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Immortalized rat proximal tubule cells produce membrane bound and soluble megalin
F F Jung1, D R Bachinsky, S S Tang
1Pediatric Renal Research Laboratory, Massachusetts General Hospital, Boston 02114, USA.
Abstract:
Megalin (gp330), a glycoprotein receptor found on renal proximal tubule cells and several other epithelial cells, is deduced to be a type I integral membrane protein, but may also exist as a cell surface form lacking a cytoplasmic domain. Furthermore, soluble megalin products have been detected in urine, and in culture medium of a rat yolk sac carcinoma cell line, combined with receptor associated protein (RAP). Permanent renal cell lines expressing megalin were unavailable until the recent description of two immortalized rat proximal tubule cell lines (IRPTC). The present study demonstrated megalin on IRPTC surface by immunofluorescence, without surface staining for RAP, which was, however, readily detected within cells. Antibodies to ectodomain megalin epitopes immunoprecipitated megalin products both from cell lysates and culture medium, whereas antibodies to cytoplasmic domain epitopes precipitated megalin only from lysates. Western blots showed two major megalin products in medium, a prominent band at approximately 200 kDa, and a fainter band above 400 kDa, slightly below intact megalin in cell lysates. Anti-receptor associated protein antibodies immunoprecipitated megalin from IRPTC lysates, but not from media. We propose that portions of megalin are spontaneously produced by IRPTC, probably either by cleavage in the ectodomain or release of forms lacking a cytoplasmic domain.
Insights
Megalin, a renal cell receptor, is shed from immortalized proximal tubule cells. These cells release soluble megalin fragments into their culture medium, indicating spontaneous production.
Area of Science:
- Biochemistry
- Cell Biology
- Nephrology
Background:
- Megalin (gp330) is a type I integral membrane glycoprotein receptor.
- It is expressed on renal proximal tubule cells and other epithelial cells.
- Soluble megalin and its binding protein, RAP, are found in urine and cell culture media.
Purpose of the Study:
- To investigate megalin expression and shedding in immortalized rat proximal tubule cell lines (IRPTC).
- To characterize the forms of megalin released by these cells.
Main Methods:
- Immunofluorescence staining for megalin and RAP on IRPTC.
- Immunoprecipitation using antibodies against megalin ectodomain and cytoplasmic domain epitopes.
- Western blot analysis of megalin products in cell lysates and culture medium.
- Immunoprecipitation of megalin with anti-RAP antibodies.
Main Results:
- Megalin was detected on the IRPTC surface, while RAP was intracellular.
- Antibodies to the megalin ectodomain precipitated megalin from both cell lysates and medium.
- Antibodies to the cytoplasmic domain precipitated megalin only from lysates.
- Western blots revealed two major megalin products in the medium (~200 kDa and >400 kDa).
- Anti-RAP antibodies immunoprecipitated megalin from lysates but not from medium.
Conclusions:
- Immortalized rat proximal tubule cells spontaneously produce and release soluble megalin fragments.
- These fragments are likely generated by ectodomain cleavage or release of forms lacking a cytoplasmic domain.
- This shedding mechanism may contribute to soluble megalin found in vivo.