EXPRESSION OF RAT RENAL GAMMA-GLUTAMYL-TRANSPEPTIDASE IN LLC-PK(1) CELLS AS A MODEL FOR APICAL TARGETING

In the rat, gamma-glutamyltranspeptidase (gammaGT) is transcribed into four unique mRNAs from a single gene by use of at least three different promoters and alternative splicing. For the first time, two distinct full-length cDNAs encoding the protein for rat renal gamma-glutamyltranspeptidase have now been isolated. Characterization by restriction enzyme mapping and nucleotide sequencing indicates that the two cDNAs, corresponding to transcripts I and II, differ only in the 5' noncoding region. However, transcription from promoter I, most proximal to the coding sequence, apparently began 20 bases upstream from the major transcription start previously reported. Since in vitro transcription and translation of these two new gammaGT cDNAs were found to produce a full-length peptide (M(r) approximately 62 000), both cDNAs were used to transfect LLC-PK1 (porcine) cells, a polarized cell line most representative of the renal proximal tubule. Rat gammaGT was expressed in transfected cells as judged by immunofluorescence analysis, direct immunoprecipitation after metabolic labeling with [S-35]methionine, and an increase in gammaGT specific enzymatic activity (up to 5-fold). When clonal cell lines (I or II) were grown on Falcon filter inserts, the increased gammaGT activity was found only at the apical surface, consistent with polarized expression of the rat gammaGT. In contrast, transfection of the same cells with cDNA of human growth hormone resulted in both apical (70%) and basal lateral (30%) secretion of the expressed hormone. Altogether the data indicate that transfection of LLC-PK1 cells with rat gammaGT and its derivatives should provide an alternative model system in which to characterize the cellular mechanisms involved in apical targeting.