I started at the Weis Center for Research, Pennsylvania State University, College of Medicine and continued my teaching program in the Department of Medicine at the University of Colorado Health Sciences Center. I was a member of an instructional team that taught the Renal Physiology Course in the Division of Renal Diseases and Hypertension. In this course, I taught Molecular and Cellular Biology of Angiotensin II. I have also participated in small group tutorials and seminar series for students and fellows. My expertise and research interests are well-suited for teaching courses in molecular and cellular biology underlying physiological and pathophysiological processes. Therefore, I also took a joint (secondary) appointment in the Department of Cellular and Structural Biology at the University of Colorado Health Sciences Center. Since coming to Texas Tech University Health Sciences Center, my instructional responsibilities at the School of Pharmacy have involved both professional (pharmacy) and graduate (pharmaceutical sciences) students. I team-teach several didactic courses such as Pharmacotherapy-Cardiovascular, Case Studies I Tutorial, Biotechnology, Biochemistry, and Research Design and Analysis, and I am the course coordinator for the Principles of Disease and Research Design and Analysis courses. All of these are required courses for the professional education. In addition, I teach the Pharmaceutical Sciences Drug Design and Discovery (receptor) elective course for the graduate students.
Medical School Courses:
- Team member, Renal Physiology Course, taught by the Division of Renal Diseases and Hypertension at the University of Colorado Health Sciences (1995-2000)
School of Pharmacy Courses:
- Team member, Pharmacotherapy-Cardiovascular (PHARM 2352; 3 Credits) Fall Semester, 2000-Present
- Team Leader, Principles of Diseases (PHARM 1221; 2 Credits) Spring Semester, 2000-Present
- Team member, Case Studies I Tutorial (PHARM 3361; 3 Credits) Spring Semester, 2000-2003
- Guest Lecturer, Endocrinology (PHARM 3257; 2 Credits) Spring Semester, Mechanism of Insulin Action, 2001-2002
Graduate School of Biomedical Sciences Courses:
- Team member, Pharmaceutical Sciences Research Design and Analysis (GPSC 5390; 3 credits) Spring Semester, 2000-Present
- Team Leader, Pharmaceutical Sciences Research Design and Analysis (GPSC 5390; 3 credits) Fall Semester, 2004-Present
- Team member, Drug Design and Discovery (GPSC 5310; 3 Credits) Spring semester, 2002- Present
- Team Member, Biochemistry (GPSC 5601: 6 Credits) Fall Semester, 2003-Present
- Team Leader, Advanced Principles of Diseases (GSPC 5356; 3 Credits) Spring Semester, 2004-Present
- Team Member, Biotechnology (GPSC 5370: 3 Credits) Spring Semester, 2004-Present
I am primarily interested in molecular mechanisms of activation and regulation of G-protein coupled receptors. I have focused on the receptor for the potent vasoconstrictor hormone, angiotensin II, as a model for the subfamily of G-protein coupled receptors. Angiotensin II is the effector of the renin-angiotensin system, which plays a critical role in blood pressure regulation and electrolyte balance and which has been implicated in many important medical disorders, including hypertension and congestive heart failure, with its associated cardiovascular and renal damage. Consequently, investigating the biochemical and molecular mechanisms of Angiotensin II receptor activation and regulation is crucial for understanding the physiology and pathophysiology of the renin-angiotensin system.
· Preparation, enzymatic manipulation and analysis of DNA and RNA
· Construction and screening of recombinant DNA and genomic libraries
· DNA footprinting and sequencing (manual and automated)
· Stable and transient expression and functional analysis of genes in mammalian cells
· Expression and characterization of proteins in Escherichia coli
· Enzymatic amplification of RNA and DNA by the Polymerase Chain Reaction
· Mobility shift DNA and RNA binding assay
· Site-directed mutagenesis
· In vitro transcription and translation of cloned genes
· Fluorescence spectroscopy
· Detection, purification, radiolabeling and analysis of proteins
· Substrate specificity assays
· Enzymatic activity and kinetic assays
· Phosphorylation assays
· Receptor/ligand binding and functional assays
· Photo affinity-labeling assays
· Polarized epithelial cell culture and sodium transport studies
· Mammalian and bacterial cell culture
RESEARCH AREA AND ACCOMPLISHMENTS
Since my appointment at the Weis Center for Research at Pennsylvania State University College of Medicine, I have developed a well-funded independent research program. Below, I have listed my research interests and major scientific achievements in the area of renin-angiotensin system.
