eyes light up when a chemical reaction they initiated results in light, gas, colors or a small explosion. I'm also a consultant for the Toxicology Section of the Cumberland County Forensic Lab, reviewing cases of alcohol and illicit drug use.
It seems that the tendency to be a Biochemist runs in our family. My wife, Debbie, has a bachelor's degree in Biochemistry with a Ph.D. in Microbiology. Our two daughters have also earned bachelor's degrees in Biochemistry… absolutely no pressure there…we just talked a lot about science at the dinner table when they were young.
In my spare time, we visit with our daughters; I bike (not often enough to be in shape), I ski (not often enough considering I really like the speed) and I brew beer in my basement (not often enough since I do enjoy biochemistry experiments that are also drinkable).