IDENTIFICATION AND SUBCELLULAR-DISTRIBUTION OF THE G(I)-PROTEINS IN THE ENTEROCYTIC-DIFFERENTIATED ADENOCARCINOMA CELL-LINE, CACO-2

As evidenced by pertussis toxin-catalysed [P-32]ADP-ribosylation, immu noblotting and Northern blot, the human adenocarcinoma intestinal cell line Caco-2 expresses G(i2) and G(i3) proteins. The localization of t hese two G(i)s within the cell was investigated by using subcellular f ractionation and confocal microscopy on intact cell layer. A brush-bor der rich fraction and a pellet containing the remaining cellular membr anes were prepared. [P-32]ADP-ribosylation and immunoblotting demonstr ated the presence of both alpha(i2) and alpha(i3) in these two prepara tions. Immunofluorescence studies performed on intact cells grown on T ranswell filters and viewed by confocal microscopy further confirmed t he localization of alpha(i3)-subunit on basolateral as well as on apic al membranes. In contrast, alpha(i2)-subunit was shown to accumulate m ainly in the intra-cellular compartment while only faint staining of t he plasma membrane was detectable. Based upon double-labelling experim ents with antibody against rough endoplasmic reticulum (RER), there is a strong possibility that intra-cellular sites of alpha(i2)-subunit c orrespond to association with RER membranes.