35 results on '"Hamiyet Unal"'
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2. Calcium Mobilization Assay to Measure the Activity of Gq-coupled Receptors
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Hamiyet Unal
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Biology (General) ,QH301-705.5 - Abstract
Calcium mobilization assay is a cell-based second messenger assay to measure the calcium flux associated with Gq-protein coupled receptor activation or inhibition. The method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. In some cell lines in which organic-anion transporters are particularly active (e.g. CHO, HeLa), addition of probenecid, an inhibitor of anion transport, is required for retention of this dye in the cells. The dye binds the calcium released from intracellular store and its fluorescence intensity increases. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of the receptor of interest. This protocol can be applied to most mammalian cell lines expressing both endogenous and transiently/stably transfected receptors. The method is sensitive enough to be used for low-expressing systems or high throughput screening of target of interest. Note: The method does not differentiate the Ca2+ mobilization induced by Gqα from the Ca2+ mobilization induced by Gβγ.
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- 2013
- Full Text
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3. Interaction of G-protein βγ complex with chromatin modulates GPCR-dependent gene regulation.
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Anushree Bhatnagar, Hamiyet Unal, Rajaganapathi Jagannathan, Suma Kaveti, Zhong-Hui Duan, Sandro Yong, Amit Vasanji, Michael Kinter, Russell Desnoyer, and Sadashiva S Karnik
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Medicine ,Science - Abstract
Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gβγ in the cytosol. In this study, we show novel nuclear functions of Gβγ through demonstrating interaction of Gβ(2) with integral components of chromatin and effects of Gβ(2) depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gβ(2) levels in the nucleus and Gβ(2) interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gβ(2) repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gβ(2) interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gβ(2)-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gβγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.
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- 2013
- Full Text
- View/download PDF
4. Mechanism of Hormone Peptide Activation of a GPCR: Angiotensin II Activated State of AT1R Initiated by van der Waals Attraction.
- Author
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Khuraijam Dhanachandra Singh, Hamiyet Unal, Russell Desnoyer, and Sadashiva S. Karnik
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- 2019
- Full Text
- View/download PDF
5. Divergent Spatiotemporal Interaction of Angiotensin Receptor Blocking Drugs with Angiotensin Type 1 Receptor.
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Kh. Dhanachandra Singh, Hamiyet Unal, Russell Desnoyer, and Sadashiva S. Karnik
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- 2018
- Full Text
- View/download PDF
6. Angiotensin receptors in GtoPdb v.2023.1
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Patrick Vanderheyden, Thomas Unger, Hamiyet Unal, Kalyan Tirupula, Pieter B. M. W. M. Timmermans, Walter G. Thomas, Jacqueline Kemp, Sadashiva Karnik, Tadashi Inagami, Ahsan Husain, László Hunyady, Mastgugu Horiuchi, Theodore L. Goodfriend, Emanuel Escher, Satoru Eguchi, Khuraijam Dhanachandra Singh, Marc De Gasparo, Kevin J. Catt, Kenneth E. Bernstein, and Wayne Alexander
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General Medicine ,General Chemistry - Abstract
The actions of angiotensin II (Ang II) are mediated by AT1 and AT2 receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Angiotensin receptors [63, 155]), which have around 30% sequence similarity. The decapeptide angiotensin I, the octapeptide angiotensin II and the heptapeptide angiotensin III are endogenous ligands. losartan, candesartan, olmesartan, telmisartan, etc. are clinically used AT1 receptor blockers.
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- 2023
7. The Concise Guide To Pharmacology 2021/22: G Protein-Coupled Receptors
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Nigel J. M. Birdsall, Stephen J Lolait, Hans-Jürgen Wester, Paul L. Chazot, Sadashiva S. Karnik, Simon D. Harding, Celine Valant, Stephen P.H. Alexander, Olivier Civelli, Zsolt Csaba, Mastgugu Horiuchi, Khuraijam Dhanachandra Singh, Theodore L. Goodfriend, Morley D. Hollenberg, Duuamene Nyimanu, Shlomo Melmed, G. Enrico Rovati, Xavier Norel, Leigh A. Stoddart, Klemens Kaupmann, Robyn Macrae, Nicholas D. Holliday, Deborah L. Segaloff, Justo P. Castaño, Tony Ngo, Gordon Dent, Jean-Martin Beaulieu, Thomas L Williams, Antonia Cianciulli, Paweł Kozielewicz, Xaria X. Li, Jyrki P. Kukkonen, Craig A. McArdle, John D. Lee, Philippe Rondard, Steven D. Douglas, Hubert Vaudry, Khaled A. Al-Hosaini, Nan Chiang, Bernhard Bettler, Giovanni Tulipano, Corinne Bousquet, Karen J. Gregory, Craig Gerard, Robert T. Jensen, Stefan Schulz, Rithwik Ramachandran, Ross A. D. Bathgate, Deepa Jonnalagadda, David M. Thal, Takio Kitazawa, Manisha Ray, Bice Chini, Thomas Unger, Marta Fumagalli, Jean-Pierre Vilardaga, Donald T. Ward, Roger G. Pertwee, Stefan Offermanns, Marvin C. Gershengorn, Marc Parmentier, Pieter Timmermans, Eamonn Kelly, Yukihiko Sugimoto, Hamiyet Unal, Vincenzo Mitolo, Alistair Mathie, Emma L. Veale, Andrew L. Gundlach, Anthony P. Davenport, Mark T. Quinn, Jérôme Leprince, Christa E. Müller, Geoffrey Burnstock, Akos Heinemann, Jacqueline R. Kemp, Richard D. Ye, Bernard Mouillac, Roger J. Summers, Raul R. Gainetdinov, Girolamo Calò, Philip N. Murphy, Amelie Lupp, Kalyan C. Tirupula, Walter G. Thomas, Julien Hanson, Ahsan Husain, Katie Leach, Lucie H. Clapp, Hans Bräuner-Osborne, Gunnar Schulte, Kenneth E. Bernstein, Jean Mazella, Torsten Schöneberg, Satoru Eguchi, Martin C. Michel, Maria Antonietta Panaro, Kevin J. Catt, Rainer K. Reinscheid, Hans-Jürgen Kreienkamp, Wayne R. Alexander, Emanuel Escher, Anne Marie O'Carroll, Magnus Bäck, Laurence J. Miller, Jane F. Armstrong, Chiara Ruzza, Trent M. Woodruff, Daniel Hoyer, Chengcan Yao, Maria P. Abbracchio, John A. Peters, Gary B. Willars, Jean-Philippe Pin, David Vaudry, Debbie L. Hay, François Boulay, Davide Lecca, Eric R. Prossnitz, Arthur Christopoulos, Victoria A. Blaho, Yasuyuki Kihara, Charles Kennedy, Christopher Southan, László Hunyady, Pascal Dournaud, Fernand Gobeil, Cyril Goudet, Charles N. Serhan, Claes Dahlgren, Jörg Hamann, Tobias Langenhan, Ralf Jockers, Nicholas M. Barnes, Jean-Louis Nahon, Richard L. Hauger, Adam J. Pawson, Gareth J. Sanger, Tung Fong, Susan E. Leeman, Elena Faccenda, Edward J. Filardo, Valerie P. Tan, Marc de Gasparo, Ji Ming Wang, Jamie A. Davies, Jerold Chun, Stefania Ceruti, Tadashi Inagami, Réjean Couture, Kenneth