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First published online 1 March 2005
doi: 10.1242/jcs.01725

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Intracellular fate of LDL receptor family members depends on the cooperation between their ligand-binding and EGF domains

Department of Biochemical Physiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

View larger version (45K): [in a new window]   Fig. 1. Construction of a three-dimensional model of the structure of the LpR ectodomain from the third cysteine-rich repeat to the EGF-C module (black). The LpR structure is superimposed onto the X-ray crystal structure of the LDLR ectodomain from the second cysteine-rich repeat to the EGF-C module at pH 5.3 (grey); this reveals that His562 in LDLR corresponds to Asn643 in LpR. Enlargement of the frame shows the side chains of His190, His562 and His586 of LDLR (grey) in the interface between cysteine-rich repeats 4 and 5 of the ligand-binding domain and the ß-propeller of the EGF domain in LDLR, and the Asn643 residue in LpR (black). CR, cysteine-rich repeats.

View larger version (14K): [in a new window]   Fig. 2. Schematic models of wt LDLR, wt LpR, LpR1-790LDLR791-839, LpR1-342LDLR293-839, LDLR1-292LpR343-850, LDLRH562N, LDLRH562Y and LDLRH190Y; LDLR domains are depicted in grey and LpR domains in black. LpR1-790LDLR791-839 is composed of the ectodomain and transmembrane domain of LpR, and the intracellular tail of LDLR. LpR1-342LDLR293-839 harbours the ligand-binding domain of LpR and the region from the EGF domain to the C-terminus of LDLR. LDLR1-292LpR343-850 is composed of the ligand-binding domain of LDLR and the region from the EGF domain to the C-terminus of LpR. In the mutant LDLR receptors, His562 is substituted by Tyr (small white circle in the ß-propeller) or Asn (small black circle in the ß-propeller), or His190 is mutated to Tyr (small white square in the fifth cysteine-rich repeat). Squares, ligand-binding domain; diamonds, EGF repeats; circle, ß-propeller; oval, O-linked glycosylation domain; short rectangle, transmembrane domain; long rectangle, intracellular tail.

View larger version (27K): [in a new window]   Fig. 3. (A,B) Western-blot analysis of membrane proteins isolated from wt CHO, CHO(LpR), CHO(LpR1-790LDLR791-839) and ldlA(LpR1-342LDLR293-839) cells (lanes 1-4, respectively). The samples were subjected to SDS-PAGE under reducing (A) or non-reducing (B) conditions and blotted onto PVDF membrane. The wt LpR, LpR1-790LDLR791-839 and LpR1-342LDLR293-839 were detected with anti-LpR antibody 2189/90. (C) Membrane proteins of LDLR-deficient ldlA (lane 1), wt CHO (lanes 2 and 8), ldlA(LDLR) (lane 3), ldlA(LDLRH562Y) (lane 4), ldlA(LDLRH562N) (lane 5), ldlA(LDLR1-292LpR343-850) (lanes 6, 7) and CHO(LpR) cells (lane 9) were subjected to SDS-PAGE under non-reducing conditions and blotted onto PVDF membrane Lanes 1-6 were incubated with anti-LDLR antibody 121 and lanes 7-9 with anti-LpR antibody 9218. The size markers (in kDa) are indicated on the left (A-C) and right (C) of the blots.

View larger version (35K): [in a new window]   Fig. 4. Fluorescence-microscopic visualization of receptor-mediated endocytic uptake of fluorescently-labelled lipoprotein. Wild-type CHO (A), CHO(LpR) (B), CHO(LpR1-790LDLR791-839) (C), ldlA(LpR1-342LDLR293-839) (D) and ldlA(LDLR1-292LpR343-850) (E) cells were incubated with OG-Lp in incubation medium for 15 minutes at 37°C, fixed and analysed with fluorescence microscopy. Similarly, ldlA(LDLR) (F), ldlA(LDLR1-292LpR343-850) (G), ldlA(LpR1-342LDLR293-839) (H), ldlA(LDLRH562N) (I) and ldlA(LDLRH562Y) cells (J) were incubated with OG-LDL. The grey dots (B-D,F,G,I,J) represent endocytic vesicles containing fluorescently labelled lipoprotein. Scale bar, 10 µm (J).

