Sadashiva S. Karnik
Cleveland Clinic
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Publication
Featured researches published by Sadashiva S. Karnik.
Proceedings of the National Academy of Sciences of the United States of America | 2003
Huijun Wei; Seungkirl Ahn; Sudha K. Shenoy; Sadashiva S. Karnik; László Hunyady; Louis M. Luttrell; Robert J. Lefkowitz
Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of β-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation ≈50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular β-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with ≈60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular β-arrestin 2 by small interfering RNA (β-arrestin dependent). These findings imply the existence of independent G protein- and β-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct “active” conformations of a seven-membrane-spanning receptor coupled to each.
Pharmacological Reviews | 2005
Dianne M. Perez; Sadashiva S. Karnik
Studies have been amassed in the past several years indicating that an agonist can conform a receptor into an activation state that is dependent upon an intrinsic property of the agonist usually based upon its chemical composition. Theoretically, each different agonist could impart its own unique activation state. Evidence for multiple signaling states for the G-protein-coupled receptors will be reviewed and is derived from many different pharmacological behaviors: efficacy, kinetics, protean agonism, differential desensitization and internalization, inverse agonism, and fusion chimeras. A recent extension of the ternary complex model is suggested by evidence that the different processes that govern deactivation, such as desensitization and internalization, is also regulated by conformers specific to the agonist. Rhodopsin may serve as a primer for the study of multiple activation states. Therapeutic implications that utilize multiple signaling states hold vast promise in the rationale design of drugs.
Science | 1996
Unnikrishnan M. Chandrasekharan; Subramaniam Sanker; Manuel J. Glynias; Sadashiva S. Karnik; Ahsan Husain
The current model of serine protease diversity theorizes that the earliest protease molecules were simple digestive enzymes that gained complex regulatory functions and restricted substrate specificities through evolution. Among the chymase group of serine proteases are enzymes that convert angiotensin I to angiotensin II, as well as others that simply degrade angiotensins. An ancestral chymase reconstructed with the use of phylogenetic inference, total gene synthesis, and protein expression had efficient and specific angiotensin II-forming activity (turnover number, about 700 per second). Thus, angiotensin II-forming activity is the more primitive state for chymases, and the loss of such activity occurred later in the evolution of some of these serine proteases.
Trends in Endocrinology and Metabolism | 2003
Sadashiva S. Karnik; Camelia Gogonea; Supriya Patil; Yasser Saad; Takanobu Takezako
G-protein-coupled receptors (GPCRs) are a large family of proteins that contain a seven transmembrane helical structural motif. They mediate responses to several ligands by binding and activating intracellular heterotrimeric G proteins. Since the cloning of the first GPCR, insights gained from structure-function studies, genetics and drug development have contributed to uncovering a common mechanism that explains the activation of diverse GPCRs by their cognate agonists. This mechanism takes into consideration the conservation of the structure-function relationship in the basic seven transmembrane structural motif, and the dynamic changes in receptor conformation that are associated with activation. Combining models derived from the X-ray structure of rhodopsin with structure-function data allows a deeper understanding of the activation mechanism of GPCRs.
Cell | 2015
Haitao Zhang; Hamiyet Unal; Cornelius Gati; Gye Won Han; Wei Liu; Nadia A. Zatsepin; Daniel James; Dingjie Wang; Garrett Nelson; Uwe Weierstall; Michael R. Sawaya; Qingping Xu; Marc Messerschmidt; Garth J. Williams; Sébastien Boutet; Oleksandr Yefanov; Thomas A. White; Chong Wang; Andrii Ishchenko; Kalyan C. Tirupula; Russell Desnoyer; Jesse Coe; Chelsie E. Conrad; Petra Fromme; Raymond C. Stevens; Vsevolod Katritch; Sadashiva S. Karnik; Vadim Cherezov
Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.
