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Identification and subcellular distribution of the G i ‐proteins in the enterocytic‐differentiated adenocarcinoma cell‐line, Caco‐2
Author(s) -
Lacombe Christiane,
Viallard Viviane,
Schaak Stéphane,
Paris Hervé
Publication year - 1996
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/j.1768-322x.1996.tb00986.x
Subject(s) - cell fractionation , biology , endoplasmic reticulum , microbiology and biotechnology , immunofluorescence , confocal microscopy , western blot , subcellular localization , cell culture , protein subunit , membrane , biochemistry , antibody , cytoplasm , immunology , genetics , gene
Summary— As evidenced by pertussis toxin‐catalysed [ 32 P]ADP‐ribosylation, immunoblotting and Northern blot, the human adenocarcinoma intestinal cell line Caco‐2 expresses G i2 and G i3 proteins. The localization of these two G i s within the cell was investigated by using subcellular fractionation and confocal microscopy on intact cell layer. A brush‐border rich fraction and a pellet containing the remaining cellular membranes were prepared. [ 32 P]ADP‐ribosylation and immunoblotting demonstrated the presence of both α i2 and α i3 in these two preparations. Immunofluorescence studies performed on intact cells grown on Transwell filters and viewed by confocal microscopy further confirmed the localization of α i3 ‐subunit on basolateral as well as on apical membranes. In contrast, α i2 ‐subunit was shown to accumulate mainly in the intra‐cellular compartment while only faint staining of the plasma membrane was detectable. Based upon double‐labelling experiments with antibody against rough endoplasmic reticulum (RER), there is a strong possibility that intra‐cellular sites of α i2 ‐subunit correspond to association with RER membranes.