A targeted deletion in the endocytic receptor gene Endo180 results in a defect in collagen uptake

The four members of the mannose receptor family (the mannose receptor, the M-type phospholipase A₂ receptor, DEC-205 and Endo180) share a common extracellular arrangement of an amino-terminal cysteine-rich domain followed by a fibronectin type II (FNII) domain and multiple C-type lectin-like domains (CTLDs). In addition, all have a short cytoplasmic domain, which mediates their constitutive recycling between the plasma membrane and the endosomal apparatus, suggesting that these receptors function to internalize ligands for intracellular delivery. We have generated mice with a targeted deletion of Endo180 exons 2–6 and show that this mutation results in the efficient expression of a truncated Endo180 protein that lacks the cysteine-rich domain, the FNII domain and CTLD1. Analysis of embryonic fibroblasts reveals that this mutation does not disrupt the C-type lectin activity that is mediated by CTLD2, but results in cells that have a defect in collagen binding and internalization and an impaired migratory phenotype.     

The mannose receptor family

The mannose receptor family comprises four glycoproteins each of which is a type I transmembrane receptor with an N-terminal cysteine-rich domain, a single fibronectin type II (FNII) domain and eight to ten C-type lectin-like domains (CTLDs). Characteristically, these proteins are able to recycle between the plasma membrane and the endosomal apparatus due to discrete motifs present within their cytoplasmic domains. This review discusses the structure and function of these four proteins-the mannose receptor (MR), the M-type receptor for secretory phospholipases A(2) (PLA(2)R), DEC-205/gp200-MR6 and Endo180/uPARAP. Despite their overall structural similarity, these four receptors have evolved to use different domains to interact with discrete ligands. In addition, they differ in their ability to mediate endocytic and phagocytic events and in their intracellular destinations. Together, they represent a unique group of multidomain, multifunctional receptors.     

Characterization of sugar binding by the mannose receptor family member,Endo180

Members of the mannose receptor family, the mannose receptor, the phospholipase A(2) receptor, DEC-205, and Endo180, contain multiple C-type lectin-like domains (CTLDs) within a single polypeptide. In addition, at their N termini, all four family members contain a cysteine-rich domain similar to the R-type carbohydrate recognition domains of ricin. However, despite the common presence of multiple lectin-like domains, these four endocytic receptors have divergent ligand binding activities, and it is clear that the majority of these domains do not bind sugars. Here the functions of the lectin-like domains of the most recently discovered family member, Endo180, have been investigated. Endo180 is shown to bind in a Ca(2+)-dependent manner to mannose, fucose, and N-acetylglucosamine but not to galactose. This activity is mediated by one of the eight CTLDs, CTLD2. Competition assays indicate that the monosaccharide binding specificity of Endo180 CTLD2 is similar to that of mannose receptor CTLD4. However, additional experiments indicate that, unlike the cysteine-rich domain of the mannose receptor, the cysteine-rich domain of Endo180 does not bind sulfated sugars. Thus, although Endo180 and the mannose receptor are now both known to be mannose binding lectins, each receptor is likely to have a distinct set of glycoprotein ligands in vivo.     

The C-type lectin receptor Endo180 displays internalization and recycling properties distinct from other members of the mannose receptor family

Endo180/urokinase plasminogen activator receptor-associated protein together with the mannose receptor, the phospholipase A(2) receptor, and DEC-205/MR6-gp200 comprise the four members of the mannose receptor family. These receptors have a unique structural composition due to the presence of multiple C-type lectin-like domains within a single polypeptide backbone. In addition, they are all constitutively internalized from the plasma membrane via clathrin-mediated endocytosis and recycled back to the cell surface. Endo180 is a multifunctional receptor displaying Ca(2+)-dependent lectin activity, collagen binding, and association with the urokinase plasminogen activator receptor, and it has a proposed role in extracellular matrix degradation and remodeling. Within their short cytoplasmic domains, all four receptors contain both a conserved tyrosine-based and dihydrophobic-based putative endocytosis motif. Unexpectedly, Endo180 was found to be distinct within the family in that the tyrosine-based motif is not required for efficient delivery to and recycling from early endosomes. By contrast, receptor internalization is completely dependent on the dihydrophobic motif and modulated by a conserved upstream acidic residue. Furthermore, unlike the mannose receptor, Endo180 does not function as a phagocytic receptor in vitro. These findings demonstrate that despite an overall structural similarity, members of this receptor family employ distinct trafficking mechanisms that may reflect important differences in their physiological functions.     

