Cross talk between engulfment receptors stabilin-2 and integrin αvβ5 orchestrates engulfment of phosphatidylserine-exposed erythrocytes

Efficient cell corpse clearance is critical for health in organisms. Apoptotic cells displaying phosphatidylserine (PS) are recognized by engulfment receptors and ingested through two conserved pathways. In one pathway, engulfment receptor brain-specific angiogenesis inhibitor 1 (BAI-1) or integrin functions upstream of ELMO/DOCK180 and activate the small GTPase Rac1. In the other pathway, engulfment receptor CED-1 or stabilin-2 acts in concert with the adaptor protein GULP to activate Rac1. Stabilin-2, a PS receptor, facilitates phagocytosis of apoptotic cells and mediates the production of anti-inflammatory cytokines. Here, we propose that the stabilin-2 extracellular domain consisting of integrin-binding fasciclin 1 (FAS1) domains coordinates the activities of the two phagocytic pathways via direct interactions with integrin. Interactions between stabilin-2 and integrin were determined using biochemical assays, including coimmunoprecipitation and fluorescence resonance energy transfer (FRET). These interactions appear to have functional relevance, since knockdown of endogenous αvβ5 expression or treatment with a function-blocking αvβ5 antibody significantly decreased stabilin-2-mediated phagocytosis in the absence of soluble factors. Our data collectively suggest that the engulfment receptors of the two phagocytic pathways communicate with each other to orchestrate engulfment of damaged erythrocytes. Coordinated phagocytic signaling would be advantageous for physiological and pathological circumstances that require rapid clearance of abnormal (apoptotic or aged) cells.     

Thymosin beta4 is involved in stabilin-2-mediated apoptotic cell engulfment

Stabilin-2 was recently identified as a novel receptor for membrane phosphatidylserine of apoptotic cells. To identify proteins that were candidates for stabilin-2 cytoplasmic domain binding, we screened a human spleen cDNA library using the yeast two-hybrid system. We found that thymosin beta4 interacts with the stabilin-2 cytoplasmic domain and is co-localized with stabilin-2 at the phagocytic cup. Knockdown of thymosin beta4 significantly decreased the phagocytic activity of stabilin-2, whereas overexpression of thymosin beta4 increased this activity. Additionally, amino acids 2504-2514 of stabilin-2 cytoplasmic domain were found to be responsible for the interaction with thymosin beta4. Taken together, these results suggest that thymosin beta4 is a downstream molecule of stabilin-2 that plays a role in stabilin-2-mediated cell corpse clearance.     

Axonin-1/TAG-1 mediates cell-cell adhesion by a cis-assisted trans-interaction.

The neural cell adhesion molecule axonin-1/TAG-1 mediates cell-cell interactions via homophilic and heterophilic contacts. It consists of six Ig and four fibronectin type III domains anchored to the membrane by glycosylphosphatidylinositol. The recently solved crystal structure indicates a module composed of the four N-terminal Ig domains as the contact site between trans-interacting axonin-1 molecules from apposed membranes. Here, we have tested domain-specific monoclonal antibodies for their capacity to interfere with homophilic binding in a cell aggregation assay. The results confirmed the existence of a binding region within the N-terminal Ig domains and identified a second region contributing to homophilic binding on the third and fourth fibronectin domains near the C terminus. The perturbation of each region alone resulted in a complete loss of cell aggregation, suggesting that axonin-1-mediated cell-cell contact results from a cooperative action of two homophilic binding regions. The data support that axonin-1-mediated cell-cell contact is formed by cis-assisted trans-binding. The N-terminal binding regions of axonin-1 establish a linear zipper-like string of trans-interacting axonin-1 molecules alternately provided by the two apposed membranes. The C-terminal binding regions strengthen the cell-cell contact by enhancing the expansion of the linear string into a two-dimensional array via cis-interactions. Cis-assisted trans-binding may be a basic binding mechanism common to many cell adhesion molecules.     