View larger version (31K): [in a new window]   Fig. 5. Colocalization of lipoprotein receptors with Tf in the ERC. Incubation of CHO(LpR) (A), CHO(LpR1-790LDLR791-839) (B), ldlA(LpR1-342LDLR293-839) (C), ldlA(LDLR) (D), ldlA(LDLR1-292LpR343-850) (E), ldlA(LDLRH562N) (F) and ldlA(LDLRH562Y) transfectants (G) for 15 minutes with OG-Tf followed by a short chase of 10 minutes in growth medium shows that Tf accumulates in the ERC (left, grey). IF using anti-LpR antibody 2189/90 (A-C) or anti-LDLR antibody C7 (D-G) reveals that all receptors (middle, grey), except LpR1-342LDLR293-839 (C), also converge in this organelle and colocalize with Tf (right – Tf, green; receptor, red; colocalization, yellow). Scale bar, 10 µm (G, right).

View larger version (34K): [in a new window]   Fig. 6. Visualization of lipoprotein distribution after receptor-mediated endocytosis. CHO(LpR) (A), CHO(LpR1-790LDLR791-839) (B), ldlA(LpR1-342LDLR293-839) (C), ldlA(LDLR) (D), ldlA(LDLR1-292LpR343-850) (E), ldlA(LDLRH562N) (F) and ldlA(LDLRH562Y) transfectants (G) were preincubated for 15 minutes with OG-Lp (A-C) or OG-LDL (D-G), followed by a 60-minute chase in growth medium without ligand. After fixation, the receptors were detected with IF using anti-LpR antibody 2189/90 (A-C) or anti-LDLR antibody C7 (D-G). Fluorescence microscopy reveals that CHO(LpR) (A) and CHO(LpR1-790LDLR791-839) (B) transfectants have become depleted of lipoprotein (left), whereas the ligand remains visible in the other cell lines (C-G, grey). In contrast to wt LpR (A), LpR1-790LDLR791-839 (B) and wt LDLR (D), the other receptors are scattered throughout the cell and do not prominently converge in the ERC (middle, grey). Although they are more diffuse, the intracellular distributions of LpR1-342LDLR293-839 (C), LDLR1-292LpR343-850 (E), LDLRH562N (F) and LDLRH562Y (G) coincide with that of the internalized ligand (right – lipoprotein, green; receptor, red; colocalization, yellow). Scale bar, 10 µm (G, right).

View larger version (42K): [in a new window]   Fig. 7. Visualization of LDL uptake and receptor distribution when LDL is continuously present. In contrast to ldlA(LDLR), LDL uptake by ldlA(LDLRH562Y), ldlA(LDLRH562N) and ldlA(LDLR1-292LpR343-850) transfectants is reduced and receptors are not prominently localized to the ERC after prolonged LDL incubation. Transfectants were preincubated for 90 minutes in growth medium with unlabelled LDL, followed by a 15-minute pulse with OG-LDL; the receptor was stained for IF using anti-LDLR antibody C7. Whereas OG-LDL uptake by wt LDLR (A) is not visibly affected upon LDL preincubation, and ligand (left, grey) and receptor (middle, grey) do not colocalize (right – ligand, green; receptor, red; colocalization, yellow), OG-LDL endocytosis by ldlA(LDLR1-292LpR343-850) (B), ldlA(LDLRH562N) (C) and ldlA(LDLRH562Y) transfectants (D) is decreased, and the receptors are not prominently concentrated in the ERC. (E) LDLRH562Y appears scattered throughout the cell interior after a 105-minute incubation with unlabelled LDL, reducing receptor visibility to some extent (middle, grey). The receptor distribution is similar to that of the lysosomal marker (left, grey; right – LysoTracker, blue; receptor, red; colocalization, magenta). Scale bar, 10 µm (E, right).

View larger version (32K): [in a new window]   Fig. 8. Quantification of constitutive RRE (black) by counting the relative numbers of transfectants in which the receptor had perceptibly converged in the ERC after a short pulse of fluorescently labelled ligand. RRE after prolonged lipoprotein incubation (grey) was determined after a similar pulse preceded by a 90-minute preincubation of unlabelled ligand. ldlA(LDLR), ldlA(LDLR1-292LpR343-850), ldlA(LDLRH562N) and ldlA(LDLRH562Y) transfectants were incubated with LDL, and CHO(LpR), CHO(LpR1-790LDLR791-839) and ldlA(LpR1-342LDLR293-839) transfectants with Lp. Error bars show s.d. of each duplicate experiment (s.d. LpR1-790LDLR791-839 < 0.1%; LDLREGF, single experiment). The total number of cells counted for each data set is indicated at the bottom of each bar.

View larger version (24K): [in a new window]   Fig. 9. LDLRH190Y can recycle constitutively and mediates endocytic uptake of LDL in vitro. The ldlA(LDLRH190Y) transfectants were incubated with OG-LDL for 15 minutes (A), after which the receptor was visualized with anti-LDLR antibody C7 (B) following fixation. The arrows indicate the ERC in transfected cells (B). Scale bar, 10 µm (B).

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