Journal of Biological Chemistry | 2009
Sathyamangala V. Naga Prasad; Zong Hui Duan; Manveen K. Gupta; Venkata Suresh K. Surampudi; Stefano Volinia; George A. Calin; Chang Gong Liu; Ashwin Kotwal; Christine S. Moravec; Randall C. Starling; Dianne M. Perez; Subha Sen; Qingyu Wu; Edward F. Plow; Carlo M. Croce; Sadashiva S. Karnik
It is well established that gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, but the reasons for such differences are not clear. MicroRNAs (miRNAs) are short noncoding RNAs that provide a novel mechanism for gene regulation. The goal of this study was to comprehensively test for alterations in miRNA expression using human heart failure samples with an aim to build signaling pathway networks using predicted targets for the miRNAs and to identify nodal molecules that control these networks. Genome-wide profiling of miRNAs was performed using custom-designed miRNA microarray followed by validation on an independent set of samples. Eight miRNAs are significantly altered in heart failure of which we have identified two novel miRNAs that are yet to be implicated in cardiac pathophysiology. To gain an unbiased global perspective on regulation by altered miRNAs, predicted targets of eight miRNAs were analyzed using the Ingenuity Pathways Analysis network algorithm to build signaling networks and identify nodal molecules. The majority of nodal molecules identified in our analysis are targets of altered miRNAs and are known regulators of cardiovascular signaling. A heart failure gene expression data base was used to analyze changes in expression patterns for these target nodal molecules. Indeed, expression of nodal molecules was altered in heart failure and inversely correlated to miRNA changes validating our analysis. Importantly, using network analysis we have identified a limited number of key functional targets that may regulate expression of the myriad proteins in heart failure and could be potential therapeutic targets.
The EMBO Journal | 2000
Shin-ichiro Miura; Sadashiva S. Karnik
Conventional models of ligand–receptor regulation predict that agonists enhance the tone of signals generated by the receptor in the absence of ligand. Contrary to this paradigm, stimulation of the type 2 (AT2) receptor by angiotensin II (Ang II) is not required for induction of apoptosis but the level of receptor protein expression is critical. We compared Ang II‐dependent and ‐independent AT2 receptor signals involved in regulating apoptosis of cultured fibroblasts, epithelial cells and vascular smooth muscle cells. We found that induction of apoptosis—blocked by pharmacological inhibition of p38 mitogen‐activated protein kinase and caspase 3—is a constitutive function of the AT2 receptor. Biochemical and genetic studies suggest that the level of AT2 receptor expression is critical for physiological ontogenesis and its expression is restricted postnatally, coinciding with cessation of developmental apoptosis. Re‐expression of the AT2 receptor in remodeling tissues in the adult is linked to control of tissue growth and regeneration. Therefore, we propose that overexpression of the AT2 receptor itself is a signal for apoptosis that does not require the renin–angiotensin system hormone Ang II.
Journal of Biological Chemistry | 2000
Walter G. Thomas; Hongwei Qian; Chang Sheng Chang; Sadashiva S. Karnik
G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT1A) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar1,Ile4,Ile8]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT1A receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT1A receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT1 receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar1,Ile4,Ile8]AngII. Paradoxically, [Sar1,Ile4,Ile8]AngII produced a robust (∼85% of AngII), dose-dependent phosphorylation of the wild type AT1A receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar1,Ile4,Ile8]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT1A receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.
Journal of Biological Chemistry | 1995
Keita Noda; Yasser Saad; Sadashiva S. Karnik
The acidic pharmacophores of selective ligands bind to Lys199 and His256 of the AT1 receptor (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M., Husain, A., and Karnik, S.(1995) J. Biol. Chem. 270, 2284-2289). In this report we examine how interactions between these residues and agonists activate inositol phosphate production in transiently transfected COS-1 cells. [Sar1] angiotensin (Ang II) II and [Sar1]Ang II-amide stimulated a 5-fold inositol phosphate response from wild-type AT1 receptor. The peptide antagonist [Sar1,Ile8]Ang II and the non-peptide agonist L-162,313 produced a partial but saturating response. Stimulation of wild-type receptor by [Sar1]Ang II-amide and the mutant K199Q and K199A receptors by [Sar1]Ang II demonstrates that AT1 receptor activation is not critically dependent on the ion-pairing of the α-COOH group of Ang II with Lys199. The mutation of His256 produced diminished inositol phosphate response without commensurate change in binding affinity of ligands. The His256 side chain is critical for maximal activation of the AT1 receptor, although isosteric Gln substitution is sufficient for preserving the affinity for Phe8-substituted analogues of [Sar1]Ang II. Therefore, AT1 receptor activation requires interaction of Phe8 side chain of Ang II with His256, which is achieved by docking the α-COOH group of Phe8 to Lys199. Furthermore, non-peptide agonists interact with Lys199 and His256 in a similar fashion.
Journal of Biological Chemistry | 2006
Shin-ichiro Miura; Masahiro Fujino; Hiroyuki Hanzawa; Yoshihiro Kiya; Satoshi Imaizumi; Yoshino Matsuo; Sayo Tomita; Yoshinari Uehara; Sadashiva S. Karnik; Hiroaki Yanagisawa; Hiroyuki Koike; Issei Komuro; Keijiro Saku
To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.