Ian S. Trowbridge

Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis

Using detailed functional studies on 24 human transferrin receptor mutants, we identified YXRF as the internalization sequence. Provided that at least 7 residues separate this tetrapeptide from the transmembrane region, changing the tetrapeptide position within the TR cytoplasmic domain does not reduce internalization activity. Thus, any conformational determinant for internalization must be localized to the YXRF sequence. Twenty-eight tetrapeptide analogs of YXRF, found by an unbiased search of all known three-dimensional protein structures, significantly favored tight turns similar to a type I turn. Of the ten tetrapeptides most closely related to YXRF, eight were surface exposed and had tight-turn conformations, as were four of five tetrapeptides with sequences related to the low density lipoprotein receptor internalization motif, NPXY. The internalization sequences of both receptors contain aromatic residues with intervening hydrogen-bonding residues. Thus, two distinct internalization sequences favor a common structural chemistry and implicate an exposed tight turn as the recognition motif for high efficiency endocytosis.

Biochemical characterization and cellular distribution of a polymorphic, murine cell-surface glycoprotein expressed on lymphoid tissues

A murine leukocyte surface glycoprotein (Mr = 95 000) has been defined by means of xenogeneic monoclonal antibodies. In normal hematopoietic tissues, the glycoprotein is found in highest amounts in the bone marrow. Flow cytometric analysis shows that essentially all bone-marrow cells express the glycoprotein and that it is a major component of a subpopulation of cells containing predominantly granulocytic precursors. In contrast, only about 5 percent of thymocytes express sufficient glycoprotein to be detected by flow cytometric analysis, although under stringent conditions up to 20 percent of thymocytes are susceptible to complement-mediated cytotoxicity using a monoclonal antibody against the glycoprotein. Functional assays showed that both prothymocytes and colony forming unit-spleen express the glycoprotein which is broadly distributed on murine hematopoietic tumor cell lines. However, although some Thy-I+ (T) cell lymphomas express large amounts of the glycoprotein, others do not express detectable quantities of the molecule. The glycoprotein is not restricted to hematopoietic cells and can be detected on lung, kidney, brain, and liver as well as cultured fibroblasts. Monoclonal antibodies against the glycoprotein cross-react with an antigen present on human cells. As described in the accompanying paper, the glycoprotein exists in two antithetical allelic forms and we show that it is identical to a polymorphic surface molecule independently characterized by Colombatti and co-workers.

Cross-linking of human T cell receptor proteins: association between the T cell idiotype β subunit and the T3 glycoprotein heavy subunit

Bifunctional cross-linking reagents DSP, DSS, and BSOCOES were used to cross-link 125I-surface-labeled viable T lymphocytes. The cross-linked cells were solubilized in Nonidet-P40, immunoprecipitated with anti-Ti (monoclonal antibody T40/25) or anti-T3 (monoclonal antibodies UCHT-1 or OKT3), and analyzed by SDS-PAGE. With all three cross-linkers, the intact cross-linked products obtained with monoclonal antibody T40/25 from HPB-ALL cells were 20-30 kd heavier than the Ti dimer (Mr 80,000). When the DSP cross-linked product was isolated using either anti-Ti or anti-T3 monoclonal antibodies and then cleaved, bands having molecular weights identical with both the Ti and T3 subunits were obtained. The two-dimensional SDS-PAGE analysis (nonreducing followed by reducing conditions) of the DSS and BSOCOES cross-linked products revealed the specifically cross-linked bands to have Mr 40,000 and Mr 28,000. These data indicate that the Ti molecule and the T3 molecule are spatially associated on the cell surface and suggest the predominant association is between the Ti beta subunit (Mr 40,000) and the T3 heavy subunit (Mr 28,000).

The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid

We have found that the transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus all contain tightly bound lipid. This modification could play a role in the binding of these proteins to cellular membranes. The lipid associated with p60src, the transforming protein of Rous sarcoma virus, is located in the NH2-terminal domain of the polypeptide. This is the region of the protein that has been shown previously to participate in binding the protein to membranes. Two mature forms of p21, the transforming protein of Harvey sarcoma virus, contain lipid. Lipid is not, however, associated with newly synthesized p21. While mature p60src and p21 are bound to cellular membranes, the newly synthesized forms of these proteins are not. The posttranslational addition of lipid may therefore be the means by which these proteins acquire an affinity for membranes.

Endocytosis and signals for internalization.

The most important recent advance in the field of endocytosis has been the identification of the internalization signals of several constitutively recycling receptors. Common structural features and chemistry of internalization sequences have been defined, and an exposed tight turn has been implicated as the conformational recognition motif for endocytosis.

