An exogenous plasma membrane protein is polarized on the apical surface of the Retinal Pigment Epithelium (RPE) in vivo and in vitro

Purpose. To determine if an exogenous plasma membrane protein expresssed in RPE in vivo is polarized and if the reversal or loss of polarity observed in cultured RPE for Na,K ATPase, N-CAM, and RET-PE2 antigen extends to other apical proteins as well. Methods. The neurotrophin receptor p75-NTR was introduced into cultured RPE-J cells or adult rat RPE in vivo and in vitro by gene transfer with a replication defective adenovirus vector. The polarity and sorting of p75-NTR was examined by biotin polarity assays and laser scanning confocal microscopy (LSCM). Results. Introduction of p75-NTR in RPE in vivo was efficiently accomplished by sub-retinal injection. Inspection en face of whole mounted tissue indicated that nearly the entire RPE was expressing p75-NTR. Examination by LSCM of 10μm cryosections revealed that p75-NTR was localized to the apical surface of the RPE with little or no choroidal staining. Co-localization studies of p75-NTR and RET-PE2 antigen confirmed the apical distribution of p75-NTR. In RPE-J cells p75-NTR was found to be apical by both LSCM and steady state biotin polarity assays. Conclusion. This is the first demonstration that a polarized plasma membrane protein expressed in RPE in vivo via adenovirus mediated gene transfer is sorted to the appropriate cell surface. p75-NTR is apically polarized both in vivo and in vitro. This suggests that some apical proteins (ie. Na,K-ATPase) in RPE either aquire and retain apical polarity through stabilizing contacts with components of the cytoskeleton and/or interphotoreceptor matrix, or, that multiple pathways to the apical surface exist in vivo, at least one of which is altered or lost when RPE are removed from their native environment.