Expression of rat renal gamma-glutamyltranspeptidase in LLC-PK1 cells as a model for apical targeting.
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)
Altman
R A
RA
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Orr
A V
AV
Lagenaur
C F
CF
Curthoys
N P
NP
Hughey
R P
RP
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007163:Immunosorbent Techniques; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D051381:Rats; D013552:Swine; D014158:Transcription, Genetic; D014162:Transfection; D005723:gamma-Glutamyltransferase / Q000235:genetics*
D004247:DNA; D005723:gamma-Glutamyltransferase