A. Jacobson, Patrick Vanderheyden, Adriaan P. IJzerman, Janet J. Maguire, Christopher S. Walker, RS: CARIM other, Alexander, Stephen Ph [0000-0003-4417-497X], Christopoulos, Arthur [0000-0003-4442-3294], Davenport, Anthony P [0000-0002-2096-3117], Mathie, Alistair [0000-0001-6094-2890], Peters, John A [0000-0002-4277-4245], Veale, Emma L [0000-0002-6778-9929], Armstrong, Jane F [0000-0002-0524-0260], Faccenda, Elena [0000-0001-9855-7103], Harding, Simon D [0000-0002-9262-8318], Pawson, Adam J [0000-0003-2280-845X], Southan, Christopher [0000-0001-9580-0446], Davies, Jamie A [0000-0001-6660-4032], Abbracchio, Maria Pia [0000-0002-7833-3388], Bäck, Magnus [0000-0003-0853-5141], Bathgate, Ross [0000-0001-6301-861X], Beaulieu, Jean-Martin [0000-0002-0446-7447], Bettler, Bernhard [0000-0003-0842-8207], Blaho, Victoria [0000-0001-8499-2278], Bousquet, Corinne [0000-0002-2501-0593], Bräuner-Osborne, Hans [0000-0001-9495-7388], Burnstock, Geoffrey [0000-0001-8152-7979], Ceruti, Stefania [0000-0003-1663-4211], Chazot, Paul [0000-0002-5453-0379], Chiang, Nan [0000-0003-1963-1585], Chini, Bice [0000-0002-1686-284X], Chun, Jerold [0000-0003-3964-0921], Clapp, Lucie H [0000-0001-7802-4481], Dent, Gordon [0000-0001-9419-2952], Singh, Khuraijam Dhanachandra [0000-0003-0506-6896], Fumagalli, Marta [0000-0002-0158-842X], Gainetdinov, Raul R [0000-0003-2951-6038], Goudet, Cyril [0000-0002-8255-3535], Gregory, Karen J [0000-0002-3833-2137], Gundlach, Andrew L [0000-0002-6066-9692], Hamann, Jörg [0000-0002-9448-1727], Hanson, Julien [0000-0001-7063-7590], Hay, Debbie L [0000-0002-9558-5122], Heinemann, Akos [0000-0002-8554-2372], Holliday, Nicholas D [0000-0002-2900-828X], Hoyer, Daniel [0000-0002-1405-7089], IJzerman, Adriaan P [0000-0002-1182-2259], Jacobson, Kenneth A [0000-0001-8104-1493], Jockers, Ralf [0000-0002-4354-1750], Jonnalagadda, Deepa [0000-0002-1511-8197], Karnik, Sadashiva [0000-0003-0746-2753], Kaupmann, Klemens [0000-0001-8903-2508], Kennedy, Charles [0000-0001-9661-5437], Kihara, Yasuyuki [0000-0001-7462-3006], Kozielewicz, Pawel [0000-0003-1414-3566], Kukkonen, Jyrki P [0000-0002-6989-1564], Langenhan, Tobias [0000-0002-9061-3809], Leach, Katie [0000-0002-9280-1803], Lecca, Davide [0000-0002-3258-363X], Lee, John D [0000-0002-9976-7396], Leprince, Jérôme [0000-0002-7814-9927], Li, Xaria X [0000-0001-5924-2977], Lolait, Stephen J [0000-0001-7228-8072], Maguire, Janet [0000-0002-9254-7040], Mazella, Jean [0000-0002-5627-0742], McArdle, Craig A [0000-0003-4836-5351], Michel, Martin C [0000-0003-4161-8467], Miller, Laurence J [0000-0002-4554-3872], Mouillac, Bernard [0000-0002-3906-8673], Müller, Christa E [0000-0002-0013-6624], Nahon, Jean-Louis [0000-0001-9572-7779], Ngo, Tony [0000-0002-6779-2546], Norel, Xavier [0000-0003-0734-3359], O'Carroll, Anne-Marie [0000-0001-5255-8506], Offermanns, Stefan [0000-0001-8676-6805], Pertwee, Roger G [0000-0003-3227-2783], Pin, Jean-Philippe [0000-0002-1423-345X], Prossnitz, Eric R [0000-0001-9190-8302], Ramachandran, Rithwik [0000-0001-9557-9905], Ray, Manisha [0000-0002-8844-6191], Rondard, Philippe [0000-0003-1134-2738], Rovati, G Enrico [0000-0002-8788-9783], Ruzza, Chiara [0000-0003-1360-202X], Sanger, Gareth J [0000-0002-4231-1945], Schöneberg, Torsten [0000-0001-5313-0237], Schulte, Gunnar [0000-0002-2700-7013], Stoddart, Leigh A [0000-0002-4469-0600], Sugimoto, Yukihiko [0000-0001-6973-932X], Summers, Roger [0000-0002-8367-4056], Tan, Valerie P [0000-0002-7308-1601], Thal, David [0000-0002-0325-2524], Valant, Celine [0000-0002-2509-7465], Walker, Christopher S [0000-0001-8151-4123], Ward, Donald T [0000-0003-1342-9458], Woodruff, Trent M [0000-0003-1382-911X], Yao, Chengcan [0000-0003-3754-2842], Apollo - University of Cambridge Repository, Department of Pharmacology, and Experimental Immunology
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RM ,Cytoplasmic and Nuclear ,Computer science ,Databases, Pharmaceutical ,Humans ,Ion Channels ,Ligands ,Receptors, Cytoplasmic and Nuclear ,Receptors, G-Protein-Coupled ,Pharmacology ,IN-VITRO CHARACTERIZATION ,NO ,RS ,law.invention ,G-Protein-Coupled ,Databases ,03 medical and health sciences ,CALCIUM-SENSING RECEPTOR ,0302 clinical medicine ,DELTA-OPIOID RECEPTOR ,law ,Summary information ,Receptors ,HISTAMINE H-3 RECEPTOR ,FATTY-ACID RECEPTOR ,METABOTROPIC GLUTAMATE-RECEPTOR ,030304 developmental biology ,G protein-coupled receptor ,GONADOTROPIN-RELEASING-HORMONE ,0303 health sciences ,Clinical pharmacology ,FORMYL PEPTIDE RECEPTOR ,MUSCARINIC ACETYLCHOLINE-RECEPTOR ,3. Good health ,317 Pharmacy ,030220 oncology & carcinogenesis ,Pharmaceutical ,NEGATIVE ALLOSTERIC MODULATOR ,Catalytic receptors - Abstract
The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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- 2021
8. Mechanism of Hormone Peptide Activation of a GPCR: Angiotensin II Activated State of AT1R Initiated by van der Waals Attraction
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Hamiyet Unal, Khuraijam Dhanachandra Singh, Sadashiva S. Karnik, and Russell Desnoyer
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Chemistry ,General Chemical Engineering ,General Chemistry ,Library and Information Sciences ,Angiotensin II ,Computer Science Applications ,symbols.namesake ,Protein structure ,Cytoplasm ,Renin–angiotensin system ,Biophysics ,symbols ,van der Waals force ,Receptor ,Intracellular ,G protein-coupled receptor - Abstract
We present a succession of structural changes involved in hormone peptide activation of a prototypical GPCR. Microsecond molecular dynamics simulation generated conformational ensembles reveal propagation of structural changes through key "microswitches" within human AT1R bound to native hormone. The endocrine octa-peptide angiotensin II (AngII) activates AT1R signaling in our bodies which maintains physiological blood pressure, electrolyte balance, and cardiovascular homeostasis. Excessive AT1R activation is associated with pathogenesis of hypertension and cardiovascular diseases which are treated by sartan drugs. The mechanism of AT1R inhibition by sartans has been elucidated by 2.8 A X-ray structures, mutagenesis, and computational analyses. Yet, the mechanism of AT1R activation by AngII is unclear. The current study delineates an activation scheme initiated by AngII binding. A van der Waals "grasp" interaction between Phe8AngII with Ile2887.39 in AT1R induced mechanical strain pulling Tyr2927.43 and breakage of critical interhelical H-bonds, first between Tyr2927.43 and Val1083.32 and second between Asn1113.35 and Asn2957.46. Subsequently changes are observed in conserved microswitches DRYTM3, Yx7K(R)TM5, CWxPTM6, and NPxxYTM7 in AT1R. Activating the microswitches in the intracellular region of AT1R may trigger formation of the G-protein binding pocket as well as exposure of helix-8 to cytoplasm. Thus, the active-like conformation of AT1R is initiated by the van der Waals interaction of Phe8AngII with Ile2887.39, followed by systematic reorganization of critical interhelical H-bonds and activation of microswitches.
