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Elucidating allosteric signal transmission in the beta1-adrenergic receptor

English title Elucidating allosteric signal transmission in the beta1-adrenergic receptor
Applicant Isogai Shin
Number 168031
Funding scheme Ambizione
Research institution Abteilung Strukturbiologie und Biophysik Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Structural Research
Start/End 01.04.2017 - 31.08.2019
Approved amount 600'000.00
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All Disciplines (2)

Discipline
Structural Research
Biophysics

Keywords (5)

G protein coupled receptor; In vitro fluorescent kinetic analysis; Protein allostery; Nuclear magnetic resonance; Thermodynamics

Lay Summary (German)

Lead
Die Signalübertragung in „G-Protein gekoppelten Rezeptoren“ (GPCRs) ist ein Schlüsselmechanismus der Informationsübermittlung vom Zelläusseren ins Zellinnere. Das Projekt leistet einen wichtigen Beitrag zum Verständnis dieses Mechanismus.
Lay summary

Inhalt und Ziele des Forschungsprojekts

„G-Protein gekoppelte Rezeptoren“ (GPCRs) sind die grösste Klasse von Proteinrezeptoren in der Zellmembran. Sie erkennen verschiedene äussere Reize (Licht, Hormone, usw.), lösen Zellsignale aus und regeln die Zellantwort.

Die molekulare Proteinstruktur von GPCRs ist flexibel und ihre Funktion wird allosterisch gesteuert. Der allosterische Mechanismus bezieht sich auf eine Wechselwirkung zwischen Teile des Rezeptors ausserhalb und innerhalb der Zelle. In statischen Molekülstrukturen, die durch Röntgenkristallographie erzeugt werden, sind diese dynamischen Mechanismen nicht sichtbar. In diesem Projekt wird der allosterische Mechanismus des beta1-Adrenozeptors durch verschiedene biophysikalische Methoden untersucht, die diese Dynamik sichtbar machen sollen, um so den komplexen Signalübertragungsmechanismus besser zu verstehen.

In der ersten Phase des Projektes wird die Erkennung der pharmazeutisch wirksamen Moleküle untersucht. In der zweiten Phase wird das eigentliche Signalnetzwerk im beta1-Adrenozeptor durch Bioinformatik untersucht. In der dritten Phase wird die allosterische Kommunikation zwischen dem GPCR und dem G-Protein untersucht. Insgesamt sollen die Resultate den Mechanismus der molekularen Erkennung, Signalübertragung und Signalausbreitung innerhalb der Zelle im beta1-Adrenozeptor erklären. Die Ergebnisse sollen dazu beitragen, diese Mechanismen auch in allen anderen GPCRs besser zu verstehen.

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Da die Proteinstrukturen der GPCRs ähnlich sind, können die Erkenntnisse des Modellrezeptors zum allgemeinen Verständnis dieser grössten Klasse von Membranrezeptoren beitragen. Dies ist wichtig für zukünftige Arzneimittelentwicklungen.

Direct link to Lay Summary Last update: 24.03.2017

Responsible applicant and co-applicants

Name Institute

Employees

Collaboration

Group / person Country
Types of collaboration
Dr. Timothy Sharpe, Biophysics Facility/Biozentrum Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Dmitry Veprintsev, Faculty of Medicine & Health Sciences, University of Nottingham Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Adnan Sljoka, Department of Informatics, Kwansei Gakuin University, Japan Japan (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
GE Healthcare Bio-Sciences AB Sweden (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Industry/business/other use-inspired collaboration
Prof. Daniel Nietlispach, Department of Biochemistry, Cambridge University Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure

Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
Solving everyday purification problems with automated multistep methods on ÄKTA pure systems Talk 27.05.2019 GE Healthcare Bio-Sciences, Uppsala, Sweden, Sweden Isogai Shin;


Communication with the public

Communication Title Media Place Year
Video/Film Automated multistep purification A user's story International 2018
New media (web, blogs, podcasts, news feeds etc.) Four purification steps with buffer dilutions in one go? No problem! GE Healthcare website International 2018

Associated projects

Number Title Start Funding scheme
141898 NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins 01.12.2012 Sinergia
173089 Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology 01.04.2017 Project funding

Abstract

G protein coupled receptors (GPCRs) are an important class of trans-membrane proteins that recognize a multitude of extracellular molecules and transmit their signal to the intracellular side. Despite recent achievements in X-ray crystallography of GPCRs, the high-resolution structures obtained do not capture their intrinsic dynamic properties, which are tightly associated with their function. NMR spectroscopy promises to provide such missing dynamic information. However so far, despite being valuable, only limited information has been obtained by NMR due to the difficult spectroscopic properties of this protein class. At this point, the potential of solution NMR analysis of GPCRs has not been fully realized.Recently, I have overcome many of the obstacles that hinder the application of solution NMR to study signal transduction in GPCRs. Using a stabilized form of the ß1-adrenergic receptor and a selective isotope labeling method in the baculovirus-insect cell expression system, I was able to acquire well-resolved backbone amide proton-nitrogen correlation spectra. These spectra revealed numerous mechanisms within the receptor that are new, or had been postulated but never observed directly. Thus I have established a system that can now be used to study many more functional mechanisms of GPCRs at atomic resolution. In addition, we have developed an economic method to produce uniformly isotope-labeled (including deuteration) GPCRs in the insect cell systems. This will allow more advanced applications of NMR spectroscopy to GPCRs such as the study of their dynamics by relaxation measurements.In the present proposal I want to use this system to obtain detailed insights into the receptor’s signal transmission mechanism with the aim to understand how the receptor recognizes ligands and passes this information to the G protein in order to modulate its activity. Using state-of-the-art methods of isothermal titration calorimetry, protein rigidity theory, coevolutionary sequence alignment, in vitro real-time observation of GDP/GTP exchange by fluorescence as well as solution NMR, I want to elucidate the underlying molecular mechanism of the thermodynamic behavior of ligand-receptor interactions, determine a high-resolution allosteric network model of signal transmission, and provide mechanical insights into how different agonists elicit varying levels of G protein activation.If successful, the results will have implications for the general understanding of GPCR function and the developed methods should be applicable to other GPCRs.
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