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

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 Å 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.     

Complex Determinants in Specific Members of the Mannose Receptor Family Govern Collagen Endocytosis

Members of the well-conserved mannose receptor (MR) protein family have been functionally implicated in diverse biological and pathological processes. Importantly, a proposed common function is the internalization of collagen for intracellular degradation occurring during bone development, cancer invasion, and fibrosis protection. This functional relationship is suggested by a common endocytic capability and a candidate collagen-binding domain. Here we conducted a comparative investigation of each member''s ability to facilitate intracellular collagen degradation. As expected, the family members uPARAP/Endo180 and MR bound collagens in a purified system and internalized collagens for degradation in cellular settings. In contrast, the remaining family members, PLA₂R and DEC-205, showed no collagen binding activity and were unable to mediate collagen internalization. To pinpoint the structural elements discriminating collagen from non-collagen receptors, we constructed a series of receptor chimeras and loss- and gain-of-function mutants. Using this approach we identified a critical collagen binding loop in the suggested collagen binding region (an FN-II domain) in uPARAP/Endo180 and MR, which was different in PLA₂R or DEC-205. However, we also found that an active FN-II domain was not a sufficient determinant to allow collagen internalization through these receptors. Nevertheless, this ability could be acquired by the transfer of a larger segment of uPARAP/Endo180 (the Cys-rich domain, the FN-II domain and two CTLDs) to DEC-205. These data underscore the importance of the FN-II domain in uPARAP/Endo180 and MR-mediated collagen internalization but at the same time uncover a critical interplay with flanking domains.     

Identification and characterization of the endocytic transmembrane glycoprotein Endo180 as a novel collagen receptor

Endo180, a member of the mannose receptor family, is constitutively recycled between clathrin-coated pits on the cell surface and intracellular endosomes. Its large extracellular domain contains an N-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. The second of these lectin-like domains has been shown to mediate Ca2+-dependent mannose binding. In addition, cross-linking studies have identified Endo180 as a urokinase plasminogen activator receptor-associated protein and this interaction can be blocked by collagen V. Here we demonstrate directly using in vitro assays, cell-based studies and tissue immunohistochemistry that Endo180 binds both to native and denatured collagens and provide evidence that this is mediated by the fibronectin type II domain. In cell culture systems, expression of Endo180 results in the rapid uptake of soluble collagens for delivery to lysosomal degradative compartments. Together with the observed restricted expression of Endo180 in both embryonic and adult tissue, we propose that Endo180 plays a physiological role in mediating collagen matrix remodelling during tissue development and homeostasis and that the observed receptor upregulation in pathological conditions may contribute to disease progression.     

An extended conformation of the macrophage mannose receptor

The macrophage mannose receptor mediates phagocytosis of pathogenic microorganisms and endocytosis of potentially harmful soluble glycoproteins by recognition of their defining carbohydrate structures. The mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of 8-10 domains related to C-type carbohydrate recognition domains (CRDs), a fibronectin type II repeat and an N-terminal cysteine-rich domain. Hydrodynamic analysis and proteolysis experiments performed on fragments of the extracellular region of the receptor have been used to investigate its conformation. Size and shape parameters derived from sedimentation and diffusion coefficients indicate that the receptor is a monomeric, elongated and asymmetric molecule. Proteolysis experiments indicate the presence of close contacts between several pairs of domains and exposed linker regions separating CRDs 3 and 6 from their neighboring domains. Hydrodynamic coefficients predicted for modeled receptor conformations are consistent with an extended conformation with close contacts between three pairs of CRDs. The N-terminal cysteine-rich domain and the fibronectin type II repeat appear to increase the rigidity of the molecule. The rigid, extended conformation of the receptor places domains with different functions at distinct positions with respect to the membrane.     

Structural determinants of the dominant conformational epitopes of phospholipase A2 receptor in primary membranous nephropathy