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- 2019
9. The non-biphenyl-tetrazole angiotensin AT1 receptor antagonist eprosartan is a unique and robust inverse agonist of the active state of the AT1 receptor
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Hamiyet Unal, Sadashiva S. Karnik, Takanobu Takezako, and Koichi Node
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0301 basic medicine ,Pharmacology ,Angiotensin II receptor type 1 ,Chemistry ,Antagonist ,Eprosartan ,Angiotensin II ,03 medical and health sciences ,Candesartan ,030104 developmental biology ,cardiovascular system ,medicine ,Inverse agonist ,Telmisartan ,Receptor ,hormones, hormone substitutes, and hormone antagonists ,circulatory and respiratory physiology ,medicine.drug - Abstract
Background and purpose Conditions such as hypertension and renal allograft rejection are accompanied by chronic, agonist-independent, signalling by angiotensin II AT1 receptors. The current treatment paradigm for these diseases entails the preferred use of inverse agonist AT1 receptor blockers (ARBs). However, variability in the inverse agonist activities of common biphenyl-tetrazole ARBs for the active state of AT1 receptors often leads to treatment failure. Therefore, characterization of robust inverse agonist ARBs for the active state of AT1 receptors is necessary. Experimental approach To identify the robust inverse agonist for active state of AT1 receptors and its molecular mechanism, we performed site-directed mutagenesis, competition binding assay, inositol phosphate production assay and molecular modelling for both ground-state wild-type AT1 receptors and active-state N111G mutant AT1 receptors. Key results Although candesartan and telmisartan exhibited weaker inverse agonist activity for N111G- compared with WT-AT1 receptors, only eprosartan exhibited robust inverse agonist activity for both N111G- and WT- AT1 receptors. Specific ligand-receptor contacts for candesartan and telmisartan are altered in the active-state N111G- AT1 receptors compared with the ground-state WT-AT1 receptors, suggesting an explanation of their attenuated inverse agonist activity for the active state of AT1 receptors. In contrast, interactions between eprosartan and N111G-AT1 receptors were not significantly altered, and the inverse agonist activity of eprosartan was robust. Conclusions and implications Eprosartan may be a better therapeutic option than other ARBs. Comparative studies investigating eprosartan and other ARBs for the treatment of diseases caused by chronic, agonist-independent, AT1 receptor activation are warranted.
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- 2018
10. Divergent Spatiotemporal Interaction of Angiotensin Receptor Blocking Drugs with Angiotensin Type 1 Receptor
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Khuraijam Dhanachandra Singh, Hamiyet Unal, Russell Desnoyer, and Sadashiva S. Karnik
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0301 basic medicine ,Angiotensin receptor ,General Chemical Engineering ,Protein Data Bank (RCSB PDB) ,Molecular Dynamics Simulation ,Library and Information Sciences ,Pharmacology ,Crystallography, X-Ray ,urologic and male genital diseases ,Receptor, Angiotensin, Type 1 ,Article ,03 medical and health sciences ,Spatio-Temporal Analysis ,Renin–angiotensin system ,Humans ,cardiovascular diseases ,Receptor ,Chemistry ,General Chemistry ,Angiotensin II ,female genital diseases and pregnancy complications ,Computer Science Applications ,Molecular Docking Simulation ,030104 developmental biology ,Docking (molecular) ,Drug Design ,Mutagenesis, Site-Directed ,Angiotensin II Type 1 Receptor Blockers ,hormones, hormone substitutes, and hormone antagonists - Abstract
Crystal structures of the human angiotensin II type 1 receptor (AT1R) complex with the antihypertensive agent ZD7155 (PDB id: 4YAY) and the blood pressure medication Benicar (PDB id: 4ZUD) showed that binding poses of both antagonists are similar. This finding implies that clinically used angiotensin receptor blocking (ARB) drugs may interact in a similar fashion. However, clinically observed differences in pharmacological and therapeutic efficacies of ARBs lead to the question of whether the dynamic interactions of AT1R with ARBs vary. To address this, we performed induced-fit docking (IFD) of eight clinically used ARBs to AT1R followed by 200 ns molecular dynamic (MD) simulation. The experimental Ki values for ARBs correlated remarkably well with calculated free energy with R2 = 0.95 and 0.70 for AT1R-ARB models generated respectively by IFD and MD simulation. The eight ARB–AT1R complexes share a common set of binding residues. In addition, MD simulation results validated by mutagenesis data discovered distinctive spatiotemporal interactions that display unique bonding between an individual ARB and AT1R. These findings provide a reasonably broader picture reconciling the structure-based observations with clinical studies reporting efficacy variations for ARBs. The unique differences unraveled for ARBs in this study will be useful for structure-based design of the next generation of more potent and selective ARBs.
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- 2017
11. Significance of angiotensin 1-7 coupling with MAS1 receptor and other GPCRs to the renin-angiotensin system: IUPHAR Review 22
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Sadashiva S. Karnik, Hamiyet Unal, Khuraijam Dhanachandra Singh, and Kalyan C. Tirupula
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0301 basic medicine ,Pharmacology ,medicine.medical_specialty ,Angiotensin receptor ,business.industry ,Endogeny ,Angiotensin II ,03 medical and health sciences ,Crosstalk (biology) ,030104 developmental biology ,Endocrinology ,Internal medicine ,Renin–angiotensin system ,medicine ,business ,Receptor ,G protein-coupled receptor ,Hormone - Abstract
Angiotensins are a group of hormonal peptides and include angiotensin II and angiotensin 1-7 produced by the renin angiotensin system. The biology, pharmacology and biochemistry of the receptors for angiotensins were extensively reviewed recently. In the review, the receptor nomenclature committee was not emphatic on designating MAS1 as the angiotensin 1-7 receptor on the basis of lack of classical G protein signalling and desensitization in response to angiotensin 1-7, as well as a lack of consensus on confirmatory ligand pharmacological analyses. A review of recent publications (2013-2016) on the rapidly progressing research on angiotensin 1-7 revealed that MAS1 and two additional receptors can function as 'angiotensin 1-7 receptors', and this deserves further consideration. In this review we have summarized the information on angiotensin 1-7 receptors and their crosstalk with classical angiotensin II receptors in the context of the functions of the renin angiotensin system. It was concluded that the receptors for angiotensin II and angiotensin 1-7 make up a sophisticated cross-regulated signalling network that modulates the endogenous protective and pathogenic facets of the renin angiotensin system.
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- 2017
12. Angiotensin receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database
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Kevin J. Catt, Kalyan C. Tirupula, Jacqueline R. Kemp, Hamiyet Unal, Walter G. Thomas, Marc de Gasparo, Patrick Vanderheyden, László Hunyady, Tadashi Inagami, Ahsan Husain, Satoru Eguchi, Thomas Unger, Wayne R. Alexander, Sadashiva S. Karnik, Theodore L. Goodfriend, Kenneth E. Bernstein, Emanuel Escher, Mastgugu Horiuchi, Khuraijam Dhanachandra Singh, and Pieter B.M.W.M. Timmermans
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Angiotensin receptor ,Angiotensin II receptor type 1 ,business.industry ,Angiotensin III ,Pharmacology ,Angiotensin II ,Candesartan ,Losartan ,cardiovascular system ,medicine ,Telmisartan ,business ,Receptor ,hormones, hormone substitutes, and hormone antagonists ,circulatory and respiratory physiology ,medicine.drug - Abstract
The actions of angiotensin II (Ang II) are mediated by AT1 and AT2 receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Angiotensin receptors [61, 152]), which have around 30% sequence similarity. The decapeptide angiotensin I, the octapeptide angiotensin II and the heptapeptide angiotensin III are endogenous ligands. losartan, candesartan, telmisartan, etc. are clinically used AT1 receptor blockers.
