The identification of a second actin-binding region in spinophilin/neurabin II

Anthony P. Barnes; F. Donelson Smith; Hendrika M. VanDongen; Antonius M.J. VanDongen; Sharon L. Milgram

doi:10.1016/j.molbrainres.2003.12.020

The identification of a second actin-binding region in spinophilin/neurabin II

Anthony P. Barnes
, F. Donelson Smith
, Hendrika M. VanDongen
, Antonius M.J. VanDongen
, Sharon L. Milgram

Research output: Contribution to journal › Article › peer-review

16 Scopus citations

Abstract

Spinophilin/neurabin II is an actin-associated scaffolding protein enriched in the dendritic spines of neurons. Previously, the actin-binding domain (ABD) of spinophilin was localized to a domain between amino acids (aa) 1 and 154. In a mass spectrometry screen for spinophilin-binding proteins, we have identified an additional actin-binding region between aa 151 and 282. F-actin co-sedimentation and GST affinity chromotography experiments further substantiate this result. Phalloidin staining of Rat2 fibroblasts transiently expressing GFP-spinophilin deletion constructs indicates co-localization with a subset of actin. Regions of spinophilin that lack the revised ABD (aa 1-230) do not co-localize with phalloidin-labeled actin, suggesting that the actin-binding domain contributes to directing the subcellular distribution of spinophilin. Targeting experiments using primary hippocampal cultures indicate that only the first actin-binding site contributes to dendritic spine localization. The second ABD targets to spines inefficiently and thus may interact with and affect actin filaments in a different manner than the first ABD.

Original language	English (US)
Pages (from-to)	105-113
Number of pages	9
Journal	Molecular Brain Research
Volume	124
Issue number	2
DOIs	https://doi.org/10.1016/j.molbrainres.2003.12.020
State	Published - May 19 2004
Externally published	Yes

Funding

Mass spectrometry was performed in collaboration with Mary Moyer and William A. Burkhart in the Department of Structural Chemistry, Glaxo Wellcome, RTP, NC. We thank Mihir Patel and Leah Means for the technical assistance, Drs. Richard Cheney and Jonathan Berg for the helpful suggestions and discussions, and the members of the Milgram lab for their critical comments and suggestions. This work was supported by NS38961 from the NIH. APB was supported by the UNC Neurodevelopmental Disorders Research Center Training Grant (5T32HD40127-02).

Funders	Funder number
Author National Institutes of Health National Institutes of Health National Institutes of Health National Institutes of Health The Bev Hartig Huntington's Disease Foundation National Institutes of Health
National Institute of Neurological Disorders and Stroke	R01NS038961
North Carolina Central University/University of North Carolina at Chapel Hill	5T32HD40127-02

Keywords

Actin cytoskeleton
Actin-binding domain
Cellular and Molecular Biology
Cytoskeleton Transport and membrane targeting
Mass spectrometry
Neurabin
Spinophilin

ASJC Scopus subject areas

Molecular Biology
Cellular and Molecular Neuroscience

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10.1016/j.molbrainres.2003.12.020

Cite this

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title = "The identification of a second actin-binding region in spinophilin/neurabin II",

abstract = "Spinophilin/neurabin II is an actin-associated scaffolding protein enriched in the dendritic spines of neurons. Previously, the actin-binding domain (ABD) of spinophilin was localized to a domain between amino acids (aa) 1 and 154. In a mass spectrometry screen for spinophilin-binding proteins, we have identified an additional actin-binding region between aa 151 and 282. F-actin co-sedimentation and GST affinity chromotography experiments further substantiate this result. Phalloidin staining of Rat2 fibroblasts transiently expressing GFP-spinophilin deletion constructs indicates co-localization with a subset of actin. Regions of spinophilin that lack the revised ABD (aa 1-230) do not co-localize with phalloidin-labeled actin, suggesting that the actin-binding domain contributes to directing the subcellular distribution of spinophilin. Targeting experiments using primary hippocampal cultures indicate that only the first actin-binding site contributes to dendritic spine localization. The second ABD targets to spines inefficiently and thus may interact with and affect actin filaments in a different manner than the first ABD.",

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author = "Barnes, \{Anthony P.\} and Smith, \{F. Donelson\} and VanDongen, \{Hendrika M.\} and VanDongen, \{Antonius M.J.\} and Milgram, \{Sharon L.\}",

note = "Funding Information: Mass spectrometry was performed in collaboration with Mary Moyer and William A. Burkhart in the Department of Structural Chemistry, Glaxo Wellcome, RTP, NC. We thank Mihir Patel and Leah Means for the technical assistance, Drs. Richard Cheney and Jonathan Berg for the helpful suggestions and discussions, and the members of the Milgram lab for their critical comments and suggestions. This work was supported by NS38961 from the NIH. APB was supported by the UNC Neurodevelopmental Disorders Research Center Training Grant (5T32HD40127-02).",

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AU - Milgram, Sharon L.

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N2 - Spinophilin/neurabin II is an actin-associated scaffolding protein enriched in the dendritic spines of neurons. Previously, the actin-binding domain (ABD) of spinophilin was localized to a domain between amino acids (aa) 1 and 154. In a mass spectrometry screen for spinophilin-binding proteins, we have identified an additional actin-binding region between aa 151 and 282. F-actin co-sedimentation and GST affinity chromotography experiments further substantiate this result. Phalloidin staining of Rat2 fibroblasts transiently expressing GFP-spinophilin deletion constructs indicates co-localization with a subset of actin. Regions of spinophilin that lack the revised ABD (aa 1-230) do not co-localize with phalloidin-labeled actin, suggesting that the actin-binding domain contributes to directing the subcellular distribution of spinophilin. Targeting experiments using primary hippocampal cultures indicate that only the first actin-binding site contributes to dendritic spine localization. The second ABD targets to spines inefficiently and thus may interact with and affect actin filaments in a different manner than the first ABD.

AB - Spinophilin/neurabin II is an actin-associated scaffolding protein enriched in the dendritic spines of neurons. Previously, the actin-binding domain (ABD) of spinophilin was localized to a domain between amino acids (aa) 1 and 154. In a mass spectrometry screen for spinophilin-binding proteins, we have identified an additional actin-binding region between aa 151 and 282. F-actin co-sedimentation and GST affinity chromotography experiments further substantiate this result. Phalloidin staining of Rat2 fibroblasts transiently expressing GFP-spinophilin deletion constructs indicates co-localization with a subset of actin. Regions of spinophilin that lack the revised ABD (aa 1-230) do not co-localize with phalloidin-labeled actin, suggesting that the actin-binding domain contributes to directing the subcellular distribution of spinophilin. Targeting experiments using primary hippocampal cultures indicate that only the first actin-binding site contributes to dendritic spine localization. The second ABD targets to spines inefficiently and thus may interact with and affect actin filaments in a different manner than the first ABD.

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KW - Cellular and Molecular Biology

KW - Cytoskeleton Transport and membrane targeting

KW - Mass spectrometry

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The identification of a second actin-binding region in spinophilin/neurabin II

Abstract

Funding

Keywords

ASJC Scopus subject areas

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