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First published online 4 December 2002
doi: 10.1242/jcs.00243

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Functional interaction of megalin with the megalinbinding protein (MegBP), a novel tetratrico peptide repeat-containing adaptor molecule

¹ Max-Delbrueck-Center for Molecular Medicine and ²Medical Faculty of the Free University of Berlin, Germany
² Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark
³ Center for Human Genetics, K. U. Leuven and Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium

View larger version (38K): [in a new window]   Fig. 1. Sequence and structural analysis of MegBP. (A) Amino-acid sequence of a partial MegBP clone isolated from a human brain library by yeast two-hybrid screening. (B) Alignment of the amino-acid sequences of human and murine MegBP assembled from ESTs. The arrow indicates the beginning of the clone identified by the yeast two-hybrid screen. Potential sites for phosphorylation by protein kinase C (*), casein kinase II (•) and tyrosine kinase () are highlighted. (C) Schematic presentation of two tetratrico peptide repeats (TPR-1, amino acids 58-91; and TPR-2, amino acids 313-333) found in MegBP (NCBI database).

View larger version (33K): [in a new window]   Fig. 2. Ligand blot analysis of the megalin tail binding to MegBP and Dab1. 10 µg of purified GST-MegBP (lanes 1, 4 and 7), GST (lanes 2, 5 and 8) and GST-Dab1 (lanes 3, 6 and 9) were subjected to reducing 10% SDS-PAGE and stained with Coomassie blue (lanes 1-3) or transferred to a nitrocellulose membrane and incubated with an anti-GST antibody (lanes 4-6) or 125I-His-megalin tail (lanes 7-9) as described in Materials and Methods. Binding of the 125I-His-megalin tail to GST-MegBP and GST-Dab1, but not to GST or GST-MegBP degradation products, was detected by autoradiography.

View larger version (20K): [in a new window]   Fig. 3. Coprecipitation of GST-MegBP and megalin from mouse renal extracts. Mouse renal membrane extracts were incubated with anti-glutathione IgG in the presence (lane 2) or absence of GST-MegBP (lane 3). Proteins bound to IgG were recovered as detailed in the Materials and Methods section and subjected to non-reducing 4-12% SDS-PAGE and immunoblot analysis using a goat anti-megalin antibody and enhanced chemoluminescence. As a positive control, 2 µg of renal membrane preparations were run in parallel (lane 1). Endogenous mouse megalin was recovered in the presence but not in the absence of GST-MegBP. The arrow indicates full-length megalin. The lower molecular weight band corresponds to a megalin degradation product often observed in membrane preparations.

View larger version (35K): [in a new window]   Fig. 4. BIAcore analysis of megalin and adaptor interaction. Immobilized rabbit megalin (A,C) or human megalin tail protein (B,D) were incubated with the indicated concentrations of purified GST-MegBP (A,B) or GST-Dab1 (C,D). Binding to the receptor polypeptide was detected by surface plasmon resonance signal (BIAcore) and indicated in response units. As a control, binding of 1 µM GST to the megalin tail was tested (B,D). From the kinetic data, the Kd values for the interactions were calculated as 1.1 µM (MegBP to megalin), 0.8 µM (MegBP to the megalin tail), 0.25 µM (Dab1 to megalin) and 0.15 µM (Dab1 to the megalin tail).

View larger version (10K): [in a new window]   Fig. 5. Mapping of the MegBP-binding site on the megalin tail. Truncations of the human megalin tail sequence were PCR-cloned as bait and tested for interaction with MegBP in the yeast two-hybrid system. The extension of various receptor tail fragments (NM_004525) and their interaction with MegBP are indicated. For clarity, the localization of NPxY, (S/T)xY and PxxP elements in the receptor tail are shown.

View larger version (44K): [in a new window]   Fig. 6. Expression of EGFP and EGFP-MegBP in mammalian cell lines. The indicated mammalian cell lines were transiently transfected with expression constructs encoding EGFP or EGFP-MegBP. Expression of the proteins was detected 24-48 hours later by fluorescence microscopy.

View larger version (26K): [in a new window]   Fig. 7. Expression of megalin in mammalian cell lines. Purified membrane proteins from the indicated cell lines (20 µg/lane) or rabbit kidney (5 µg/lane) were subjected to non-reducing 4-15% SDS-PAGE and immunoblot analysis using a goat anti-megalin antibody and enhanced chemoluminescence.

View larger version (48K): [in a new window]   Fig. 8. Localization of megalin and MegBP in BN16 cells. BN16 cells were transiently transfected with the EGFP-MegBP expression constructs. Four hours later, the cells were fixed and incubated with a goat anti-rabbit megalin antibody followed by Cy5-labeled rabbit anti-goat IgG. Subcellular localization of megalin (A) and EGFP-MegBP (B) was detected by confocal fluorescence microscopy. Micrographs in A and B were merged to produce panel C. Colocalization of both proteins is indicated in yellow.

View larger version (111K): [in a new window]   Fig. 9. Endocytic uptake of GST-DsRed-RAP in BN16 cells. (A,B) Replicate monolayers of BN16 cells were incubated for 2 hours on ice with phenol-red-free DMEM containing 30 µg/ml of purified GST-DsRed-RAP. Then, the cells were shifted to 37°C and the subcellular localization of GST-DsRed-RAP tested by confocal fluorescence microscopy. At 4°C, all of the GST-DsRed-RAP is bound to the cell surface (A). Internalization of the ligand is seen 20 minutes (B) after shift to 37°C. (C,D) BN16 cells were transiently transfected with an EGFP- MegBP expression construct and incubated in DMEM containing 30 µg/ml of purified GST-DsRed-RAP. After 4 hours, the subcellular localization of EGFP- MegBP and DsRed-GST-RAP was analyzed by confocal fluorescence microscopy. Examples for endocytic uptake of GST-DsRed-RAP in MegBP expressing cells are shown.

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