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- 2019
13. Luciferase reporter assay for unlocking ligand-mediated signaling of GPCRs
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Hamiyet, Unal
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Genetic Vectors ,CHO Cells ,Ligands ,Rats ,Receptors, G-Protein-Coupled ,Cricetulus ,HEK293 Cells ,Genes, Reporter ,Cricetinae ,Animals ,Humans ,Biological Assay ,Luciferases ,Signal Transduction - Abstract
G protein-coupled receptors (GPCRs) play an active role in numerous cellular processes, from cell proliferation to differentiation, by modulating gene transcription through various signal transduction pathways. Transcriptional regulation coupled to reporter gene expression may be used to study both G protein-dependent and G protein-independent responses activated by GPCR ligands. Reporter genes are typically used to monitor changes in receptor-mediated cellular responses at the transcription/translation level. Genetic reporter assays are based on reporter gene expression in response to activation of specific signaling cascade, followed by monitoring the presence of the reporter protein by directly measuring its enzymatic activity. These optimized genes are expressed under the control of a response element to assess its transcriptional activity that can be readily detected by a luminescent signal. Firefly luciferase gene has been widely used as a genetic reporter that responds rapidly to modulation of a GPCR by agonists or antagonists. Luciferase assays have been successfully developed for deorphanization of GPCRs, high-throughput screening (HTS) applications for drug discovery and deciphering both canonical and non-canonical signaling of numerous GPCRs. The protocol outlined for STAT3-driven luciferase assay could be adapted with appropriate changes to any aspect of GPCR signaling.
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- 2019
14. Mechanism of Hormone Peptide Activation of a GPCR: Angiotensin II Activated State of AT
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Khuraijam Dhanachandra, Singh, Hamiyet, Unal, Russell, Desnoyer, and Sadashiva S, Karnik
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Models, Molecular ,Protein Conformation ,Angiotensin II ,Entropy ,Humans ,Receptor, Angiotensin, Type 1 ,Article - Abstract
We present a succession of structural changes involved in hormone peptide activation of a prototypical GPCR. Microsecond molecular dynamics simulation generated conformational ensembles reveal propagation of structural changes through key “microswitches” within human AT(1)R bound to native hormone. The endocrine octa-peptide angiotensin II (AngII) activates AT(1)R signaling in our bodies which maintains physiological blood pressure, electrolyte balance, and cardiovascular homeostasis. Excessive AT(1)R activation is associated with pathogenesis of hypertension and cardiovascular diseases which are treated by sartan drugs. The mechanism of AT(1)R inhibition by sartans has been elucidated by 2.8 Å X-ray structures, mutagenesis, and computational analyses. Yet, the mechanism of AT(1)R activation by AngII is unclear. The current study delineates an activation scheme initiated by AngII binding. A van der Waals “grasp” interaction between Phe8(AngII) with Ile288(7.39) in AT(1)R induced mechanical strain pulling Tyr292(7.43) and breakage of critical interhelical H-bonds, first between Tyr292(7.43) and Val108(3.32) and second between Asn111(3.35) and Asn295(7.46). Subsequently changes are observed in conserved microswitches DRY(TM3), Yx7K(R)(TM5), CWxP(TM6), and NPxxY(TM7) in AT(1)R. Activating the microswitches in the intracellular region of AT(1)R may trigger formation of the G-protein binding pocket as well as exposure of helix-8 to cytoplasm. Thus, the active-like conformation of AT(1)R is initiated by the van der Waals interaction of Phe8(AngII) with Ile288(7.39), followed by systematic reorganization of critical interhelical H-bonds and activation of microswitches.
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- 2019
15. Luciferase Reporter Assay For Unlocking Ligand-Mediated Signaling Of Gpcrs
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Hamiyet Unal
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0303 health sciences ,03 medical and health sciences ,Reporter gene ,Drug discovery ,Response element ,Transcriptional regulation ,Luciferase ,Biology ,Signal transduction ,Gene ,030304 developmental biology ,G protein-coupled receptor ,Cell biology - Abstract
G protein-coupled receptors (GPCRs) play an active role in numerous cellular processes, from cell proliferation to differentiation, by modulating gene transcription through various signal transduction pathways. Transcriptional regulation coupled to reporter gene expression may be used to study both G protein-dependent and G protein-independent responses activated by GPCR ligands. Reporter genes are typically used to monitor changes in receptor-mediated cellular responses at the transcription/translation level. Genetic reporter assays are based on reporter gene expression in response to activation of specific signaling cascade, followed by monitoring the presence of the reporter protein by directly measuring its enzymatic activity. These optimized genes are expressed under the control of a response element to assess its transcriptional activity that can be readily detected by a luminescent signal. Firefly luciferase gene has been widely used as a genetic reporter that responds rapidly to modulation of a GPCR by agonists or antagonists. Luciferase assays have been successfully developed for deorphanization of GPCRs, high-throughput screening (HTS) applications for drug discovery and deciphering both canonical and non-canonical signaling of numerous GPCRs. The protocol outlined for STAT3-driven luciferase assay could be adapted with appropriate changes to any aspect of GPCR signaling.
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- 2019
16. Effect of novel GPCR ligands on blood pressure and vascular homeostasis
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Dulce Elena Casarini, Hamiyet Unal, Khuraijam Dhanachandra Singh, Zaira Palomino Jara, Sadashiva S. Karnik, Russell Desnoyer, Rodrigo Yokota, and Jorge Luís Pesquero
- Subjects
0303 health sciences ,Vascular homeostasis ,Blood Pressure ,Pharmacology ,Biology ,Ligands ,Angiotensin II ,Article ,Receptor, Angiotensin, Type 1 ,Receptors, G-Protein-Coupled ,Mice ,03 medical and health sciences ,Blood pressure ,Renin–angiotensin system ,Animals ,Blood Vessels ,Homeostasis ,Humans ,Receptor ,030304 developmental biology ,G protein-coupled receptor ,Hormone - Abstract
Maintenance of normal blood pressure under conditions of drug treatment is a measure of system-wide neuro-hormonal controls and electrolyte/fluid volume homeostasis in the body. With increased interest in designing and evaluating novel drugs that may functionally select or allosterically modulate specific GPCR signaling pathways, techniques that allow us to measure acute and long-term effects on blood pressure are very important. Therefore, this chapter describes techniques to measure acute and long-term impact of novel GPCR ligands on blood pressure regulation. We will use the angiotensin type 1 receptor, a powerful blood pressure regulating GPCR, in detailing the methodology. Normal blood pressure maintenance depends upon dynamic modulation of angiotensin type 1 receptor activity by the hormone peptide angiotensin II. Chronic activation of angiotensin type 1 receptor creates hypertension and related cardiovascular disease states which are treated with angiotensin type 1 receptor blockers (ARBs). Thus, a prototype for evaluation of blood pressure control under experimental evaluation of novel drugs.
- Published
- 2019
17. Structural Basis for Ligand Recognition and Functional Selectivity at Angiotensin Receptor
- Author
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Raymond C. Stevens, Gye Won Han, Vadim Cherezov, Vsevolod Katritch, Nilkanth Patel, Haitao Zhang, Hamiyet Unal, Sadashiva S. Karnik, and Russell Desnoyer
- Subjects
Agonist ,Angiotensin receptor ,medicine.drug_class ,Allosteric regulation ,Tetrazoles ,Crystallography, X-Ray ,Ligands ,Binding, Competitive ,Biochemistry ,Partial agonist ,Receptor, Angiotensin, Type 1 ,Cell Line ,Chlorocebus aethiops ,Sf9 Cells ,medicine ,Functional selectivity ,Animals ,Humans ,Inverse agonist ,Computer Simulation ,Molecular Biology ,Antihypertensive Agents ,Ions ,Chemistry ,Cell Membrane ,Sodium ,Imidazoles ,Cell Biology ,Angiotensin II ,Protein Structure, Tertiary ,Protein Structure and Folding ,COS Cells ,Mutation ,Mutagenesis, Site-Directed ,Olmesartan ,Oligopeptides ,Allosteric Site ,Protein Binding ,medicine.drug - Abstract
Angiotensin II type 1 receptor (AT(1)R) is the primary blood pressure regulator. AT(1)R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT(1)R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT(1)R in complex with an inverse agonist olmesartan (Benicar (TM)), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-35(1.39), Trp-84(2.60), and Arg-167(ECL2), similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT(1)R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N111(3.35)A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the beta-arrestin-biased peptide TRV120027.
