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An EH-domain switching mechanism regulates stable membrane association of EHD2
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Richard Lundmark)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Richard Lundmark)
Max-Delbrück-Center for Molecular Medicine.
Max-Delbrück-Center for Molecular Medicine.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

EHD2 is a dimeric ATPase known to stabilise the surface connection of the characteristic small invaginations of the cell surface termed caveolae. EHD2 oligomerises into rings around lipid membranes thereby controlling their shape. Here, we have analysed the domain interactions and mechanism that control the stable membrane association of EHD2 at caveolae. We have found that the N-terminus of EHD2, which is buried in the core protein and obstruct assembly, has to be relieved by an EH domain dependent mechanism. The binding between the EH domain and a loop in the GTPase domain of EHD2 was required for stable membrane association, but the loop in itself was not sufficient for specific recruitment to caveolae. A positively charged stretch in the EH domain is proposed to bind to lipids and thereby influence the exchange rate of EHD2. Taken together, we propose a stringent regulatory mechanism for the assembly of EHD2 involving switching of the EH domain position to release the N-terminus and facilitate oligomerisation.

National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-92494OAI: oai:DiVA.org:umu-92494DiVA: diva2:741051
Available from: 2014-08-27 Created: 2014-08-27 Last updated: 2014-09-08Bibliographically approved
In thesis
1. Caveolae associated proteins and how they effect caveolae dynamics
Open this publication in new window or tab >>Caveolae associated proteins and how they effect caveolae dynamics
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Caveolae-associerade proteiner och hur dom påverkar dynamiken hos caveolae
Abstract [en]

Caveolae are a type of invaginated membrane domain that has been shown to be involved in several disease states, including lipodystrophy, muscular dystrophies and cancer. Several of these diseases are caused by the lack of caveolae or caveolae-related signaling deficiencies in the tissues in which the caveolar domain are abundant such as lung, adipose, muscle and their related endothelial cells. Caveolae are formed through the assembly of the membrane inserted protein caveolin, cholesterol and the recently described family of cavin proteins, which together form the caveolae coat. The work in this thesis focuses on understanding the protein components and mechanisms that control the biogenesis and dynamics of caveolae.

We have found that the protein EHD2 is an important regulator and stabilizer of the caveolar domain at the cell membrane. EHD2 is a dimeric ATPase known to oligomerize into ring-like structures around lipid membranes to control their shape. We have characterized the domain interactions involved in the specific targeting and assembly of this protein at caveolae. We propose a stringent regulatory mechanism for the assembly of EHD2 involving ATP binding and switching of the EH domain position to release the N-terminus and facilitate oligomerization in the presence of membrane species. We show that loss of EHD2 in cells results in hyper- dynamic caveolae and that caveolae stability at the membrane can be restored by reintroducing EHD2 into these cells.

In a study of the protein cavin-3, which is known to be an integral component of the caveolar coat, we showed that this protein is targeted to caveolae via direct binding to the caveolar core protein caveolin1. Furthermore, we show that cavin-3 is enriched at deeply invaginated caveolae and regulate the duration time of caveolae at the cell surface.

In combination with a biochemical and cellbiological approach, the advanced fluorescence microscopy techniques, like Fluorescence Recovery After Photobleaching (FRAP), Total Internal Reflection microscopy (TIRF), combined with correlative Atomic Force Microscopy (AFM) have allowed us to characterize distinct caveolae-associated proteins and their respective functions at caveolae.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2014. 54 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1668
Keyword
Caveolae, caveolin, EHD2, cavin, microdomain, microscopy, TIRF, AFM
National Category
Cell and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-92500 (URN)978-91-7601-114-0 (ISBN)
Public defence
2014-09-19, N420, Naturvetarhuset, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2014-08-29 Created: 2014-08-27 Last updated: 2014-08-29Bibliographically approved

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Larsson, ElinMorén, BjörnLundmark, Richard
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