EVIDENCE FOR BETA(1)-INTEGRINS ON BOTH APICAL AND BASAL SURFACES OF XENOPUS RETINAL-PIGMENT EPITHELIUM

The retinal pigment epithelium (RPE) is a transporting epithelium with polarized membrane domains. A unique characteristic of these cells is that their apical surface does not face a lumenal space, but is direc tly apposed to a layer of neurons (photoreceptors) and their associate d extracellular matrix. Because the interaction occurring at this site is important for retinal attachment and particle phagocytosis, an att empt was made to identify epithelial molecules which potentially could mediate cell-cell or cell-matrix adhesion. In the present report, the subcellular localization of beta(1)-integrins, the main receptors for extracellular matrix ligands, has been examined within Xenopus RPE. S everal previously characterized antibodies were used in this analysis including: two rabbit polyclonal antibodies directed against purified chick muscle fibronectin receptor (pAbs No. 3818 and No. 2999), and a monoclonal antibody specific for Xenopus beta(1)-integrin subunit (mAb 8C8). In Western blots of whole epithelial cell extracts, each of the antibodies intensely labeled a 115 kDa band, consistent with beta(1)- integrin reactivity. One of the reagents (pAb No. 3818) also weakly st ained unidentified bands of 50 and 100 kDa. Pre-clearing experiments d emonstrated that pAb No. 3818 and mAb 8C8 both recognize the same dete rgent-soluble integrin: when cell extracts were depleted of beta(1)-in tegrin by immunoprecipitation with mAb 8C8, the 115 kDa antigen recogn ized by pAb No. 3818 was not observed. Consistent with their similar i mmunochemical reactivities, each of the antibodies produced equivalent immunocytochemical staining of many eyecup tissues, including extraoc ular skeletal muscle cells, scleral and choroidal fibroblasts and vasc ular endothelium of the choroid and neural retina. In the native RPE, and isolated sheets of epithelium, however, qualitative differences in labeling between these antibodies were evident. Analysis by confocal microscopy showed that, while all three antibodies stained the basal s urface of the epithelium, pAb No. 3818 also strongly labeled the apica l microvillar surface. As the adjacent photoreceptors did not crossrea ct with this antibody in control experiments, the apical RPE staining could not be accounted for as contamination with retinal tissues durin g isolation. Furthermore, when the apical cell surface was selectively biotinylated in situ, and biotinylated proteins precipitated by strep tavidin-agarose, beta(1)-integrin was detected by immunoblotting with both mAb 8C8 and pAb No. 3818. This domain-specific material, however, represented only a fraction of the whole cell surface integrin: subst antially greater amounts of tagged molecules could be detected when is olated epithelial sheets were biotinylated, most likely representing t he basal protein. Based on these results, it can be concluded that bet a(1)-integrin is present in both basal and apical RPE plasma membranes . Molecules present in the apical membrane may represent components of adhesion receptors responsible for retina-epithelium interactions. (C ) 1997 Academic Press Limited