Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180

Abstract

Endo180, also known as the urokinase plasminogen activator receptor (uPAR)-associated protein (uPARAP), is one of the four members of the mannose receptor family, and is implicated in extracellular-matrix remodelling through its interactions with collagens, sugars and uPAR. The extracellular portion of Endo180 contains an amino-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. We have purified a soluble version of Endo180 and analysed it by single-particle electron microscopy to obtain a three-dimensional structure of the N-terminal part of the protein at a resolution of 17 A and reveal, for the first time, the interactions between non-adjacent domains in the mannose receptor family. We show that for Endo180, the cysteine-rich domain contacts the second C-type lectin-like domain, thus providing structural insight into how modulation of its several ligand interactions may regulate Endo180 receptor function.

Figure 1

Purification of the Endo180 extracellular domain. (A) Domain structure of Endo180. The arrangement of the extracellular domains reflects the model proposed for the mannose receptor (Napper et al., 2001). (B) Purified sEndo180–HA–His₆ (a soluble Endo180 extracellular-domain construct that contains a carboxy-terminal haemagglutinin (HA) tag and a six-histidine (His₆) tag) was immunoblotted using the monclonal antibody E1/183 (directed against the human Endo180 cysteine-rich domain) or the anti-HA monoclonal 3F10, or was silver stained. (C) Purified sEndo180–HA–His₆ protein was loaded onto a GlcNAc–sepharose column. The column was washed eight times with 1 ml of loading buffer (containing 25 mM Ca²⁺) and then eight times with 1 ml of elution buffer (containing 10 mM EDTA). Fractions were precipitated and analysed by immunoblotting with the anti-Endo180 monoclonal antibody A5/158. CTLD, C-type lectin-like domain; Cys, cysteine-rich domain; cyto, cytoplasmic domain; FNII, fibronectin type II domain.

Figure 2

Electron microscopy of purified Endo180. (A) Micrograph. Particles of sEndo180–HA–His₆ (a soluble Endo180 extracellular-domain construct that contains a carboxy-terminal haemagglutinin (HA) tag and a six-histidine (His₆) tag) are visible in negative contrast. (B) Extracted images after contrast inversion. (C) Classes obtained from the input images. Rows 1b, 2b and 3b are averages of the images of a given class. Images in rows 1a, 2a and 3a are the corresponding projections of the final volume. (D) Resolution estimation by Fourier shell correlation. (E) Angular coverage of Endo180 particles. The number of particles (y axis) for each class average (x axis) are shown. The average images of four of the most highly populated classes are shown (insets). The numbers at the bottom right-hand corners of the images indicate the class number.

Figure 3

Endo180 volume. (A–C) Views of the reconstructed volume for sEndo180–HA–His₆ (a soluble Endo180 extracellular-domain construct that contains a carboxy-terminal haemagglutinin (HA) tag and a six-histidine (His₆) tag). CTLD, C-type lectin-like domain; Cys, cysteine-rich domain; FNII, fibronectin type II domain. Scale bar, 32 Å.

Figure 4

The Endo180ΔCTLD3–8–Fc construct. (A) The Endo180ΔCTLD3–8–Fc protein. (B) Left and middle panels show immunoblotting of purified Endo180ΔCTLD3–8–Fc protein with the anti-Endo180 monoclonal antibody E1/183 and an anti-human-Fc monoclonal antibody. Right panel shows a silver-stained SDS–polyacrylamide gel containing the purified material. Note that the size of the protein on the non-reducing SDS gel reflects Fc-mediated dimerization. (C) Images obtained for the construct using an electron microscope. (D) Views of the reconstructed volume. CTLD, C-type lectin-like domain; Cys, cysteine-rich domain; FNII, fibronectin type II domain.

Figure 5

Fitting the atomic structures into the Endo180 electron microscopic model. (A) Tube representation of each domain. (B) Fitting the atomic structures shown in (A) into the electron microscopic volume for sEndo180–HA–His₆ (a soluble Endo180 extracellular-domain construct that contains a carboxy-terminal haemagglutinin (HA) tag and a six-histidine (His₆) tag; shown as a black grid). CTLD, C-type lectin-like domain; Cys, cysteine-rich domain; FNII, fibronectin type II domain.