The conserved histidine in epidermal growth factor-like domains of stabilin-2 modulates pH-dependent recognition of phosphatidylserine in apoptotic cells

Clearance of apoptotic cells is involved in the resolution of inflammation, and this mechanism is controlled by the regulation of pro- and anti-inflammatory cytokine production during the ingestion of apoptotic cells. Inflamed areas show extracellular acidity, and low pH stimulates cellular functions of immune cells. However, little is known about the influence of extracellular acidic pH on the function of phagocytic cells. In this study, we showed that stabilin-2-mediated phagocytosis is activated in low pH media (pH 6.8) and examined the molecular mechanisms underlying this pH-dependent enhancement of phagocytic activity. Stabilin-2, which is expressed in human monocyte derived macrophages (HMDM), is a phosphatidylserine (PS) receptor that mediates phagocytosis of apoptotic cells, and releases the anti-inflammatory cytokine, TGF-β. The PS binding activity of stabilin-2 is enhanced in low pH, and a conserved histidine¹⁴⁰³ in close proximity to the PS binding loop is critical for pH-dependent activity. We propose that protonation of His¹⁴⁰³ may rearrange the PS binding loop to enhance binding affinity in low pH, indicating that acidic pH might act as a danger signal to stimulate stabilin-2-mediated phagocytosis to resolve inflammation. Considering that phosphatidylserine is an important target molecule for apoptotic cells in the acidic microenvironment of inflammation and tumors, our results also have implications for pH sensitive targeting of apoptotic cells.     

Stabilin-1, a homeostatic scavenger receptor with multiple functions

The multifunctional scavenger receptor stabilin-1 (STAB1, FEEL-1, CLEVER-1, KIAA0246) was originally identified as the MS-1 antigen, expressed by sinusoidal endothelial cells in human spleen. Extensive histological studies revealed that stabilin-1 is also expressed by tissue macrophages and sinusoidal endothelial cells in the healthy organism; its expression on both macrophages and different subtypes of endothelial cells is induced during chronic inflammation and tumorigenesis. In vitro induction of stabilin-1 in macrophages requires the presence of glucocorticoids. Stabilin-1 is involved in two intracellular trafficking pathways: receptor mediated endocytosis and recycling; and shuttling between the endosomal compartment and trans-Golgi network (TGN). The latter intracellular pathway of stabilin-1 trafficking is mediated by GGAs, clathrin adaptors that interact with the DDSLL motif in the cytoplasmic tail of stabilin-1. When expressed by alternatively activated macrophages, stabilin-1 mediates the uptake and targeting for degradation of acLDL and SPARC, a regulator of tissue remodeling. Likewise, stabilin-1 in macrophages is involved in intracellular sorting and lysosomal delivery of the novel stabilin- 1-interacting chitinase-like protein (SI-CLP). Indirect evidence suggests that stabilin-1 is involved in adhesion and transmigration in various cell types (including tumor cells, leukocytes, and lymphocytes); however, its rapid recycling and scant level of surface expression argue against its universal role in cell adhesion. In summary, stabilin-1 is a homeostatic receptor which links signals from the extracellular environment to intracellular vesicular processes, creating a potential impact on the macrophage secretion profile.     

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.     

Characterization of the Adhesive Properties of the Type IIb Subfamily Receptor Protein Tyrosine Phosphatases

Abstract

Receptor protein tyrosine phosphatases (RPTPs) have cell adhesion molecule–like extracellular domains coupled to cytoplasmic tyrosine phosphatase domains. PTPμ is the prototypical member of the type IIb subfamily of RPTPs, which includes PTPρ, PTPκ, and PCP-2. The authors performed the first comprehensive analysis of the subfamily in one system, examining adhesion and antibody recognition. The authors evaluated if antibodies that they developed to detect PTPmu also recognized other subfamily members. Notably, each antibody recognizes distinct subsets of type IIb RPTPs. PTPμ, PTPρ, and PTPκ have all been shown to mediate cell-cell aggregation, and prior work with PCP-2 indicated that it can mediate bead aggregation in vitro. This study reveals that PCP-2 is unique among the type IIb RPTPs in that it does not mediate cell-cell aggregation via homophilic binding. The authors conclude from these experiments that PCP-2 is likely to have a distinct biological function other than cell-cell aggregation.     

