ABSTRACT
The primary structure of polycystin predicts a large integral membrane protein with multiple cell recognition motifs, but its function remains unknown. Insight into polycystin's normal function and its role in the development of autosomal dominant polycystic kidney disease (PKD1) requires the assembly of an extensive collection of molecular reagents to examine its expression and create model systems for functional studies. Development of these crucial reagents has been complicated due to the presence of transcriptionally active homologous loci. We have assembled the authentic full-length PKD1 cDNA and demonstrated expression of polycystin in vitro. Polyclonal antibodies directed against distinct extra- and intracellular domains specifically immunoprecipitated in vitro translated polycystin. The panel of antibodies was used to determine localization of polycystin in renal epithelial and endothelial cell lines and tissues of fetal, adult, and cystic origins. In normal adult kidney and maturing fetal nephrons, polycystin expression was confined to epithelial cells of the distal nephron and vascular endothelial cells. Expression in the proximal nephron was only observed after injury-induced cell proliferation. Polycystin expression was confined to ductal epithelium in liver, pancreas, and breast, and restricted to astrocytes in normal brain. We report clear evidence for the membrane localization of polycystin by both tissue sections and by confocal microscopy in cultured renal and endothelial cells. Interestingly, when cultured cells made cell-cell contact, polycystin was localized to the lateral membranes of cells in contact. These data suggest that polycystin is likely to have a widespread role in epithelial cell differentiation and maturation and in cell-cell interactions.
Subject(s)
Kidney/metabolism , Protein Biosynthesis , Adult , Brain/embryology , Brain/metabolism , Cell Line , Cells, Cultured , DNA, Complementary , Endothelium, Vascular/metabolism , Epithelium/metabolism , Fetus , Gene Library , Humans , Nephrons/embryology , Nephrons/metabolism , Organ Specificity , Polycystic Kidney, Autosomal Dominant , Polymerase Chain Reaction , Proteins/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Subcellular Fractions/metabolism , TRPP Cation ChannelsABSTRACT
The ATP binding cassette (ABC) transporters, or traffic ATPases, constitute a large family of proteins responsible for the transport of a wide variety of substrates across cell membranes in both prokaryotic and eukaryotic cells. We describe a human ABC protein with regions of strong homology to the recently described murine ABC1 and ABC2 transporters. The gene for this novel protein, human ABC3, maps near the polycystic kidney disease type 1 (PKD1) gene on chromosome 16p13.3. The ABC3 gene is expressed at highest levels in lung compared to other tissues.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Chromosome Mapping , Chromosomes, Human, Pair 16 , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Animals , Gene Expression , Humans , Lung/metabolism , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
A pyrimidine-rich element (PyRE), present in the 21st intron of the PKD1 gene, posed a significant obstacle in determining the primary structure of the gene. Only cycle sequencing of nested, single-stranded phage templates of the CT-rich strand enabled complete and accurate sequence data. Similar attempts on the GA-rich strand were unsuccessful. The resulting primary structure showed the 3 kb 21st intron to contain a 2.5 kb PyRE, whose sense-strand is 97% C + T. The PKD1 PyRE does not appear to be polymorphic based on RFLP analysis of DNA from 6 unrelated individuals digested with 9 different restriction enzymes. This is the largest pyrimidine tract sequenced to date, being over twice as large as those previously identified and shows little homology to other polypyrimidine tracts. Additional analysis of this PyRE revealed the presence of 23 mirror repeats with stem lengths of at least 10 nucleotides. The 23 H-DNA-forming sequences in the PKD1 PyRE exceed the cumulative total of 22 found in 157 human genes that have been completely sequenced. The mirror repeats confer this region of the PKD1 gene with a strong probability of forming H-DNA or triplex structures under appropriate conditions. Based on studies with PyRE found in other eukaryotic genes, the PKD1 PyRE may play a role in regulating PKD1 expression, and its potential for forming an extended triplex structure may explain some of the observed instability in the PKD1 locus.
Subject(s)
DNA/genetics , Proteins/genetics , Repetitive Sequences, Nucleic Acid/genetics , Base Sequence , Humans , Introns , Molecular Sequence Data , Polycystic Kidney, Autosomal Dominant/genetics , Polymorphism, Restriction Fragment Length , TRPP Cation ChannelsABSTRACT
The complete genomic sequence of the gene responsible for the predominant form of polycystic kidney disease, PKD1, was determined to provide a framework for understanding the biology and evolution of the gene, and to aid in the development of molecular diagnostics. The DNA sequence of a 54 kb interval immediately upstream of the poly(A) addition signal sequence of the PKD1 transcript was determined, and then analyzed using computer methods. A leucine-rich repeat (LRR) motif was identified within the resulting predicted protein sequence of the PKD1 gene. By analogy with other LRR-containing proteins, this may explain some of the disease-related renal alterations such as mislocalization of membrane protein constituents and changes in the extracellular matrix organization. Finally, comparison of the genomic sequence and the published partial cDNA sequence showed several differences between the two sequences. The most significant difference detected predicts a novel carboxy-terminus for the PKD1 gene product.
