Internalization of glycosyl‐phosphatidylinositol (GPI)‐anchored lymphocyte proteins II. GPI‐anchored and transmembrane molecules internalize through distinct pathways

Bamezai, Anil; Goldmacher, Victor S.; Rock, Kenneth L.

doi:10.1002/eji.1830220104

Cited by 54 publications

(30 citation statements)

References 39 publications

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“…In lymphocytes. both types of proteins form caps when crosslinked, but only transmembrane proteins are endocytosed by coated pits (Bamezai et al, 1992). The structure through which GPI-anchored proteins are internalized has not been identified, but it might be a counterpart ofcaveolae.…”

Section: Discussionmentioning

confidence: 99%

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

Fujimoto

1996

J Histochem Cytochem.

113

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GPI-anchored proteins, glpicosphingolipids, and sphhgomyelin are all enriched in the detergent-insoluble complex which has been suggested to be purified caveolae. I studied the relationship of the molecules with caveolae in cultured cells by i"unOcgt0chemical methods. In cells reacted with antibodies to various membrane proteins and lipids on ice and fmed More applying secondary antibodies, labeling did not show concentration in caveolae. In contrast, when cells were incubated with the primary and secondary antibodies on ice and then transferred to 37'C without fmtion, labeled Thy-1.2, p2-miaoglobulin, lactosyl ceramide, ceramide tetrahexose, Fomman antigen, and sphingomyelin became concentrated in caveolae, whereas labeled transferrin receptor did were sequestered in the same caveolae when crosslinked with not. Thy-1.2 and sphingolipids formed COIIUIIOII patches and

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Section: Discussionmentioning

confidence: 99%

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

Fujimoto

1996

J Histochem Cytochem.

113

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“…However, a dependence on internalization cannot be completely ruled out by any of the experiments to date. Even some GPI-anchored proteins become internalized upon treatment with activating antibodies (38 …”

Section: Discussionmentioning

confidence: 99%

Human immunodeficiency virus infection is efficiently mediated by a glycolipid-anchored form of CD4.

Diamond

Finberg

Chaudhuri

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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Two broad roles have been revealed for the CD4 molecule. It serves as a receptor for both class II major histocompatibility complex molecules and human immunodeficiency virus (HIV). Upon binding class II major histocompatibility molecules, CD4 functions to enhance T-cell activation. By binding to CD4, HIV gains entry into the cell. We have used a chimeric molecule of CD4 and lymphocyte functionassociated antigen 3 (LFA-3), CD4PI, which lacks a membrane-spanning domain and is instead anchored in the membrane by linkage to glycosyl-phosphatidylinositol. (10)(11)(12), and perhaps induce inhibitory signals (12). Previous work has shown that alteration or replacement of various cytoplasmic and membrane-spanning regions of CD4, as well as deletions of the cytoplasmic domain, can disrupt modulation by phorbol 12-myristate 13-acetate (PMA) (13-15) but not the inhibitory effects ofgpl20 (10) or the ability to mediate infection by HIV (13,14). While these mutants show that the wild-type CD4 sequence of these regions is not required by HIV, they do all have membrane-spanning and, at least, rudimentary cytoplasmic domains, thus leaving open the possibility of a generic requirement for such structures in order to serve as a viral receptor. The retention of membrane-spanning sequences may allow continued interactions with other membrane-spanning regions. Likewise, even the few cytoplasmic amino acids that are retained may allow interactions with cytoskeletal or other proteins at the inner surface of the membrane. Any of these interactions could be important for the uptake of virus, which must follow binding.Here we demonstrate the ability of CD4PI, a lipidanchored form of CD4, to mediate productive infection by HIV, thereby proving that neither the membrane-spanning nor the cytoplasmic domain of CD4 is required, specifically or as a structural element, in order for CD4 to function as a viral receptor. These results serve to identify the class of naturally glycosyl-phosphatidylinositol (GPI)-anchored proteins as potential viral receptors. MATERIALS AND METHODSMedia. All experiments were carried out in RPMI 1640 supplemented with 10% calf serum, 10 mM Hepes, 2 mM L-glutamine, 50 ,RM 2-mercaptoethanol, 100 units ofpenicillin per ml, and 100 Ag of streptomycin per ml except as noted otherwise. Once established, the HSB2-derived lines were carried in the same medium but with only 5% serum.Expression of CD4 and CD4PI in HSB-2 Cells. Expression of the CD4 constructs in the human T-cell line HSB-2 was obtained by coculture with the retroviral producer lines MNST4 DAMP (6) and MNCT4PI DAMP as described (6,15,16). In brief, the retroviral producer lines were generated by transfection, by calcium phosphate coprecipitation, of the amphotropic helper line DAMP with defective proviral vector DNA carrying neomycin resistance and the cDNA encoding CD4 or CD4PI, followed by selection with the antibiotic G418 (1 mg/ml). Following coculture the HSB-2 infectants were separated from the adherent producer line by repeated plating and subj...

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“…In some experiments, the microfilamentdisrupting agent cytochalasin D (Sigma) (3 M), or the phosphoinositide-3 kinase inhibitor wortmannin (Calbiochem, San Diego, CA) (1 M) were added to the cells 30 min prior to the addition of LPS (33,34). After various times of LPS incubation, cells were washed and treated at 4°C with 200 g/ml Pronase (Boehringer Mannheim) for 1 h (35,36). Supernatants and cells were separated by centrifugation and transferred into scintillation liquid.…”

Section: Methodsmentioning

confidence: 99%

CD14-dependent Endotoxin Internalization via a Macropinocytic Pathway

Poussin¹,

Foti²,

Carpentier³

et al. 1998

Journal of Biological Chemistry

104

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Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from "classical" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.

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Internalization of glycosyl‐phosphatidylinositol (GPI)‐anchored lymphocyte proteins II. GPI‐anchored and transmembrane molecules internalize through distinct pathways

Cited by 54 publications

References 39 publications

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

Human immunodeficiency virus infection is efficiently mediated by a glycolipid-anchored form of CD4.

CD14-dependent Endotoxin Internalization via a Macropinocytic Pathway

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