The identification of a second actin-binding region in spinophilin/neurabin II.
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.
Barnes
Anthony P
AP
Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27707, USA. abarnes@med.unc.edu
Smith
F Donelson
FD
VanDongen
Hendrika M
HM
VanDongen
Antonius M J
AM
Milgram
Sharon L
SL
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002451:Cell Compartmentation / Q000502:physiology; D002460:Cell Line; D003712:Dendrites / Q000378:metabolism* / Q000648:ultrastructure; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D000099273:Neurabins
D000199:Actins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D000099273:Neurabins