Abstract. The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the ctl-chain of collagen IV (ctl [IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the otl(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for cd(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding. BASEMENT membrane is a thin extracellular matrix that forms soon after cytodifferentiation on the basal surface of epithelial and endothelial cells, and on all surfaces of muscle cells (20). Current evidence using components derived from Englebreth-Holm-Swarm basement membrane suggests that this sheet is formed by the coassembly of its main components (8, 28) particularly collagen IV, a triple helical filament of two otl-chains and one c~2-chain (48); laminin, a cruciform-shaped glycoprotein (13) composed ofa B1, B2, and A chain; heparan sulfate proteoglycan, consisting of one chain with two to three glycosaminoglycans attached to one end (29, 38); and entactin (nidogen), a dumbbell-shaped molecule formed of one chain (6, 48).Various functions have been attributed to parts of these molecules (1, 48), for example, certain laminin regions are involved in interactions with collagen IV, heparan sulfate proteoglycan, and entactin (nidogen). Also, cells bind to laminin, collagen IV (48), and heparan sulfate proteoglycan (8a) through specific cell surface receptors. Basement membrane molecules through cell receptors thereby influence cell adhesion, migration, differentiation, and growth (26).The appearance of basement membrane molecules appear to represent key events in the differentiation of cells of the kidney (12). During nephrogenesis, undifferentiated cortical mesenchymal cells are locally induced by branching ureter bud epithelium to aggregate and transform into epithelial comma-and S-shaped bodies through the acquisition of indentations. S-shaped bodies give rise to nephrons consisting of glomeruli, proximal and distal tubules, ...
Abstract. The interaction of hepatocytes with the basement membrane glycoprotein laminin was studied using synthetic peptides derived from laminin sequences. Rat hepatocytes bind to laminin and three different sites within the A and B1 chains of laminin were identified. Active laminin peptides include the PA22-2 peptide (close to the carboxyl end of the long arm in the A chain), the RGD-containing peptide, PA21 (in the short arm of the A chain) and the pentapeptide YIGSR (in the short arm of the B1 chain). PA22-2 was the most potent peptide, whereas the other two peptides had somewhat lower activity. Furthermore, hepatocyte attachment to laminin was inhibited by the three peptides, with PA22-2 being the most active. Various proteins from isolated membranes of cell-surface iodinated hepatocytes bound to a laminin affinity column including three immunologically related binding proteins : M~ = 67,000, 45,000, and 32,000. Several proteins-M~ = 80,000, 55,000, and 38,000-36,000-with a lower affinity for laminin were also identified. Affinity chromatography on peptide columns revealed that the PA22-2 peptide specifically bound the Mr = 80000, 67,000, 45,000, and 32,000 proteins, the PA21 peptide bound the Mr = 45,000 and 38,000-36,000 proteins and the YIGSR peptide column bound the 38,000-36,000 protein. Antisera to a bacterial fusion protein of the 32-kD laminin-binding protein (LBP-32) reacted strongly with the three laminin-binding proteins, M~ = 67000, 45,000, and 32,000, showing that they are immunologically related. Immunoperoxidase microscopy studies confirmed that these proteins are present within the plasma membrane of the hepatocyte. The antisera inhibited the adhesion of hepatocytes to laminin by 30%, supporting the finding that these receptors and others mediate the attachment of hepatocytes to several regions of laminin.
Transport of aminopeptidase I (API) to the vacuole appears to be insensitive to blockage of the secretory pathway. Here we show that the N‐terminal extension of the 61 kDa precursor of API (pAPI) is proteolytically processed in two sequential steps. The first step involves proteinase A (PrA) and produces a 55 kDa unstable intermediate (iAPI). The second step involves proteinase B (PrB) and converts iAPI into the 50 kDa stable, mature enzyme (mAPI). Reversion of the cup1 growth phenotype by a pAPI‐CUP1 chimera indicates that pAPI is transported to the vacuole by a post‐translational mechanism. Deletion of the first 16 amino acids results in accumulation of the truncated protein in the cytosol, indicating that pAPI is actively transported to the vacuole. The chimera pAPI‐myc, constructed by fusing a myc tag to the C‐terminus of pAPI, was exploited to dissect the mechanism of pAPI transport. Cell fractionation studies show the presence of iAPI‐myc and mAPI in a fraction of vacuoles purified by density centrifugation. This and the sequential conversion of pAPI‐myc into iAPI‐myc and mAPI lacking the myc tag is consistent with insertion of pAPI into the vacuolar membrane through its N‐terminal extension. The specific mechanism of API sorting demonstrates a new pathway of protein transport in vacuolar biogenesis.
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