GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface

Hiscox, Stephen Edward ORCID: https://orcid.org/0000-0003-0105-2702, Hallett, Maurice Bartlett ORCID: https://orcid.org/0000-0001-8197-834X, Morgan, Bryan Paul ORCID: https://orcid.org/0000-0003-4075-7676 and Van Den Berg, Carmen Wilma 2002. GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface. Biochemical and Biophysical Research Communications 293 (2) , pp. 714-721. 10.1016/S0006-291X(02)00280-2

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Abstract

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP–GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP–GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP–GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP–GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Medicine Pharmacy
Subjects:	Q Science > QP Physiology R Medicine > R Medicine (General)
Publisher:	Elsevier
ISSN:	0006-291X
Last Modified:	14 Nov 2022 07:45
URI:	https://orca.cardiff.ac.uk/id/eprint/66514

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