Philip D. Stahl

THE MANNOSE RECEPTOR IS A PATTERN RECOGNITION RECEPTOR INVOLVED IN HOST DEFENSE

The mannose receptor recognizes the patterns of carbohydrates that decorate the surfaces and cell walls of infectious agents. This macrophage and dendritic cell pattern-recognition receptor mediates endocytosis and phagocytosis. The mannose receptor is the prototype of a new family of multilectin receptor proteins (membrane-spanning receptors containing eight-ten lectin-like domains, which appear to play a key role in host defense) and provides a link between innate and adaptive immunity. Recent advances include the identification of three new members of the mannose receptor family, additional work on defining the molecular requirements for sugar binding, a role for the mannose receptor in antigen presentation of lipoglycan antigens and evidence that the mannose receptor is associated with a signal transduction pathway leading to cytokine production.

Receptor-mediated endocytosis

In 1934, Lewis described the process ofpinocytosis an invagination of the plasma membrane enclosing a small droplet of fluid. Pinocytosis appeared to be a constitutive process, whereas phagocytosis described some years earlier by Metchnikoffwas regulated. Only within the last two decades has it been recognized that (i) during pinocytosis cells internalize vast amounts of membrane. indeed, more membrane than they synthesize [which led to the concept ofmembrane recycling (Steinman et al., 1983; refer to Schneider et al., 1979 a, b)] and (ii) that receptors mediate selective uptake of both macromolecules and particles. While the term endocytosis has been used to describe these processes, the term receptor-mediated endocytosis is usually reserved for specific uptake of soluble molecules. Broadly speaking, receptor-mediated endocytosis involves the movement of bound ligands from the cell surface to the interior. Whereas some ligands (e.g. ligands specific for the galactose, mannose, mannose 6-phosphate and LDL receptors) are targeted to lysosomes often subserving the nutritional needs of the cell, others (e.g. transferrin and IgG) are recycled to the plasma membrane or targeted to specific plasma membrane domains. Ligands may also be modified on entering the cell [e.g. proteolysis in macrophage endosomes (Diment & Stahl, 1984) or lysosomes, resialylation by passage through the Golgi or removal of iron in the case of transferrin (Klausner et al., 1983; Dautry-Varsat et al., 1983; Harding & Stahl, 1983)]. Intracellular transport and processing vary markedly between different receptor-ligand systems and different cell types, but these systems may be placed in four general categories (Fig. 1, Table 1). 1. Receptors that recycle but target their ligand to lysosomes. This category includes the LDL (Goldstein et al., 1979), asialoglycoprotein (Gal/GalNAc) (Schwartz et al., 1982), mannose 6-phosphate (Fischer et al., 1980 a, b), and mannose receptor systems (Stahl et al., 1980). 2. Receptors that recycle but do not target their ligand to lysosomes. The ligand remains attached to the receptor during its transit through the cell. In polarized cells this may result in transport of ligands between the apical and basolateral surfaces (transcytosis or diacytosis). Examples include transferrin and IgG (in suckling rat ileum) (Abrahamson & Rodewald, 1981). 3. Receptors that do not recycle and target their ligand to lysosomes, including the EGF receptor (Carpenter & Cohen, 1976), the insulin receptor (Kasuga et al., 1981, Carpentier et al., 1978, 1979) (insulin receptors may recycle in some cells) and the Fc receptor (in macrophages) (Mellman & Plutner, 1984) (Fc receptors may recycle under different conditions in some cells). 4. Receptors,that do not recycle and do not transport ligands to lysosomes. Transport of IgA represents a unique mechanism, transcytosis ofIgA results in cleavage and loss ofthe IgA receptor (Kuhn & Kraehenbuhl, 1979; Mostov & Blobel, 1982).

Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: Characterization and evidence for receptor recycling

125I-Mannose--BSA is taken up by alveolar macrophages by receptor-mediated endocytosis. Uptake is macrophage-specific and does not occur in polymorphonuclear leukocytes. Binding (4 degrees C) and uptake (37 degrees C) of 125I--Man--BSA are time- and ligand concentration-dependent [Kuptake = 40 nM; Kd (4 degrees C) = 10 nM]. When adjusted for ligand degradation, ligand uptake is linear with time. Binding saturates at 60 min and requires Ca++. Following binding, ligand remains on the cell surface where it can be released by EGTA and trypsin. Internalization of prebound ligand occurs very rapidly (t 1/2 less than 5 min) when cells are warmed to 37 degrees C. Following internalization of prebound ligand, binding activity is rapidly recovered (t 1/2 less than 5 min). Trypsin treatment (4 degrees C) substantially reduces binding activity (greater than 70% per 30 min). However, binding activity is rapidly recovered in cells treated with trypsin at 4 degrees C by warming to 37 degrees C in the absence of added ligand. Trypsin treatment at 37 degrees C rapidly destroys binding and uptake. On the contrary, 4 degrees C trypsin treatment produces only a modest reduction in subsequent ligand uptake. These results, taken together with the observation that cycloheximide has no effect on ligand uptake, suggest that receptors must be spared from degradation and that reutilization of receptors probably occurs.

A regulatory role for ARF6 in receptor-mediated endocytosis

Adenosine diphosphate-ribosylation factor 6 (ARF6), ARF6 mutants, and ARF1 were transiently expressed in Chinese hamster ovary cells, and the effects on receptor-mediated endocytosis were assessed. Overexpressed ARF6 localized to the cell periphery and led to a redistribution of transferrin receptors to the cell surface and a decrease in the rate of uptake of transferrin. Similar results were obtained when a mutant defective in guanosine triphosphate hydrolysis was expressed. Expression of a dominant negative mutant, ARF6(T27N), resulted in an intracellular distribution of transferrin receptors and an inhibition of transferrin recycling to the cell surface. In contrast, overexpression of ARF1 had little or no effect on these parameters of endocytosis.

A Salmonella virulence protein that inhibits cellular trafficking.

Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI‐2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI‐2‐encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild‐type Salmonella inhibited fusion of Salmonella‐containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome–endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.

Ras-Activated Endocytosis Is Mediated by the Rab5 Guanine Nucleotide Exchange Activity of RIN1

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.

Modulation of Rab5 and Rab7 Recruitment to Phagosomes by Phosphatidylinositol 3-Kinase

ABSTRACT Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.

A role for POR1, a Rac1‐interacting protein, in ARF6‐mediated cytoskeletal rearrangements

The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Here we report that expression of the GTPase‐defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement was inhibited by co‐expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1‐interacting protein involved in membrane ruffling, but not with the dominant‐negative mutant of Rac1, Rac1(S17N). A synergistic effect between POR1 and ARF6 for the induction of actin polymerization was detected. Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent. These findings indicate that ARF6 and Rac1 function on distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1.

The mannose receptor and other macrophage lectins

Abstract The macrophage expresses a variety of cell surface lectins with activities that support specific functional roles and correspond to various differentiation states characteristic of this cell type. Recently, research has been carried out to investigate the mannose receptor, the advanced glycosylation end products receptor, the mannose-6-phosphate-receptor, the β-glucan receptor, sialoadhesin and several galactose-specific binding proteins.

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P2 hydrolysis or increased PI(4,5)P2 generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P2 dictates the remodeling of actin necessary for completion of phagocytosis.