- Published
- 2015
18. Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography
- Author
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Gye Won Han, Vadim Cherezov, Sadashiva S. Karnik, Marc Messerschmidt, Qingping Xu, Sébastien Boutet, Andrii Ishchenko, Cornelius Gati, Russell Desnoyer, Petra Fromme, Garrett Nelson, Vsevolod Katritch, Dingjie Wang, Haitao Zhang, Jesse Coe, Nadia A. Zatsepin, Thomas A. White, Chelsie E. Conrad, Kalyan C. Tirupula, Daniel James, Chong Wang, Wei Liu, Garth J. Williams, Michael R. Sawaya, Oleksandr Yefanov, Hamiyet Unal, Uwe Weierstall, and Raymond C. Stevens
- Subjects
Angiotensin receptor ,Biochemistry, Genetics and Molecular Biology(all) ,Regulator ,Biology ,Angiotensin II ,Article ,General Biochemistry, Genetics and Molecular Biology ,3. Good health ,Crystallography ,Docking (molecular) ,ddc:570 ,Femtosecond ,Receptor ,Peptide sequence ,G protein-coupled receptor - Abstract
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-angstrom 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 antihypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.
- Published
- 2015
19. Current Topics in Angiotensin II Type 1 Receptor Research: Focus on Inverse Agonism, Receptor Dimerization and Biased Agonism
- Author
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Takanobu Takezako, Sadashiva S. Karnik, Hamiyet Unal, and Koichi Node
- Subjects
0301 basic medicine ,Pharmacology ,Drug Inverse Agonism ,Chemistry ,Protein Conformation ,Angiotensin II ,Article ,Receptor, Angiotensin, Type 1 ,03 medical and health sciences ,030104 developmental biology ,Drug development ,Functional selectivity ,Animals ,Humans ,Signal transduction ,Protein Multimerization ,Receptor ,Neuroscience ,Angiotensin II Type 1 Receptor Blockers ,Homeostasis ,Function (biology) ,G protein-coupled receptor - Abstract
Although the octapeptide hormone angiotensin II (Ang II) regulates cardiovascular and renal homeostasis through the Ang II type 1 receptor (AT1R), overstimulation of AT1R causes various human diseases, such as hypertension and cardiac hypertrophy. Therefore, AT1R blockers (ARBs) have been widely used as therapeutic drugs for these diseases. Recent basic research and clinical studies have resulted in the discovery of interesting phenomena associated with AT1R function. For example, ligand-independent activation of AT1R by mechanical stress and agonistic autoantibodies, as well as via receptor mutations, has been shown to decrease the inverse agonistic efficacy of ARBs, though the molecular mechanisms of such phenomena had remained elusive until recently. Furthermore, although AT1R is believed to exist as a monomer, recent studies have demonstrated that AT1R can homodimerize and heterodimerize with other G-protein coupled receptors (GPCR), altering the receptor signaling properties. Therefore, formation of both AT1R homodimers and AT1R-GPCR heterodimers may be involved in the pathogenesis of human disease states, such as atherosclerosis and preeclampsia. Finally, biased AT1R ligands that can preferentially activate the β-arrestin-mediated signaling pathway have been discovered. Such β-arrestin-biased AT1R ligands may be better therapeutic drugs for cardiovascular diseases. New findings on AT1R described herein could provide a conceptual framework for application of ARBs in the treatment of diseases, as well as for novel drug development. Since AT1R is an extensively studied member of the GPCR superfamily encoded in the human genome, this review is relevant for understanding the functions of other members of this superfamily.
- Published
- 2017
20. Angiotensin II-regulated microRNA 483-3p directly targets multiple components of the renin–angiotensin system
- Author
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Jacqueline R. Kemp, Hamiyet Unal, Anushree Bhatnagar, Hong Yue, Russell Desnoyer, and Sadashiva S. Karnik
- Subjects
Cell signaling ,Molecular Sequence Data ,Myocytes, Smooth Muscle ,Biology ,Article ,Muscle, Smooth, Vascular ,Receptor, Angiotensin, Type 1 ,Renin-Angiotensin System ,Mice ,microRNA ,medicine ,Animals ,Humans ,Molecular Biology ,Regulation of gene expression ,Genome ,Angiotensin II receptor type 1 ,Base Sequence ,Reverse Transcriptase Polymerase Chain Reaction ,Angiotensin II ,Gene Expression Profiling ,Reproducibility of Results ,Angiotensin-converting enzyme ,Molecular biology ,Rats ,Cell biology ,Mice, Inbred C57BL ,Gene expression profiling ,MicroRNAs ,HEK293 Cells ,Losartan ,Gene Expression Regulation ,Organ Specificity ,cardiovascular system ,biology.protein ,Cardiology and Cardiovascular Medicine ,medicine.drug - Abstract
Improper regulation of signaling in vascular smooth muscle cells (VSMCs) by angiotensin II (AngII) can lead to hypertension, vascular hypertrophy and atherosclerosis. The extent to which the homeostatic levels of the components of signaling networks are regulated through microRNAs (miRNA) modulated by AngII type 1 receptor (AT(1)R) in VSMCs is not fully understood. Whether AT(1)R blockers used to treat vascular disorders modulate expression of miRNAs is also not known. To report differential miRNA expression following AT(1)R activation by AngII, we performed microarray analysis in 23 biological and technical replicates derived from humans, rats and mice. Profiling data revealed a robust regulation of miRNA expression by AngII through AT(1)R, but not the AngII type 2 receptor (AT(2)R). The AT(1)R-specific blockers, losartan and candesartan antagonized >90% of AT(1)R-regulated miRNAs and AngII-activated AT(2)R did not modulate their expression. We discovered VSMC-specific modulation of 22 miRNAs by AngII, and validated AT(1)R-mediated regulation of 17 of those miRNAs by real-time polymerase chain reaction analysis. We selected miR-483-3p as a novel representative candidate for further study because mRNAs of multiple components of the renin-angiotensin system (RAS) were predicted to contain the target sequence for this miRNA. MiR-483-3p inhibited the expression of luciferase reporters bearing 3'-UTRs of four different RAS genes and the inhibition was reversed by antagomir-483-3p. The AT(1)R-regulated expression levels of angiotensinogen and angiotensin converting enzyme 1 (ACE-1) proteins in VSMCs are modulated specifically by miR-483-3p. Our study demonstrates that the AT(1)R-regulated miRNA expression fingerprint is conserved in VSMCs of humans and rodents. Furthermore, we identify the AT(1)R-regulated miR-483-3p as a potential negative regulator of steady-state levels of RAS components in VSMCs. Thus, miRNA-regulation by AngII to affect cellular signaling is a novel aspect of RAS biology, which may lead to discovery of potential candidate prognostic markers and therapeutic targets. (C) 2014 Elsevier Ltd. All rights reserved.
