Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Mar 20;380(4):825-31.
doi: 10.1016/j.bbrc.2009.01.177. Epub 2009 Feb 4.

Membrane Targeting of the EF-hand Containing Calcium-Sensing Proteins CaBP7 and CaBP8

Affiliations
Free PMC article

Membrane Targeting of the EF-hand Containing Calcium-Sensing Proteins CaBP7 and CaBP8

Hannah V McCue et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

The CaBP family of EF-hand containing small Ca(2+)-binding proteins have recently emerged as important regulators of multiple targets essential to normal neuronal function in the mammalian central nervous system. Of particular interest are CaBP7 and CaBP8, abundantly expressed brain proteins that exhibit the greatest sequence divergence from other family members. In this study, we have analysed their sub-cellular localisations in a model neuronal (Neuro2A) cell line and show that both proteins exhibit a membrane distribution distinct from the other CaBPs and consistent with localisation to the trans-Golgi network (TGN). Furthermore, we show that their localisation to the TGN critically depends upon an unusual predicted C-terminal transmembrane domain that if deleted or disrupted has dramatic consequences for protein targeting. CaBP7 and 8, therefore, possess a targeting mechanism that is unique amongst the CaBPs that may contribute to differential functional Ca(2+)-sensing by these family members.

Figures

Fig. 1
Fig. 1
Intracellular distribution of fluorescently tagged human CaBP isoforms. The indicated constructs were used to determine the localisation of CaBPs expressed in retinoic acid differentiated N2A cells. The scale bar represents 10 μm.
Fig. 2
Fig. 2
Co-localisation of CaBP7 and 8 with TGN markers in differentiated N2A cells. Localisation of CaBP7 and 8 (red) with an EYFP tagged cis/medial Golgi marker (A,B, green) or the TGN specific markers syntaxin 6 (C,D, green) and ts045 VSVG-GFP (E,F, green) was examined. Regions of co-localisation appear yellow in the merged images. The scale bar represents 10 μm. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
Fig. 3
Fig. 3
Analysis of the function of the putative TMD of CaBP7 and 8 TMD in differentiated N2A cells. Cells were transfected with ts045 VSVG-GFP (green) as a TGN marker and either CaBP71–188 or CaBP81–192 TMD deletion mutants (A,B, red); CaBP7TMD–KKK or CaBP8TMD–KKK TMD triple lysine mutants (C,D, red). Co-localisation appears yellow in merged images. The scale bar represents 10 μm. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
Fig. 4
Fig. 4
Analysis of CaBP5 and mCherry chimaeric constructs in retinoic acid differentiated N2A cells and effect of the position of the colour tag in CaBP7 and 8. The figure shows CaBP5CaBP7TMD or CaBP5CaBP8TMD chimaeras (A,B, red), or mCherryCaBP7TMD (C, red) co-expressed with ts045 VSVG-GFP (green) as a TGN marker. C-terminally mCherry tagged CaBP7 and 8 (red) were co-expressed with N-terminally EYFP tagged variants of the same proteins (green, D,E). Regions of co-localisation appear yellow in merged images. The scale bar represents 10 μm. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles

References

    1. Bootman M.D., Collins T.J., Peppiatt C.M., Prothero L.S., MacKenzie L., De Smet P., Travers M., Tovey S.C., Seo J.T., Berridge M.J., Ciccolini F., Lipp P. Calcium signalling – an overview. Semin. Cell Dev. Biol. 2001;12:3–10. - PubMed
    1. Miller R.J. Calcium signalling in neurons. Trends Neurosci. 1988;11:415–419. - PubMed
    1. Dash P.K., Moore A.N., Kobori N., Runyan J.D. Molecular activity underlying working memory. Learn Mem. 2007;14:554–563. - PubMed
    1. Burgoyne R.D. Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling. Nat. Rev. Neurosci. 2007;8:182–193. - PMC - PubMed
    1. Haeseleer F., Sokal I., Verlinde C.L., Erdjument-Bromage H., Tempst P., Pronin A.N., Benovic J.L., Fariss R.N., Palczewski K. Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin. J. Biol. Chem. 2000;275:1247–1260. - PMC - PubMed

Publication types

MeSH terms

Feedback