Anti-phospholipase A2 receptor autoantibody (PLA2R-Ab) plays a critical role in the pathogenesis of primary membranous nephropathy (PMN), an autoimmune kidney disease characterized by immune deposits in the glomerular subepithelial spaces and proteinuria. However, the mechanism of how PLA2R-Abs interact with the conformational epitope(s) of PLA2R has remained elusive. PLA2R is a single transmembrane helix receptor containing ten extracellular domains that begin with a CysR domain followed by a FnII and eight CTLD domains. Here, we examined the interactions of PLA2R-Ab with the full PLA2R protein, N-terminal domain truncations, and C-terminal domain deletions under either denaturing or physiological conditions. Our data demonstrate that the PLA2R-Abs against the dominant epitope (the N-terminal CysR-CTLD1 triple domain) possess weak cross-reactivities to the C-terminal domains beyond CTLD1. Moreover, both the CysR and CTLD1 domains are required to form a conformational epitope for PLA2R-Ab interaction, with FnII serving as a linker domain. Upon close examination, we also observed that patients with newly diagnosed PMN carry two populations of PLA2R-Abs in sera that react to the denatured CysR-CTLD3 (the PLA2R-Ab₁) and denatured CysR-CTLD1 (the PLA2R-Ab₂) domain complexes on Western blots, respectively. Furthermore, the PLA2R-Ab₁ appeared at an earlier time point than PLA2R-Ab₂ in patients, whereas the increased levels of PLA2R-Ab₂ coincided with the worsening of proteinuria. In summary, our data support that an integrated folding of the three PLA2R N-terminal domains, CysR, FnII, and CTLD1, is a prerequisite to forming the PLA2R conformational epitope and that the dominant epitope-reactive PLA2R-Ab₂ plays a critical role in PMN clinical progression.     

Molecular architecture of mouse activating NKR-P1 receptors

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.     

Design and synthesis of novel multivalent mannosides targeting the mannose receptor

According to the characteristics of C-type lectin-like domains in the mannose receptor (MR), a novel design of multivalent mannosides targeting the MR was accomplished. Beginning with a divalent mannoside as the sugar unit, a series of multivalent mannosides with variations in both valence and space were synthesized in a convergent approach. The synthetic multivalent mannosides are to be explored to study MR-sugar binding events.     

Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor.

The extracellular portion of the macrophage mannose receptor is composed of several cysteine-rich domains, including a fibronectin type II repeat and eight segments related in sequence to Ca(2+)-dependent carbohydrate-recognition domains (CRDs) of animal lectins. Expression of portions of the receptor in vitro, in fibroblasts and in bacteria, has been used to determine which of the extracellular domains are involved in binding and endocytosis of ligand. The NH2-terminal cysteine-rich domain and the fibronectin type II repeat are not necessary for endocytosis of mannose-terminated glycoproteins. CRDs 1-3 have at most very weak affinity for carbohydrate, so the carbohydrate binding activity of the receptor resides in CRDs 4-8. CRD 4 shows the highest affinity binding and has multispecificity for a variety of monosaccharides. However, CRD 4 alone cannot account for the binding of the receptor to glycoproteins. At least 3 CRDs (4, 5, and 7) are required for high affinity binding and endocytosis of multivalent glycoconjugates. In this respect, the mannose receptor is like other carbohydrate-binding proteins, in which several CRDs, each with weak affinity for single sugars, are clustered to achieve high affinity binding to oligosaccharides. In the mannose receptor, these multiple weak interactions are achieved through several active CRDs in a single polypeptide chain rather than by oligomerization of polypeptides each containing a single CRD.     

Collagen binding by the mannose receptor mediated through the fibronectin type II domain

The macrophage mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of an N-terminal cysteine-rich domain related to the R-type carbohydrate-recognition domain of ricin, a fibronectin type II domain and eight to ten domains related to C-type carbohydrate-recognition domains. The mannose receptor acts as a molecular scavenger, clearing harmful glycoconjugates or micro-organisms through recognition of their defining carbohydrate structures. Cell-adhesion assays, as well as collagen-binding assays, have now been used to show that the mannose receptor can also bind collagen and that the fibronectin type II domain mediates this activity. Neither of the two types of sugar-binding domain in the receptor is involved in collagen binding. Fibroblasts expressing the mannose receptor adhere to type I, type III and type IV collagens, but not to type V collagen, and the adherence is inhibited by isolated mannose receptor fibronectin type II domain. The fibronectin type II domain shows the same specificity for collagen as the whole receptor, binding to type I, type III and type IV collagens. This is the first activity assigned to the fibronectin type II domain of the mannose receptor. The results suggest additional roles for this multifunctional receptor in mediating collagen clearance or cell-matrix adhesion.     

Glycosylation influences the lectin activities of the macrophage mannose receptor

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man5-GlcNAc2 glycans (Man5 -MR) or non-sialylated complex glycans (SA0-MR) did not bind mannosylated glycans, but could recognize SO4-3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA0-MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands.     

The non-phagocytic route of collagen uptake: a distinct degradation pathway

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.     

Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains

Macrophages express a cell surface receptor which mediates phagocytosis and pinocytosis of particles and solutes containing mannose (fucose and N-acetylglucosamine are also ligands for the receptor). An apparently identical protein has been isolated from human placenta. Proteolytic fragments of the placental receptor were sequenced so that oligonucleotide probes complementary to the receptor cDNA could be generated. These probes were used to isolate cDNA clones covering the entire coding portion of the mRNA for the receptor. Confirmation that these clones encode the mannose receptor was obtained by expression in rat fibroblasts. The expressed protein mediates uptake and degradation of mannose-conjugated serum albumin. The deduced amino acid sequence of the receptor reveals that it is most likely to be a type I transmembrane protein (COOH terminus on the cytoplasmic side of the membrane) since the mature polypeptide is preceded by a signal sequence and a hydrophobic stop transfer sequence is located 45 amino acids from the COOH terminus. The extracellular portion of the receptor polypeptide consists of three types of domains. The first 139 amino acids constitute a cysteine-rich segment which does not resemble other known sequences. There follows a domain which closely resembles fibronectin type II repeats. The remainder of the extracellular portion of the receptor is composed of eight segments homologous with the C-type carbohydrate-recognition domains of the asialoglycoprotein receptor, mannose binding proteins, and other Ca2(+)-dependent animal lectins. This structure suggests that the receptor may contain multiple ligand-binding domains thus accounting for its tight binding to highly multivalent ligands.     

46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor

We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.     

Antigenic targeting of the human mannose receptor induces tumor immunity

Pattern recognition receptors are preferentially expressed on APCs allowing selective uptake of pathogens for the initiation of antimicrobial immunity. In particular, C-type lectin receptors, including the mannose receptor (MR), facilitate APC-mediated adsorptive endocytosis of microbial glyconjugates. We have investigated the potential of antigenic targeting to the MR as a means to induce Ag-specific humoral and cellular immunity. hMR transgenic (hMR Tg) mice were generated to allow specific targeting with the anti-hMR Ab, B11. We show that hMR targeting induced both humoral and cellular antigenic specific immunity. Immunization of hMR Tg mice with B11 mAbs induced potent humoral responses independent of adjuvant. Injection of hMR Tg mice with mouse anti-hMR Ab clone 19.2 elicited anti-Id-specific humoral immunity while non-Tg mice were unresponsive. B11-OVA fusion proteins (B11-OVA) were efficiently presented to OVA-specific CD4 and CD8 T cells in MR Tg, but not in non-Tg, mice. Effector differentiation of responding T cells in MR Tg mice was significantly enhanced with concomitant immunization with the TLR agonist, CpG. Administration of both CpG and B11-OVA to hMR Tg mice induced OVA-specific tumor immunity while WT mice remained unprotected. These studies support the clinical development of immunotherapeutic approaches in cancer using pattern recognition receptor targeting systems for the selective delivery of tumor Ags to APCs.     

Pulmonary surfactant protein A up-regulates activity of the mannose receptor,a pattern recognition receptor expressed on human macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.     

A di-aromatic motif in the cytosolic tail of the mannose receptor mediates endosomal sorting

The mannose receptor (MR), the prototype of a new family of multilectin receptor proteins important in innate immunity, undergoes rapid internalization and recycling from the endosomal system back to the cell surface. Sorting of the MR in endosomes prevents the receptor from entering lysosomes where it would be degraded. Here, we focused on a diaromatic sequence (Tyr(18)-Phe(19)) in the MR cytoplasmic tail as an endosomal sorting signal. The subcellular distribution of chimeric constructs between the MR and the cation-dependent mannose 6-phosphate receptor was assessed by Percoll density gradients and cell surface assays. Unlike the wild type constructs, mutant receptors with alanine substitutions of Tyr(18)-Phe(19) were highly missorted to lysosomes, indicating that the di-aromatic motif of the MR cytoplasmic tail mediates sorting in endosomes. Within this sequence Tyr(18) is the key residue with Phe(19) contributing to this function. Moreover, Tyr(18) was also found to be essential for internalization, consistent with the presence of overlapping signals for internalization and endosomal sorting in the cytosolic tail of the MR. A di-aromatic amino acid sequence in the cytosolic tail has now been shown to function in two receptors known to be internalized from the plasma membrane, the MR and the cation-dependent mannose 6-phosphate receptor. This feature therefore appears to be a general determinant for endosomal sorting.