Identification and cDNA cloning of angiotensin-converting enzyme isozymes: We were the first group to report the isolation of a genomic clone that encoded both the pulmonary and testicular forms of angiotensin converting enzyme (ACE). [ Nucleic Acids Res. (1992) 20:683-7., J Biol Chem. (1991) 266:3854-62., J Cardiovasc Pharmacol. (1990) 16 Suppl 4:S14-8]. We identified the transcription start site of pulmonary angiotensin converting enzyme mRNA and showed that the testicular specific mRNA are spliced out from the mature pulmonary angiotensin converting enzyme mRNA. Testicular specific mRNA, on the other hand, initiated within an intron of the pulmonary angiotensin converting enzyme mRNA transcription unit and thus contained unique sequences in its 5’-region. We also determined that the choice of the two alternative transcription initiation sites appears to be regulated by a tissue-specific mechanism. Our observation facilitated the first determination of the molecular basis of tissue specific expression and function of testicular specific angiotensin-converting isozyme mRNA.
Angiotensin II receptor internalization and signal transduction: In addition to my work on the molecular mechanisms behind tissue specific expression of angiotensin converting enzyme isozymes, I made major contributions to our understanding of the mechanisms involved in angiotensin receptor signaling and function. We isolated and functionally characterized an angiotensin II receptor (AT1) in CHO-K1 and proximal tubule epithelial cells [ Mol Cell Biochem. (1995) 146:79-89, Mol Cell Biochem. (1995) 152:77-86, Am J Physiol. (1998) 274: F897-905]. Using recombinant DNA technology, we determined for the first time the essential amino acids required for receptor internalization [ J Biol Chem. (1995) 270: 207-13. J Biol. Chem. (1995) 270: 22153-9. Eur J Pharmacol. (1996) 295: 119-22]. Deletion of these essential sequences inhibited agonist-induced receptor internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins and activation of signal transduction pathways. We also showed rapid desensitization of the angiotensin II receptor in which putative carboxyl-terminal phosphorylation sites are absent, suggesting that the mechanism of AT1 receptor desensitization differs from that of other prototypical G-protein coupled receptors. However, internalization pathways are important for AT1 receptor function in polarized proximal tubule cells [ Am J Physiol. (2002) 282: F623-9]. These results are important because they support the idea that different subclasses of G-protein coupled receptors have evolved divergent mechanisms for controlling desensitization and function. Currently, we are investigating the role of receptor expression and internalization in proximal tubule epithelial cell function.
Moreover, we were the first to demonstrate that a G-protein coupled receptor, such as the angiotensin II AT1 receptor, stimulates sis-inducing factor like DNA binding activity. This observation was the first evidence that angiotensin AT1A receptors activate STAT transcription factors. [J Biol. Chem. (1994) 269: 31443-9, J Biol. Chem. (1995) 270: 19059-65]. Because it was the first report of a G-protein coupled receptor coupling to the JAK-STAT signaling pathway, investigators studying other G-protein coupled receptors were also very interested in these findings. We also showed that angiotensin Il can cross-regulate and inhibit interleukin-6 induced Stat3 signaling, in a MAP kinase kinase 1 dependent process. The inhibitory cross talk with interleukin-6 is particularly interesting, because this suggests the capacity of angiotensin II to regulate cytokine induced JAK-STAT signaling. The capacity of angiotensin II to inhibit cytokine-induced JAK-STAT signaling may have physiological as well as clinical implications in tissue injury, repair, and inflammation. We are currently investigating the central role of angiotensin II in cell proliferation.
Angiotensin II receptor regulation by mRNA binding protein(s): Angiotensin II acts on a variety of target tissues through cell surface guanine nucleotide regulatory protein (G-protein) coupled receptors, and while multiple receptor subtypes have been identified, most known functions are mediated through the AT1A receptor subtype. AT1A expression and signaling pathways through which receptor couple occurs in a tissue specific manner. The cellular control of these processes is not known. Recently, the messenger RNA 3'untranslated region of many genes has been identified as an important regulator of the mRNA transcript itself, as well as the translated product. We showed for the first time that the 3’untranslated region of the angiotensin AT1 receptor can control specific receptor functions, via selective recognition of the 3’untranslated region by mRNA binding protein(s) [ Biochem J. 2003 Mar 1;370(Pt 2):631-9, Biochem J. 1998 Jan 15;329 ( Pt 2):255-64.See publication 24 & 30]. Previous to this point, the 3’untranslated region of a mRNA was thought only to modulate the stability of mRNA. Thus, my discovery is highly innovative and has had a dramatic influence on the way investigators think about the 3’untranslated region. This study has opened a novel and potentially important mode of receptor regulation for future investigation not only in the angiotensin field but also in the vast numbers of G-protein coupled receptor area. Understanding the mRNA binding proteins and their role in the regulation of biological functions of specific proteins as the AT1A will provide insight into the coordinate actions responsible for regulating cellular functions and coupling to signaling pathways. Our current studies utilize a variety of different methodologies to elucidate the biochemical and molecular mechanisms involved in mRNA binding protein-mediated receptor function(s).
