Identification of low density lipoprotein receptor-related protein-2/megalin as an endocytic receptor for seminal vesicle secretory protein II.

The low density lipoprotein receptor-related protein-2/megalin (LRP-2) is an endocytic receptor that is expressed on the apical surfaces of epithelial cells lining specific regions of the male and female reproductive tracts. In the present study, immunohistochemical staining revealed that LRP-2 is also expressed by epithelial cells lining the ductal region and the ampulla of the rat seminal vesicle. To identify LRP-2 ligands in the seminal vesicle, we probed seminal vesicle fluid with 125I-labeled LRP-2 in a gel-blot overlay assay. A 100-kDa protein (under non-reducing conditions) was found to bind the radiolabeled receptor. The protein was isolated and subjected to protease digestion, and the proteolytic fragments were subjected to mass spectroscopic sequence analysis. As a result, the 100-kDa protein was identified as the seminal vesicle secretory protein II (SVS-II), a major constituent of the seminal coagulum. Using purified preparations of SVS-II and LRP-2, solid-phase binding assays were used to show that the SVS-II bound to the receptor with high affinity (Kd = 5.6 nM). The binding of SVS-II to LRP-2 was inhibited using a known antagonist of LRP-2 function, the 39-kDa receptor-associated protein RAP. Using a series of recombinant subfragments of SVS-II, the LRP-2 binding site was mapped to a stretch of repeated 13-residue modules located in the central portion of the SVS-II polypeptide. To evaluate the ability of LRP-2 to mediate 125I-SVS-II endocytosis and lysosomal degradation, ligand clearance assays were performed using differentiated mouse F9 cells, which express high levels of LRP-2. Radiolabeled SVS-II was internalized and degraded by the cells, and both processes were inhibited by antibodies to LRP-2 or by RAP. The results indicate that LRP-2 binds SVS-II and can mediate its endocytosis leading to lysosomal degradation.

Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins.

Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.

Plasmid library for the transcription of RNA probes complementary to the entire genome of the human immunodeficiency virus type 1 (HIV-1).

A plasmid (pBH10R3) containing a 9-kb Sst I fragment of HIV-1 (clone BH-10) inserted in pSP64, an in vitro expression vector, has been used for the transcription of anti-sense HIV-1 RNA. With this system, the transcripts obtained in vitro were not usually full length (1 to 2 kb long) and they predominantly span the 3' end ORF and ENV regions of the viral genome. We have rearranged the HIV-1 genomic sequences with respect to the SP6 promoter in the pSP64 vector and have obtained a series of new constructs allowing the expression in vitro of RNA transcripts complementary to other regions in the HIV-1 genome, including the 5' end of the ENV region as well as the TAT, POL, and GAG regions. In fact, the combined use of these constructs as templates for in vitro transcription allows the production of RNA probes spanning the entire viral genome. Compared with the 1- to 2-kb probes mentioned above, the combined use of such probes results in a several-fold increase in the sensitivity of molecular hybridization for the detection of HIV-1 nucleic acid sequences. Also, these constructs enable the preparation of RNA probes that have the potential to detect restriction polymorphisms throughout the HIV-1 genome.

Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility.

Fibulin-1 is an extracellular matrix protein often associated with fibronectin (FN) in vivo. In this study, the ability of fibulin-1 to modulate adhesion, spreading and motility-promoting activities of FN was investigated. Fibulin-1 was found to have pronounced inhibitory effects on the cell attachment and spreading promoted by FN. Fibulin-1 was also found to inhibit the motility of a variety of cell types on FN substrata. For example, the FN-dependent haptotactic motility of breast carcinoma (MDA MB231) cells, epidermal carcinoma (A431), melanoma (A375 SM), rat pulmonary aortic smooth muscle cells (PAC1) and Chinese hamster ovary (CHO) cells was inhibited by the presence of fibulin-1 bound to FN-coated Boyden chamber membranes. Cells transfected to overproduce fibulin-1 displayed reduced velocity, distance of movement and persistence time on FN substrata. Similarly, the incorporation of fibulin-1 into FN-containing type I collagen gels inhibited the invasion of endocardial cushion mesenchymal cells migrating from cultured embryonic heart explants. By contrast, incorporation of fibulin-1 into collagen gels lacking FN had no effect on the migration of endocardial cushion cells. These results suggest that the motility-suppressive effects of fibulin-1 might be FN specific. Furthermore, such effects are cell-type specific, in that the migration of gingival fibroblasts and endothelial cells on FN substrata is not responsive to fibulin-1. Additional studies found that the mechanism for the motility-suppressive effects of fibulin-1 does not involve perturbations of interactions between alpha5beta1 or alpha4 integrins, or heparan sulfate proteoglycans with FN. However, fibulin-1 was found to inhibit extracellular signal regulated kinase (ERK) activation and to suppress phosphorylation of myosin heavy chain. This ability to influence signal transduction cascades that modulate the actin-myosin motor complex might be the basis for the effects of fibulin-1 on adhesion and motility.