Expression of rat renal .gamma.-glutamyltranspeptidase in LLC-PK1 cells as a model for apical targeting

Abstract

In the rat, gamma-glutamyltranspeptidase (gamma GT) 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 gamma GT 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 gamma GT was expressed in transfected cells as judged by immunofluorescence analysis, direct immunoprecipitation after metabolic labeling with [35S]methionine, and an increase in gamma GT specific enzymatic activity (up to 5-fold). When clonal cell lines (I or II) were grown on Falcon filter inserts, the increased gamma GT activity was found only at the apical surface, consistent with polarized expression of the rat gamma GT. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of cytochromeP-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys

“…Antibody against ␥-GT was kindly provided by Dr. R. P. Hughey, University of Pittsburgh Medical School. Western analysis for ␥-GT was performed as described previously (2).…”

γ‐glutamyltranspeptidase expression regulates the growth‐inhibitory activity of the anti‐tumor prodrug γ‐L‐glutaminyl‐4‐hydroxy‐3‐iodobenzene

“…Chinese hamster ovary cells were transfected with the expression vector pSFFVneo-GGTP (I) as previously described by Altman et al (1993). This vector includes the rat renal cDNA encoding the full-length y-glutamyltranspeptidase transcript I, downstream from the LTR of the Friend spleen focus-forming virus, and the neomycin-resistance gene with the SV40 early-region promoter.…”

Overexpression of the gamma‐glutamyltranspeptidase transgene does not alter the gamma irradiation sensitivity of the IB3–1 normal bronchoepithelial or A549 human lung carcinoma cell line

“…The rat renal cDNA encoding the full-length GGTP transcript I [15] was subcloned into the pRc/ CMVneo vector (Invitrogen Co., San Diego, CA). Human A549 lung carcinoma cells or IB3-1 cells [ 101 were transfected with pRc/CMVneo-GGTP by mixing 8 pg of linearized plasmid with 96 pg lipofectamine (Gibco BRL) in a final volume of 1.6 ml RPMI( -FCS) at room temperature for 15 min.…”

“…As a control for expression of GGTP, IB3-1 cells [lo] were transfected with a plasmid construct, pRK5-MnSOD, containing the human Mn-SOD gene [16]. IB3-1 cells were passaged into 6-well plates and when 80% confluent, were washed twice in serum-free Ham's F12 media, 1 ml of serum-free Ham's F12 containing 10 pg of lipofectamine, and 1.0 pg of pRc/CMVneo-GGTP [15], or 10 pg pRK5-MnSOD [17,18] was added and incubated at 37°C for 5 hr at which time 1 ml of Ham's F12 containing 20% FCS was added to each well. Beginning 48 hr later, the cells were grown in Ham's F12 media containing 200 pg/ml of G418 with subsequent G418 resistant colonies isolated and expanded.…”