Neural cell adhesion molecule (N-CAM) homophilic binding mediated by the two N-terminal Ig domains is influenced by intramolecular domain-domain interactions

The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains. Here, we have used a panel of recombinant proteins in a bead binding assay, as well as transfected and primary cells, to clarify the molecular mechanism of N-CAM homophilic binding. The entire extracellular region of N-CAM mediated bead aggregation in a concentration- and temperature-dependent manner. Interactions of the N-terminal Ig domains, Ig1 and Ig2, were essential for bead binding, based on deletion and mutation experiments and on antibody inhibition studies. These findings were largely in accord with aggregation experiments using transfected L cells or primary chick brain cells. Additionally, maximal binding was dependent on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding. Our results suggest that the role of Ig3 in homophilic binding is largely structural. Several Ig3-specific reagents failed to affect N-CAM binding on beads or on cells, while an inhibitory effect of an Ig3-specific monoclonal antibody is probably due to perturbations at the Ig2-Ig3 boundary. Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions.     

Cancer-Derived Mutations in the Fibronectin III Repeats of PTPRT/PTPρ Inhibit Cell-Cell Aggregation

Abstract

The receptor protein tyrosine phosphatase T PTPρ is the most frequently mutated tyrosine phosphatase in human cancer. PTPρ mediates homophilic cell-cell aggregation. In its extracellular region, PTPρ has cell adhesion molecule–like motifs, including a MAM domain, an immunoglobulin domain, and four fibronectin type III (FNIII) repeats. Tumor-derived mutations have been identified in all of these extracellular domains. Previously, the authors determined that tumor-derived mutations in the MAM and immunoglobulin domains of PTPρ reduce homophilic cell-cell aggregation. In this paper, the authors describe experiments in which the contribution of the FNIII repeats to PTPρ-mediated cell-cell adhesion was evaluated. The results demonstrate that deletion of the FNIII repeats of PTPρ result in defective cell-cell aggregation. Furthermore, all of the tumor-derived mutations in the FNIII repeats of PTPρ also disrupt cell-cell aggregation. These results further support the hypothesis that mutational inactivation of PTPρ may lead to cancer progression by disrupting cell-cell adhesion.     

Homophilic and heterophilic binding activities of Nr-CAM, a nervous system cell adhesion molecule

Nr-CAM is a membrane glycoprotein that is expressed on neurons. It is structurally related to members of the N-CAM superfamily of neural cell adhesion molecules having six immunoglobulin-like domains and five fibronectin type III repeats in the extracellular region. We have found that the aggregation of chick brain cells was inhibited by anti-Nr-CAM Fab' fragments, indicating that Nr-CAM can act as a cell adhesion molecule. To clarify the mode of action of Nr-CAM, a mouse fibroblast cell line L-M(TK-) (or L cells) was transfected with a DNA expression construct encoding an entire chicken Nr-CAM cDNA sequence. After transfection, L cells expressed Nr-CAM on their surface and aggregated. Aggregation was specifically inhibited by anti-Nr-CAM Fab' fragments. To check the specificity of this aggregation, a fusion protein (FGTNr) consisting of glutathione S-transferase linked to the six immunoglobulin domains and the first fibronectin type III repeat of Nr-CAM was expressed in Escherichia coli. Addition of FGTNr to the transfected cells blocked their aggregation. Further analysis using a combination of cell aggregation assays, binding of cells to FGTNr-coated substrates, aggregation of FGTNr-coated Covaspheres and binding of FGTNr-coated Covaspheres to FGTNr-coated substrates revealed that Nr-CAM mediates two types of cell interactions: a homophilic, divalent cation-independent binding, and a heterophilic, divalent cation-dependent binding. Homophilic binding was demonstrated between transfected L cells, between chick embryo brain cells and FGTNr, and between Covaspheres to which FGTNr was covalently attached. Heterophilic binding was shown to occur between transfected and untransfected L cells, and between FGTNr and primary chick embryo fibroblasts; in all cases, it was dependent on the presence of either calcium or magnesium. Primary chick embryo glia or a human glial cell line did not bind to FGTNr-coated substrates. The results indicate that Nr-CAM is a cell adhesion molecule of the nervous system that can bind by two distinct mechanisms, a homophilic mechanism that can mediate interactions between neurons and a heterophilic mechanism that can mediate binding between neurons and other cells such as fibroblasts.     