Angiotensin II receptor expression by glucose and growth hormone/factors: Hypertension and diabetes are two major risk factors in the pathogenesis of cardiovascular and renal diseases . Diabetes is characterized by perturbations of systemic and renal vascular tone and systemic and glomerular capillary hypertension. Diabetes induced nephropathy is a major health problem, and an urgent need exists to decipher the underlying molecular mechanism(s) and devise appropriate therapies. Several systemic and/or intrarenal networks of growth factors and cytokines can be modulated by diabetes. A potential link between vasoconstrictor angiotensin II (AngII) and progressive diabetic pathology has been demonstrated in both experimental and clinical studies with angiotensin converting enzyme (ACE) inhibitors and angiotensin type 1 receptor (AT1) blockade. Treatment of patients with ACE inhibitors is an established protective strategy in the management of diabetic nephropathy, even in the absence of systemic hypertension. However, down-regulation of AngII receptors is one of the major abnormalities of both proximal tubules and glomeruli in diabetic kidney disease. Reduction in AngII receptors could not be reversed by ACE inhibitors, demonstrating that the receptor downregulation was not mediated by the up-regulation of AngII. Ongoing research in our laboratory has shown that hAT1 is primarily regulated at the transcriptional level and a fine interplay between glucose and insulin (growth factors) mediates regulation of gene expression. Importantly, we have identified a specific sequence in the hAT1 gene promoter required for its basal transcription, and this may function as a growth factor enhancer element in hPTEC [Mol. End. (2000) 25: 97-108]. Additional studies revealed a repressor element upstream of the enhancer that can respond to normal/high levels of extracellular glucose [ Mol. Biol. Cell. (2004) 15: 4347-4355]. Our observation is that in the presence of high glucose, insulin has no effect towards enhanced transcriptional activation, whereas in the absence of glucose or presence of low glucose, insulin can activate the hAT1 gene transcription. In addition, we have evidence that these regulatory elements recognize specific nuclear transacting factors induced by glucose and insulin/growth factors. How these factors control hAT1 gene expression is not clear. While it has been suggested that multiple transcription factors may be involved in the regulation of the hAT1, our observation is the first evidence that normal levels of hAT1 gene transcription are controlled by a repressor element, perhaps through cross talk between glucose and insulin (growth factors) regulated mediators. Our hypothesis is that in normal physiology, expression of the hAT1 receptor is achieved by normalized interactions between glucose and insulin (growth factors) on hAT1 gene transcription. Alternatively, in pathophysiology such as diabetes, when extracellular glucose levels are high the equilibrium interaction between glucose and growth factors will change, and the end-result will be decreased expression of hAT1 gene. The overall focus in our laboratory is to identify the mechanism(s) by which glucose controls hAT1 gene transcription and identify specific transacting factors associated with glucose signaling, in order to understand the molecular and biochemical mechanisms involved in the hAT1 gene transcription in hyperglycemia.
Weidanz JA, Jacobson LM, Muehrer RJ, Djamali A, Hullett DA, Sprague J, Chiriva-Internati M, Wittman V, Thekkumkara TJ, Becker BN.
ATR blockade reduces IFN-gamma production in lymphocytes in vivo and in vitro. Kidney Int. 2005 Jun;67(6):2134-42.
Thomas BE, Thekkumkara TJ
Glucose mediates transcriptional repression of the human angiotensin type-1 receptor gene: role for a novel cis-acting element. Mol Biol Cell. 2004 Oct;15(10):4347-55. Epub 2004 Jul 21.
Bhat GJ, Samikkannu T, Thomas JJ, Thekkumkara TJ
Alpha thrombin rapidly induces tyrosine phosphorylation of a novel, 74-78 kDa stress response protein(s) in lung fibroblast cells. J Biol Chem. 2004 Sep 13 [Epub ahead of print]
Thekkumkara TJ, Linas SL.
Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein. Biochem J. 2003 Mar 1;370(Pt 2):631-9.
Thekkumkara T, Linas SL.
Role of internalization in AT(1A) receptor function in proximal tubule epithelium. Am J Physiol Renal Physiol. 2002 Apr;282(4):F623-9.
Fierensa FL, Vanderheyden PM, Roggeman C, De Backer J, Thekkumkara TJ, Vauquelin G
Tight binding of the angiotensin AT(1) receptor antagonist. Biochem Pharmacol. 2001 May 15;61(10):1227-35.