The major histocompatibility complex-restricted antigen receptor on T cells in mouse and man: Identification of constant and variable peptides

The variability of the MHC restricted receptor on murine T cells was examined by comparing tryptic peptide fingerprints of the receptor isolated fom three T cell hybridomas and a T cell tumor. Both variable and constant peptides were seen. Constant peptides were most apparent when comparing receptors from the same mouse strain. Peptide fingerprints of receptors from two independent T cell hybridomas with the same idiotype and specificity were identical. We also describe a molecule detected on the surface of a human T cell leukemia whose properties were identical to those reported for the MHC receptor on normal human T cells. The molecule was a dimer of 85,000-90,000 MW containing a 46,000 MW acidic alpha-chain and an unrelated 40,000 MW neutral beta-chain.

Monoclonal antibody to a membrane glycoprotein inhibits the acrosome reaction and associated Ca2+ and H+ fluxes of sea urchin sperm

Monoclonal antibodies were prepared using sea urchin (Strongylocentrotus purpuratus) sperm or isolated sperm plasma membrane vesicles as immunogen. Two monoclonal antibodies, J4/4 and J10/14, react with different epitopes on external domains of the same Mr = 210,000 integral-membrane glycoprotein. At Fab fragment concentrations of equivalent cell surface binding, J10/14 inhibits the egg jelly-induced exocytotic acrosome reaction and associated Ca2+ influx and H+ efflux, whereas J4/4 does not. Immunofluorescent localization shows that both monoclonal antibodies react with a narrow collar of plasma membrane over the acrosomal complex and also with the entire flagellum.

The synthesis and properties of T25 glycoprotein in thy-1-negative mutant lymphoma cells

The synthesis and properties of T25 glycoprotein which bears the serological specificity Thy-1 have been studied in mutants of cultured mouse lymphoma cells that do not express Thy-1 on their surface. Five complementation classes of mutant cells were previously characterized by somatic genetic analysis. Synthesis of abnormal T25 glycoproteins was detected in four classes of mutants. Each of these aberrant products was degraded move rapidly than T25 glycoprotein of wild-type cells. Defects in the oligosaccharide units of T25 glycoprotein were demonstrated in three classes of mutants. In one of these mutant classes, evidence for a general defect in glycosylation of cell surface glycoproteins was obtained. These data indicate that normal glycosylation of T25 glycoprotein is probably essential for the molecule to be incorporated into the plasma membrane and expressed on the cell surface.

Transferrin receptors promote the formation of clathrin lattices

Gold conjugates have been used to quantitate human transferrin receptors (hTfnRs) on transfected chick embryo fibroblasts. No relationship could be found between the number of hTfnRs and the number of clathrin-coated pits. However, hTfnRs are also associated with flat clathrin lattices that lie outside invaginated pits. With increasing levels of receptor expression, the density of hTfnRs within flat lattices increases, and at the highest levels of expression the total area of flat lattice increases up to 3-fold. These results show that increased receptor numbers can promote clathrin lattice growth and suggest that the recruitment of receptors like hTfnRs is an essential step in lattice construction. We conclude that the process of invagination, which gives rise to coated pits, is regulated separately.

Structural heterogeneity of human Pgp-1 and its relationship with p85

Pgp-1 is a major cell-surface phosphoglycoprotein (relative mass approximately 95 000) that has been identified in mouse and human cell lines and tissues (Isacke et al. 1986, Trowbridge et al. 1982, Hughes et al. 1983). Although monoclonal antibodies (mAb) that recognize this glycoprotein were initially raised against plasma membranes from NIH/3T3 fibroblast cells (Hughes and August 1981), its designation of Pgp-1 (phagocyte glycoprotein-1) was based on its marked abundance on macrophages, monocytes, and polymorphonuclear cells (Colombatti et al. 1982). Despite that the function of Pgp-1 is unknown, the mouse homolog is a useful differentiation antigen in that it is expressed on prothymocytes in the bone marrow (Trowbridge et al. 1982) and on a proportion of mouse thymocytes, including cells thought to be the immediate descendants of bone marrow prothymocytes (Lesley et al. 1985a, Hyman et al. 1986). Many cells found in an adult mouse thymocyte subpopulation enriched in Pgp-1 ÷ cells contain an unrearranged Tcell antigen receptor B-chain gene (Trowbridge et al. 1985). Further, Pgp-1 is expressed on more than 90% of fetal thymocytes on days 13 and 14 of gestation before the fraction of Pgp1 ÷ cells declines to adult levels shortly before birth (Lesley et al. 1985b). In contrast to the mouse, 50-60 % of human thymocytes express Pgp-1. The Pgp-1 ÷ subpopulation in the human comprises primarily medullary thymocytes, although some Pgp-1 ÷ cells scattered throughout the cortex can be detected by immunofluorescence staining (Isacke et al. 1986). p85 is a human glycoprotein with an approximate relative mass of 85 000 which was initially isolated from human chronic lymphocytic leukemia cells using mAb 50B4 and 50E6 (Letarte et al. 1985). More recently, the p85 glycoprotein was shown to carry all the antigenic deter-