In the first extracellular domain of E-cadherin,heterophilic interactions,but not the conserved His-Ala-Val motif,are required for adhesion

The classical cadherins, definitive proteins of the cadherin superfamily, are characterized functionally by their ability to mediate calcium-dependent cell aggregation in vitro. To test hypothetical mechanisms of adhesion, we have constructed two mutants of the chicken E-cadherin protein, one with the highly conserved His-Ala-Val (HAV) sequence motif reversed to Val-Ala-His (VAH), the other lacking the first extracellular domain (EC1). The inversion of HAV to VAH has no effect on the capacity of E-cadherin to mediate adhesion. Deletion of EC1 completely eliminates the ability of E-cadherin to mediate homophilic adhesion, but the deletion mutant is capable of adhering heterophilically to both unmutated E-cadherin and to the HAV/VAH mutant. These results demonstrate that the conserved HAV sequence motif is not involved in cadherin-mediated adhesion as has been suggested previously and supports the idea that in the context of the cell surface, cadherin-mediated cell-cell adhesion involves an interaction of EC1 with other domains of the cadherin extracellular moiety and not the "linear zipper" model, which posits trans interactions only between EC1 on apposing cell surfaces.     

Heterophilic and homophilic cadherin interactions in intestinal intermicrovillar links are species dependent

Enterocytes are specialized epithelial cells lining the luminal surface of the small intestine that build densely packed arrays of microvilli known as brush borders. These microvilli drive nutrient absorption and are arranged in a hexagonal pattern maintained by intermicrovillar links formed by 2 nonclassical members of the cadherin superfamily of calcium-dependent cell adhesion proteins: protocadherin-24 (PCDH24, also known as CDHR2) and the mucin-like protocadherin (CDHR5). The extracellular domains of these proteins are involved in heterophilic and homophilic interactions important for intermicrovillar function, yet the structural determinants of these interactions remain unresolved. Here, we present X-ray crystal structures of the PCDH24 and CDHR5 extracellular tips and analyze their species-specific features relevant for adhesive interactions. In parallel, we use binding assays to identify the PCDH24 and CDHR5 domains involved in both heterophilic and homophilic adhesion for human and mouse proteins. Our results suggest that homophilic and heterophilic interactions involving PCDH24 and CDHR5 are species dependent with unique and distinct minimal adhesive units.

Structures of the extracellular tips of PCDH24 and CDHR5, the proteins that form functionally important links between gut microvilli, reveal the minimum units used by these proteins to mediate adhesion, and provide insights into species-dependent interactions and functionality in the cadherin superfamily.     

Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.     

Cadherin Expression in the Developing Vertebrate CNS: From Neuromeres to Brain Nuclei and Neural Circuits

Cadherins are a family of cell surface glycoproteins which mediate cell-cell adhesion by a Ca²⁺-dependent mechanism. Results from in vitro studies with cadherin-transfected cell lines show that cadherins preferentially bind to each other in a homophilic fashion. In the developing vertebrate brain, at least 10 cadherins are found. Some of these cadherins are expressed in a restricted fashion in particular developing brain nuclei and neural circuits. Based on these results, specific morphogenetic roles for cadherins during CNS development have been proposed. This review focuses on the possible role of cadherin-mediated sorting and aggregation of early neurons and neurites in the formation of brain nuclei, fiber tracts, and neural circuits. Moreover, at least 1 cadherin is also expressed in a segmental ("neuromeric") fashion in the early chicken forebrain, suggesting that this cadherin regulates developmental processes involved in the transformation from the neuromeric organization of the early neuroepithelium to the functional organization of the mature brain.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments of FAS1-IV domain of human periostin,a component of extracellular matrix proteins