Huszar T, Mucsi I, Antus B, Terebessy T, Jeney C, Masszi A, Hunyady L, Mihalik B, Goldberg HJ, Thekkumkara TJ, Rosivall L.
Extracellular signal-regulated kinase and the small GTP-binding protein p21Rac1 are involved in the regulation of gene transcription by angiotensin II. Exp Nephrol. 2001 Mar-Apr;9(2):142-9.
Wyse BD, Linas SL, Thekkumkara TJ.
Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription. J Mol Endocrinol. 2000 Aug;25(1):97-108.
Thekkumkara TJ, Cookson R, Linas SL.
Angiotensin (AT1A) receptor-mediated increases in transcellular sodium transport in proximal tubule cells. Am J Physiol. 1998 May;274(5 Pt 2):F897-905.
Thekkumkara TJ, Thomas WG, Motel TJ, Baker KM.
Functional role for the angiotensin II receptor (AT1A) 3'-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins. Biochem J. 1998 Jan 15;329 ( Pt 2):255-64.
Thomas WG, Baker KM, Booz GW, Thekkumkara TJ.
Evidence against a role for protein kinase C in the regulation of the angiotensin II (AT1A) receptor. Eur J Pharmacol. 1996 Jan 4;295(1):119-22.
Thomas WG, Thekkumkara TJ, Baker KM.
Molecular mechanisms of angiotensin II (AT1A) receptor endocytosis. Clin Exp Pharmacol Physiol Suppl. 1996;3:S74-80. Review.
Thomas WG, Thekkumkara TJ, Baker KM.
Cardiac effects of AII. AT1A receptor signaling, desensitization, and internalization. Adv Exp Med Biol. 1996;396:59-69. Review.
Thekkumkara TJ, Du J, Zwaagstra C, Conrad KM, Krupinski J, Baker KM.
A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells. Mol Cell Biochem. 1995 Nov 8;152(1):77-86.
Thomas WG, Baker KM, Motel TJ, Thekkumkara TJ.
Angiotensin II receptor endocytosis involves two distinct regions of the cytoplasmic tail. A role for residues on the hydrophobic face of a putative amphipathic helix. J Biol Chem. 1995 Sep 22;270(38):22153-9.
Bhat GJ, Thekkumkara TJ, Thomas WG, Conrad KM, Baker KM.
Activation of the STAT pathway by angiotensin II in T3CHO/AT1A cells. Cross-talk between angiotensin II and interleukin-6 nuclear signaling. J Biol Chem. 1995 Aug 11;270(32):19059-65.
Lin C, Baker KM, Thekkumkara TJ, Dostal DE.
Sensitive bioassay for the detection and quantification of angiotensin II in tissue culture medium. Biotechniques. 1995 Jun;18(6):1014-20.
Thekkumkara TJ, Du J, Dostal DE, Motel TJ, Thomas WG, Baker KM.
Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: rapid desensitization by angiotensin II.
Mol Cell Biochem. 1995 May 10;146(1):79-89.
Korotchkina LG, Tucker MM, Thekkumkara TJ, Madhusudhan KT, Pons G, Kim H, Patel MS.
Overexpression and characterization of human tetrameric pyruvate dehydrogenase and its individual subunits. Protein Expr Purif. 1995 Feb;6(1):79-90.
Thomas WG, Thekkumkara TJ, Motel TJ, Baker KM.
Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization. J Biol Chem. 1995 Jan 6;270(1):207-13.
Bhat GJ, Thekkumkara TJ, Thomas WG, Conrad KM, Baker KM.
Angiotensin II stimulates sis-inducing factor-like DNA binding activity. Evidence that the AT1A receptor activates transcription factor-Stat91 and/or a related protein. J Biol Chem. 1994 Dec 16;269(50):31443-9.
Schworer CM, Rothblum LI, Thekkumkara TJ, Singer HA.
Identification of novel isoforms of the delta subunit of Ca2+/calmodulin-dependent protein kinase II. Differential expression in rat brain and aorta. J Biol Chem. 1993 Jul 5;268(19):14443-9
Thekkumkara TJ, Livingston W 3rd, Kumar RS, Sen GC.
Use of alternative polyadenylation sites for tissue-specific transcription of two angiotensin-converting enzyme mRNAs. Nucleic Acids Res. 1992 Feb 25;20(4):683-7.
Kumar RS, Thekkumkara TJ, Sen GC.
The mRNAs encoding the two angiotensin-converting isozymes are transcribed from the same gene by a tissue-specific choice of alternative transcription initiation sites. J Biol Chem. 1991 Feb 25;266(6):3854-62.