- Published
- 2014
21. Structure-Function Basis of Attenuated Inverse Agonism of Angiotensin II Type 1 Receptor Blockers for Active-State Angiotensin II Type 1 Receptor
- Author
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Takanobu Takezako, Sadashiva S. Karnik, Hamiyet Unal, and Koichi Node
- Subjects
Pharmacology ,Binding Sites ,Drug Inverse Agonism ,Chemistry ,Molecular Sequence Data ,Mutation, Missense ,Regulator ,Inverse ,Articles ,Angiotensin II Type 1 Receptor Blockers ,Angiotensin II ,Receptor, Angiotensin, Type 1 ,Rats ,COS Cells ,Chlorocebus aethiops ,Extracellular ,Animals ,Molecular Medicine ,Inverse agonist ,Agonism ,Amino Acid Sequence ,Receptor ,Protein Binding - Abstract
Ligand-independent signaling by the angiotensin II type 1 receptor (AT1R) can be activated in clinical settings by mechanical stretch and autoantibodies as well as receptor mutations. Transition of the AT1R to the activated state is known to lower inverse agonistic efficacy of clinically used AT1R blockers (ARBs). The structure-function basis for reduced efficacy of inverse agonists is a fundamental aspect that has been understudied not only in relation to the AT1R but also regarding other homologous receptors. Here, we demonstrate that the active-state transition in the AT1R indeed attenuates an inverse agonistic effect of four biphenyl-tetrazole ARBs through changes in specific ligand-receptor interactions. In the ground state, tight interactions of four ARBs with a set of residues (Ser109(TM3), Phe182(ECL2), Gln257(TM6), Tyr292(TM7), and Asn295(TM7)) results in potent inverse agonism. In the activated state, the ARB-AT1R interactions shift to a different set of residues (Val108(TM3), Ser109(TM3), Ala163(TM4), Phe182(ECL2), Lys199(TM5), Tyr292(TM7), and Asn295(TM7)), resulting in attenuated inverse agonism. Interestingly, V108I, A163T, N295A, and F182A mutations in the activated state of the AT1R shift the functional response to the ARB binding toward agonism, but in the ground state the same mutations cause inverse agonism. Our data show that the second extracellular loop is an important regulator of the functional states of the AT1R. Our findings suggest that the quest for discovering novel ARBs, and improving current ARBs, fundamentally depends on the knowledge of the unique sets of residues that mediate inverse agonistic potency in the two states of the AT1R.
- Published
- 2015
22. International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli [corrected]
- Author
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Sadashiva S. Karnik, Walter G. Thomas, Kalyan C. Tirupula, Satoru Eguchi, Jacqueline R. Kemp, Hamiyet Unal, Patrick Vanderheyden, Experimental Pharmacology, Molecular and Biochemical Pharmacology, and Department of Bio-engineering Sciences
- Subjects
Angiotensin receptor ,Receptor Protein-Tyrosine Kinases ,Review ,Biology ,Receptor, Angiotensin, Type 2 ,Receptor, Angiotensin, Type 1 ,Renin-Angiotensin System ,Mice ,Structure-Activity Relationship ,Mediator ,Research Support, N.I.H., Extramural ,GTP-Binding Proteins ,Animals ,Humans ,Endothelium ,Receptor ,Cell Proliferation ,Pharmacology ,ATP6AP2 ,Inflammation ,Angiotensin II receptor type 1 ,Polymorphism, Genetic ,Receptors, Angiotensin ,Protein-Tyrosine Kinases ,Angiotensin II ,IUPHAR Nomenclature Report ,Cardiovascular Diseases ,Immunology ,Molecular Medicine ,Kidney Diseases ,Signal transduction ,Nervous System Diseases ,Reactive Oxygen Species ,Neuroscience ,Signal Transduction - Abstract
The renin angiotensin system (RAS) produced hormone peptides regulate many vital body functions. Dysfunctional signaling by receptors for RAS peptides leads to pathologic states. Nearly half of humanity today would likely benefit from modern drugs targeting these receptors. The receptors for RAS peptides consist of three G-protein-coupled receptors-the angiotensin II type 1 receptor (AT(1) receptor), the angiotensin II type 2 receptor (AT(2) receptor), the MAS receptor-and a type II trans-membrane zinc protein-the candidate angiotensin IV receptor (AngIV binding site). The prorenin receptor is a relatively new contender for consideration, but is not included here because the role of prorenin receptor as an independent endocrine mediator is presently unclear. The full spectrum of biologic characteristics of these receptors is still evolving, but there is evidence establishing unique roles of each receptor in cardiovascular, hemodynamic, neurologic, renal, and endothelial functions, as well as in cell proliferation, survival, matrix-cell interaction, and inflammation. Therapeutic agents targeted to these receptors are either in active use in clinical intervention of major common diseases or under evaluation for re-purposing in many other disorders. Broad-spectrum influence these receptors produce in complex pathophysiological context in our body highlights their role as precise interpreters of distinctive angiotensinergic peptide cues. This review article summarizes findings published in the last 15 years on the structure, pharmacology, signaling, physiology, and disease states related to angiotensin receptors. We also discuss the challenges the pharmacologist presently faces in formally accepting newer members as established angiotensin receptors and emphasize necessary future developments.
- Published
- 2015
23. Computational tools for fitting the Hill equation to dose–response curves
- Author
-
Sadashiva S. Karnik and Hamiyet Unal
- Subjects
Angiotensin II receptor type 1 ,G protein ,Chemistry ,Angiotensin II ,Cell biology ,Serine ,Transmembrane domain ,cardiovascular system ,General Earth and Planetary Sciences ,Inverse agonist ,Receptor ,hormones, hormone substitutes, and hormone antagonists ,circulatory and respiratory physiology ,General Environmental Science ,G protein-coupled receptor - Abstract
The pathophysiological actions of the renin–angiotensin system hormone, angiotensin II (AngII), are mainly mediated by the AngII type 1 (AT1) receptor, a GPCR. The intrinsic spontaneous activity of the AT1 receptor in native tissues is difficult to detect due to its low expression levels. However, factors such as the membrane environment, interaction with autoantibodies, and mechanical stretch are known to increase G protein signaling in the absence of AngII. Naturally occurring and disease-causing activating mutations have not been identified in AT1 receptor. Constitutively active mutants (CAMs) of AT1 receptor have been engineered using molecular modeling and site-directed mutagenesis approaches among which substitution of Asn 111 in the transmembrane helix III with glycine or serine results in the highest basal activity of the receptor. Transgenic animal models expressing the CAM AT1 receptors that mimic various in vivo disease conditions have been useful research tools for discovering the pathophysiological role of AT1 receptor and evaluating the therapeutic potential of inverse agonists. This chapter summarizes the studies on the constitutive activity of AT1 receptor in recombinant as well as physiological systems. The impact of the availability of CAM AT1 receptors on our understanding of the molecular mechanisms underlying receptor activation and inverse agonism is described.
- Published
- 2015
24. Angiotensin II Receptor–Induced Cardiac Remodeling in Mice Without Angiotensin II
- Author
-
Sadashiva S. Karnik and Hamiyet Unal
- Subjects
Male ,medicine.medical_specialty ,Angiotensin II receptor type 1 ,Ventricular Remodeling ,Myocardium ,Biphenyl Compounds ,Tetrazoles ,Biology ,Angiotensin II ,Receptor, Angiotensin, Type 1 ,5-HT7 receptor ,Endocrinology ,Gene Expression Regulation ,Internal medicine ,Constitutive androstane receptor ,Ventricular Dysfunction ,Internal Medicine ,medicine ,Animals ,RNA ,Benzimidazoles ,5-HT5A receptor ,Estrogen-related receptor gamma ,Coagulation factor II receptor ,Protease-activated receptor 2 - Abstract
See related article, pp 627–633 Cell surface receptors and their ligands cooperatively regulate physiological processes. The receptor activity is regulated positively when agonists bind and negatively when antagonists displace the agonists. Complete absence of a hormone should abrogate physiological and pathogenic functions regulated by the cognate receptor. However, awareness of the constitutive activity, the ability of native receptors to become functionally active in absence of hormone, is changing our view of the robustness of ligand-regulated receptor mechanisms. Paradigms of constitutive activity of G protein–coupled receptors (GPCRs) and inverse agonist activity of GPCR-targeted drugs are firmly established. The GPCR, angiotensin II (Ang II) type 1 receptor (AT1R), can be spontaneously active.1 Ways such as membrane environment, interacting proteins, receptor autoantibodies, and single nucleotide polymorphisms that increase expression can increase G-protein signaling in the absence of Ang II using the potential energy of the receptor.2 Inverse agonists can suppress the constitutive activity of a receptor; however, classic antagonists cannot perform this action1,2 (Figure). Figure. Mechanism of the constitutively active angiotensin II type 1 receptor (AT1R) signaling is shown in blue. The classic agonist-activated AT1R signaling is shown in black and white. GRK indicates G protein–coupled receptor kinase; P, phosphorylation. Constitutive activity is an inherent property of a GPCR in all, including human and animal, species.1–3 Wild-type AT1R stimulates significant G protein signaling in the absence of Ang II, when 1 to 10 pmol/mg of receptor is expressed in cell lines. The constitutively active pool of wild-type AT1R is
- Published
- 2012
25. Critical role for lysine 685 in gene expression mediated by transcription factor unphosphorylated STAT3
- Author
-
Sadashiva S. Karnik, Hamiyet Unal, George Stark Stark, Jinbo Yang, Belinda Willard, and Maupali Dasgupta
- Subjects
STAT3 Transcription Factor ,Transcriptional Activation ,Mutation, Missense ,Cardiomegaly ,Biology ,Biochemistry ,complex mixtures ,Cell Line ,SOX4 ,Gene expression ,Humans ,Phosphorylation ,STAT3 ,Molecular Biology ,Gene ,Transcription factor ,Interleukin-6 ,Angiotensin II ,Lysine ,Acetylation ,Cell Biology ,Cancer research ,biology.protein ,bacteria ,Protein Processing, Post-Translational ,Signal Transduction - Abstract
STAT3 is a pleiotropic transcription factor that is activated by the phosphorylation of tyrosine 705 in response to many cytokines and growth factors. STAT3 without Tyr-705 phosphorylation (U-STAT3) is also a potent transcription factor, and its concentration in cells increases greatly in response to STAT3 activation because the STAT3 gene can be driven by phosphorylated STAT3 dimers. We have now searched for post-translational modifications of U-STAT3 that might have a critical role in its function. An analysis by mass spectroscopy indicated that U-STAT3 is acetylated on Lys-685, and the integrity of Lys-685 is required for the expression of most U-STAT3-dependent genes. In contrast, we found only a very minor role for Lys-685 in gene expression induced in response to tyrosine-phosphorylated STAT3. U-STAT3 plays an important role in angiotensin II-induced gene expression and in the consequent development of cardiac hypertrophy and dysfunction. Mutation of Lys-685 inhibits this function of STAT3, providing new information on the role of U-STAT3 in augmenting the development of heart failure.
- Published
- 2014
26. Constitutive activity in the angiotensin II type 1 receptor: discovery and applications
- Author
-
Hamiyet, Unal and Sadashiva S, Karnik
- Subjects
Biomedical Research ,Drug Inverse Agonism ,Drug Discovery ,Mutation ,cardiovascular system ,Animals ,Humans ,hormones, hormone substitutes, and hormone antagonists ,Receptor, Angiotensin, Type 1 ,Article ,circulatory and respiratory physiology - Abstract
The pathophysiological actions of the renin–angiotensin system hormone, angiotensin II (AngII), are mainly mediated by the AngII type 1 (AT1) receptor, a GPCR. The intrinsic spontaneous activity of the AT1 receptor in native tissues is difficult to detect due to its low expression levels. However, factors such as the membrane environment, interaction with autoantibodies, and mechanical stretch are known to increase G protein signaling in the absence of AngII. Naturally occurring and disease-causing activating mutations have not been identified in AT1 receptor. Constitutively active mutants (CAMs) of AT1 receptor have been engineered using molecular modeling and site-directed mutagenesis approaches among which substitution of Asn(111) in the transmembrane helix III with glycine or serine results in the highest basal activity of the receptor. Transgenic animal models expressing the CAM AT1 receptors that mimic various in vivo disease conditions have been useful research tools for discovering the pathophysiological role of AT1 receptor and evaluating the therapeutic potential of inverse agonists. This chapter summarizes the studies on the constitutive activity of AT1 receptor in recombinant as well as physiological systems. The impact of the availability of CAM AT1 receptors on our understanding of the molecular mechanisms underlying receptor activation and inverse agonism is described.
- Published
- 2014
27. Constitutive Activity in the Angiotensin II Type 1 Receptor
- Author
-
Sadashiva S. Karnik and Hamiyet Unal
- Subjects
Pharmacology ,Biology ,Cell biology ,Interleukin-21 receptor ,Constitutive androstane receptor ,cardiovascular system ,Enzyme-linked receptor ,5-HT5A receptor ,Estrogen-related receptor gamma ,Coagulation factor II receptor ,hormones, hormone substitutes, and hormone antagonists ,Protease-activated receptor 2 ,circulatory and respiratory physiology ,Insulin-like growth factor 1 receptor - Abstract
The pathophysiological actions of the renin–angiotensin system hormone, angiotensin II (AngII), are mainly mediated by the AngII type 1 (AT1) receptor, a GPCR. The intrinsic spontaneous activity of the AT1 receptor in native tissues is difficult to detect due to its low expression levels. However, factors such as the membrane environment, interaction with autoantibodies, and mechanical stretch are known to increase G protein signaling in the absence of AngII. Naturally occurring and disease-causing activating mutations have not been identified in AT1 receptor. Constitutively active mutants (CAMs) of AT1 receptor have been engineered using molecular modeling and site-directed mutagenesis approaches among which substitution of Asn 111 in the transmembrane helix III with glycine or serine results in the highest basal activity of the receptor. Transgenic animal models expressing the CAM AT1 receptors that mimic various in vivo disease conditions have been useful research tools for discovering the pathophysiological role of AT1 receptor and evaluating the therapeutic potential of inverse agonists. This chapter summarizes the studies on the constitutive activity of AT1 receptor in recombinant as well as physiological systems. The impact of the availability of CAM AT1 receptors on our understanding of the molecular mechanisms underlying receptor activation and inverse agonism is described.
- Published
- 2014
28. Inducing Conformational Changes in G Protein-Coupled Receptors by Domain Coupling
- Author
-
Sadashiva S. Karnik and Hamiyet Unal
- Subjects
Coupling (electronics) ,Chemistry ,Domain (ring theory) ,Biophysics ,G protein-coupled receptor - Published
- 2013
29. Interaction of G-protein βγ complex with chromatin modulates GPCR-dependent gene regulation
- Author
-
Rajaganapathi Jagannathan, Anushree Bhatnagar, Zhong-Hui Duan, Michael Kinter, Amit Vasanji, Sandro L. Yong, Hamiyet Unal, Suma Kaveti, Sadashiva S. Karnik, and Russell Desnoyer
- Subjects
Amino Acid Motifs ,Immunoblotting ,Molecular Sequence Data ,GTP-Binding Protein beta Subunits ,lcsh:Medicine ,Biology ,Receptor, Angiotensin, Type 1 ,Receptors, G-Protein-Coupled ,Histones ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Heterotrimeric G protein ,GTP-Binding Protein gamma Subunits ,Animals ,Humans ,Gene Regulatory Networks ,Amino Acid Sequence ,lcsh:Science ,Transcription factor ,Cells, Cultured ,030304 developmental biology ,G protein-coupled receptor ,Regulation of gene expression ,Cell Nucleus ,0303 health sciences ,Multidisciplinary ,Sequence Homology, Amino Acid ,MEF2 Transcription Factors ,Angiotensin II ,Gene Expression Profiling ,lcsh:R ,Correction ,Molecular biology ,Chromatin ,Cell biology ,Mice, Inbred C57BL ,Histone ,HEK293 Cells ,Gene Expression Regulation ,Myogenic Regulatory Factors ,biology.protein ,RNA Interference ,lcsh:Q ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, G alpha and G beta gamma in the cytosol. In this study, we show novel nuclear functions of G beta gamma through demonstrating interaction of G beta(2) with integral components of chromatin and effects of G beta(2) depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented G beta(2) levels in the nucleus and G beta(2) interacted with specific nucleosome core histones and transcriptional modulators. Depletion of G beta(2) repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. G beta(2) interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the G beta(2)-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of G beta gamma with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.
- Published
- 2013
30. Long range effect of mutations on specific conformational changes in the extracellular loop 2 of angiotensin II type 1 receptor
- Author
-
Kalyan C. Tirupula, Anushree Bhatnagar, Rajaganapathi Jagannathan, Sadashiva S. Karnik, Russell Desnoyer, and Hamiyet Unal
- Subjects
Agonist ,Conformational change ,medicine.drug_class ,Stereochemistry ,Protein Conformation ,Molecular Conformation ,Biotin ,Ligand Binding Protein ,Molecular Dynamics Simulation ,Ligands ,Biochemistry ,Receptor, Angiotensin, Type 1 ,Receptors, G-Protein-Coupled ,Protein structure ,Chlorocebus aethiops ,medicine ,Inverse agonist ,Animals ,Cysteine ,skin and connective tissue diseases ,Molecular Biology ,G protein-coupled receptor ,Chemistry ,Cell Biology ,Ligand (biochemistry) ,Angiotensin II ,Protein Structure, Tertiary ,Rats ,Mutagenesis ,COS Cells ,Mutation ,Calcium ,sense organs ,Signal Transduction - Abstract
The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific "lid" conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar(1), Gln(2), Ile(8)] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.
- Published
- 2012
31. Correction: Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation
- Author
-
Zhong-Hui Duan, Suma Kaveti, Michael Kinter, Amit Vasanji, Sandro L. Yong, Rajaganapathi Jagannathan, Hamiyet Unal, Anushree Bhatnagar, Sadashiva S. Karnik, and Russell Desnoyer
- Subjects
STAT signaling family ,Multidisciplinary ,DNA transcription ,Signaling in cellular processes ,lcsh:R ,G-protein signaling ,lcsh:Medicine ,Signal transduction ,Biology ,Chromatin ,Combinatorics ,Molecular cell biology ,Genetics ,lcsh:Q ,Gene expression ,lcsh:Science ,Research Article - Abstract
Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gβγ in the cytosol. In this study, we show novel nuclear functions of Gβγ through demonstrating interaction of Gβ2 with integral components of chromatin and effects of Gβ2 depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gβ2 levels in the nucleus and Gβ2 interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gβ2 repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gβ2 interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gβ2-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gβγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.
- Published
- 2016
32. Mechanism of GPCR-directed autoantibodies in diseases
- Author
-
Hamiyet, Unal, Rajaganapathi, Jagannathan, and Sadashiva S, Karnik
- Subjects
Humans ,Autoantibodies ,Autoimmune Diseases ,Receptors, G-Protein-Coupled - Published
- 2012
33. Mechanism of GPCR-Directed Autoantibodies in Diseases
- Author
-
Sadashiva S. Karnik, Hamiyet Unal, and Rajaganapathi Jagannathan
- Subjects
Cell signaling ,business.industry ,Idiopathic dilated cardiomyopathy ,Immunology ,Autoantibody ,Medicine ,Muscarinic acetylcholine receptor M2 ,Receptor ,business ,Angiotensin II ,G protein-coupled receptor ,Thyrotropin receptor - Abstract
Receptor-activating autoantibodies targeting different G-protein-coupled receptors (GPCRs) have been discovered that exhibit agonist-like activity in several human pathologies. For example, autoimmune pathogenesis of Graves’ disease is attributed to autoantibody-mediated activation of the thyrotropin receptor, a GPCR. Likewise, diseases such as preeclampsia and vascular allograft rejection are caused by autoantibodies against angiotensin II type 1 receptor (AT1R). The serum of patients with Chagas disease causing congestive heart failure contains an autoantibody for the β1-adrenergic receptor. Autoantibodies against α1- and β1- and β2-adrenergic receptors found in serum from patients are linked to malignant hypertension and idiopathic dilated cardiomyopathy, respectively. Additional examples of GPCR-activating antibodies include those against the mGluR, GABA, 5HT4, calcium-sensing receptor, muscarinic M1 and M2 receptors, which have been identified in various chronic neurological diseases patients. The GPCR-directed autoantibodies may actually initiate the cellular signaling responsible for the disease since each disorder is associated with a specific GPCR-directed autoantibody. Empirical evidence suggests that the autoantibody induces GPCR activation without the endogenous ligand; however, the mechanism of antibody mediated receptor activation is not known. We show that the conformational dynamics of the extracellular domain of the AT1R generates the epitope for an autoantibody on the plasma membrane surface. This allows the antibody to bind and stabilize the activated state of AT1R, thus providing a molecular basis for the autoantibody action.
- Published
- 2012
34. Domain coupling in GPCRs: the engine for induced conformational changes
- Author
-
Hamiyet Unal and Sadashiva S. Karnik
- Subjects
Pharmacology ,Stereochemistry ,Allosteric regulation ,Cooperativity ,Plasma protein binding ,Biology ,Toxicology ,Ligands ,Small molecule ,Transmembrane protein ,Article ,Protein Structure, Tertiary ,Receptors, G-Protein-Coupled ,Coupling (electronics) ,Biophysics ,Animals ,Humans ,Function (biology) ,hormones, hormone substitutes, and hormone antagonists ,G protein-coupled receptor ,Protein Binding - Abstract
Recent solved structures of G protein-coupled receptors (GPCRs) provide insights into variation of the structure and molecular mechanisms of GPCR activation. In this review we provide evidence for the emerging paradigm of domain coupling facilitated by intrinsic disorder of the ligand-free state in GPCRs. The structure-function and dynamic studies suggest that ligand-bound GPCRs exhibit multiple active conformations in initiating cellular signals. Long-range intra-molecular and inter-molecular interactions at distant sites on the same receptor are crucial factors that modulate signaling function of GPCRs. Positive or negative coupling between the extracellular, the transmembrane and the intracellular domains facilitates cooperativity of activating “switches” as requirements for the functional plasticity of GPCRs. Awareness that allosteric ligands robustly affect domain coupling provides a novel mechanistic basis for rational drug development, small molecule antagonism and GPCR regulation by classical, as well as non-classical modes.
- Published
- 2011
35. Ligand-specific Conformation of Extracellular Loop-2 in the Angiotensin II Type 1 Receptor
- Author
-
Hamiyet Unal, Rajaganapathi Jagannathan, Sadashiva S. Karnik, and Manjunatha B. Bhat
- Subjects
Agonist ,Rhodopsin ,Stereochemistry ,medicine.drug_class ,Mutation, Missense ,Plasma protein binding ,Ligands ,Peptide Mapping ,Biochemistry ,Protein Structure, Secondary ,Receptor, Angiotensin, Type 1 ,Chlorocebus aethiops ,medicine ,Animals ,Biotinylation ,Disulfides ,Receptor ,Molecular Biology ,G protein-coupled receptor ,biology ,Chemistry ,Ligand ,Cell Biology ,Angiotensin II ,Protein Structure, Tertiary ,Structural Homology, Protein ,COS Cells ,biology.protein ,Rabbits ,Streptavidin ,Angiotensin II Type 1 Receptor Blockers ,Protein Binding ,Signal Transduction - Abstract
The orientation of the second extracellular loop (ECL2) is divergent in G-protein coupled receptor (GPCR) structures determined. This discovery provoked the question, is the ECL2 conformation differentially regulated in the GPCRs that respond to diffusible ligands? We have determined the conformation of the ECL2 of the angiotensin II type 1 receptor by reporter-cysteine accessibility mapping in different receptor states (i.e. empty, agonist-bound and antagonist-bound). We introduced cysteines at each position of ECL2 of an N-terminal epitope-tagged receptor surrogate lacking all non-essential cysteines and then measured reaction of these with a cysteine-reactive biotin probe. The ability of biotinylated mutant receptors to react with a steptavidin-HRP-conjugated antibody was used as the basis for examining differences in accessibility. Two segments of ECL2 were accessible in the empty receptor, indicating an open conformation of ECL2. These segments were inaccessible in the ligand-bound states of the receptor. Using the accessibility constraint, we performed molecular dynamics simulation to predict ECL2 conformation in different states of the receptor. Analysis suggested that a lid conformation similar to that of ECL2 in rhodopsin was induced upon binding both agonist and antagonist, but exposing different accessible segments delimited by the highly conserved disulfide bond. Our study reveals the ability of ECL2 to interact with diffusing ligands and to adopt a ligand-specific lid conformation, thus, slowing down dissociation of ligands when bound. Distinct conformations induced by the bound agonist and the antagonist around the conserved disulfide bond suggest an important role for this disulfide bond in producing different functional states of the receptor.
- Published
- 2010
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