Periostin, an extracellular matrix protein, is secreted by fibroblasts and is overexpressed in various types of cancers. The four internal repeat fasciclin 1 (FAS1) domains of human periostin play crucial roles in promoting tumor metastasis and progression via interaction with cell surface integrins. Among four FAS1 domains of human periostin, the fourth FAS1 domain (FAS1-IV) was prepared for NMR study, since only FAS1-IV was highly soluble, and showed a well-dispersed 2D ¹H-¹⁵N HSQC spectrum. Here, we report nearly complete backbone and side chain resonance assignments and a secondary structural analysis of the FAS1-IV domain as first steps toward the structure determination of FAS1-IV of human periostin.     

Requirement of adaptor protein GULP during stabilin-2-mediated cell corpse engulfment

The prompt clearance of cells undergoing apoptosis is critical during embryonic development and normal tissue turnover, as well as during inflammation and autoimmune responses. We recently demonstrated that stabilin-2 is a phosphatidylserine receptor that mediates the clearance of apoptotic cells, thereby releasing the anti-inflammatory cytokine, transforming growth factor-beta. However, the downstream signaling components of stabilin-2-mediated phagocytosis are not known. Here, we provide evidence that the adaptor protein, GULP, physically and functionally interacts with the stabilin-2 cytoplasmic tail. Using fluorescent resonance energy transfer analysis and biochemical approaches, we show that GULP directly binds to the cytoplasmic tail of stabilin-2. Knockdown of endogenous GULP expression significantly decreased stabilin-2-mediated phagocytosis. Conversely, overexpression of GULP caused an increase in aged cell engulfment. The phosphotyrosine binding (PTB) domain of GULP was sufficient for the interaction with stabilin-2; therefore, transduction of TAT fusion PTB domain acts as a dominant negative, resulting in impaired engulfment of aged red blood cells in stabilin-2 expressing cells. In addition, the PTB domain of GULP was able to specifically interact with the NPXY motif of the stabilin-2 cytoplasmic tail. Taken together, these results indicate that GULP is a likely downstream molecule in the stabilin-2-mediated signaling pathway and plays an important role in stabilin-2-mediated phagocytosis.     

Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-1₂/NgCAM₂ tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-1₂/NgCAM₂ complexes and, thus, neurite fasciculation in DRG neurons.     

Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion

Type IIB receptor protein tyrosine phosphatases (RPTPs) are bi-functional cell surface molecules. Their ectodomains mediate stable, homophilic, cell-adhesive interactions, whereas the intracellular catalytic regions can modulate the phosphorylation state of cadherin/catenin complexes. We describe a systematic investigation of the cell-adhesive properties of the extracellular region of RPTPmu, a prototypical type IIB RPTP. The crystal structure of a construct comprising its N-terminal MAM (meprin/A5/mu) and Ig domains was determined at 2.7 A resolution; this assigns the MAM fold to the jelly-roll family and reveals extensive interactions between the two domains, which form a rigid structural unit. Structure-based site-directed mutagenesis, serial domain deletions and cell-adhesion assays allowed us to identify the four N-terminal domains (MAM, Ig, fibronectin type III (FNIII)-1 and FNIII-2) as a minimal functional unit. Biophysical characterization revealed at least two independent types of homophilic interaction which, taken together, suggest that there is the potential for formation of a complex and possibly ordered array of receptor molecules at cell contact sites.     

Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1)

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.     

Novel function of alternatively activated macrophages: stabilin-1-mediated clearance of SPARC

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1-mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin-1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin-1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration.