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
· 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
· 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
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
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
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
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
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
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.