Effects of transferrin receptor blockade on cancer cell proliferation and hypoxia-inducible factor function and their differential regulation by ascorbate.

Cellular iron is needed for cell survival and hydroxylation of hypoxia-inducible factor-1alpha (HIF-alpha) by prolyl hydroxylases (PHD). One mechanism of iron uptake is mediated by the cell surface transferrin receptor (TfR). Because iron is required for cell growth and suppression of HIF-alpha levels, we tested the effects of the two anti-TfR monoclonal antibodies (mAb) E2.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR.

In polarized MDCK cells basolateral vesicles arise from clathrin-gamma-adaptin-coated domains on endosomal tubules.

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices.

Analysis of the structural requirements for lysosomal membrane targeting using transferrin receptor chimeras.

The sorting of membrane proteins to the lysosome requires tyrosine- or dileucine-based targeting signals. Recycling receptors have similar signals, yet these proteins seldom enter the latter stages of the endocytic pathway. To determine how lysosomal and internalization signals differ, we prepared chimeric molecules consisting of the cytoplasmic tails of CD3 gamma-chain, lysosomal acid phosphatase, and lysosomal-associated membrane glycoprotein-1, each fused to the transmembrane and extracellular domains of the transferrin receptor (TR).

Signal-dependent trafficking of beta-amyloid precursor protein-transferrin receptor chimeras in madin-darby canine kidney cells.

We have investigated the intracellular trafficking of a chimeric molecule consisting of the cytoplasmic domain of the beta-amyloid precursor protein (APP) and the transmembrane region and external domain of the human transferrin receptor (TR) in Madin-Darby canine kidney cells. Newly synthesized APP-TR chimeras are selectively targeted to the basolateral surface by a tyrosine-dependent sorting signal in the APP cytoplasmic tail. APP-TR chimeras are then rapidly internalized from the basolateral surface and a significant fraction ( approximately 20-30%) are degraded.

Structural requirements for basolateral sorting of the human transferrin receptor in the biosynthetic and endocytic pathways of Madin-Darby canine kidney cells.

In polarized Madin-Darby canine kidney (MDCK) cells, the transferrin receptor (TR) is selectively delivered to the basolateral surface, where it internalizes transferrin via clathrin-coated pits and recycles back to the basolateral border. Mutant tailless receptors are sorted randomly in both the biosynthetic and endocytic pathways, indicating that the basolateral sorting of TR is dependent upon a signal located within the 61-amino acid cytoplasmic domain. To identify the basolateral sorting signal of TR, we have analyzed a series of mutant human TR expressed in MDCK cells.

Structural requirements for major histocompatibility complex class II invariant chain trafficking in polarized Madin-Darby canine kidney cells.

The invariant chain (Ii) targets major histocompatibility complex class II molecules to an endocytic processing compartment where they encounter antigenic peptides. Analysis of Ii-transferrin receptor chimeras expressed in polarized Madin-Darby canine kidney (MDCK) cells shows that the Ii cytoplasmic tail contains a dihydrophobic basolateral sorting signal, Met16-Leu17, which is recognized in both the biosynthetic and endocytic pathways. Pro15-Met16-Leu17 has previously been identified as one of two dihydrophobic Ii internalization signals active in non-polarized cells.

Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism.

We have evaluated transcytotic routes in MDCK cells for their ability to generate a polarized surface distribution of trafficking proteins by following the intracellular sorting of transferrin receptors (TRs). We find that the selective basolateral expression of TRs is maintained in the face of extensive trafficking between the apical and basolateral surfaces. Biochemical studies of receptors loaded with tracer under conditions approaching steady state indicate that TRs internalized from the two surfaces are extensively colocalized within MDCK cells and that both populations of receptors are selectively delivered to the basolateral surface.

Functional analysis of human/chicken transferrin receptor chimeras indicates that the carboxy-terminal region is important for ligand binding.

Chimeric human/chicken transferrin receptors have been constructed using the polymerase chain reaction. Different regions of the 671-residue external domain of the human transferrin receptor were replaced by the corresponding sequences from the chicken transferrin receptor. As chicken transferrin receptors do not bind human transferrin, functional analysis of such chimeric receptors provides an approach to define the ligand-binding site of the human transferrin receptor.

Transmembrane domain of CD44 is required for its detergent insolubility in fibroblasts.

The hyaluronan receptor CD44 is an abundant glycoprotein expressed on a variety of different cell types. In fibroblasts a significant portion of receptor molecules remain in the detergent-insoluble fraction after Triton X-100 extraction. Detergent insolubility of these CD44 molecules has been interpreted to reflect their association with the cytoskeleton.

Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain.

The beta-amyloid precursor protein (APP) is proteolytically processed to generate beta-amyloid protein, the principal protein component of neuropathological lesions characteristic of Alzheimer's disease. To investigate potential sorting signals in the cytoplasmic tail of APP, we transplanted APP cytoplasmic tail sequences into the cytoplasmic tail of the human transferrin receptor (TR) and showed that two sequence motifs from the APP cytoplasmic tail promote TR internalization. One sequence, GYENPTY, is related to the low density lipoprotein receptor internalization signal, FDNPVY, but also involves a critical glycine residue; the other, YTSI, conforms to the 4-residue tyrosine-based internalization signal consensus sequence.

Role of CD44 cytoplasmic domain in hyaluronan binding.

The hyaluronan (HA) binding activity of mutant CD44 constructs expressed in AKR1 T-lymphoma cells was evaluated by flow cytometry using fluorescein-conjugated HA (Fl-HA). Previous studies showed that wild-type hematopoietic CD44 bound Fl-HA when expressed in AKR1, but that truncated "tailless" CD44, lacking all but six amino acids of the cytoplasmic domain, did not bind. Here, we show that a disulfide-bonded dimer of CD44, formed by substituting the transmembrane region of CD3 zeta chain for that of CD44, binds Fl-HA, even when the cytoplasmic domain of the CD44 dimer is absent.

Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment.

Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (Ii). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis.

High level expression and purification of the enzymatically active cytoplasmic region of human CD45 phosphatase from yeast.

The cytoplasmic region of human CD45 corresponding to residues 584-1281 was inserted downstream of the alcohol dehydrogenase promoter and transfected into a haploid strain of yeast. Expression of recombinant CD45 in yeast reached as high as 5% of the soluble protein. Following removal of cellular debris by centrifugation and an ammonium sulfate precipitation step, the enzyme was purified using phenyl-Sepharose chromatography, preparative gel filtration, Mono Q anion exchange chromatography and a final analytical gel filtration step.

In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella.

By following the intracellular processing of recycling transferrin receptors and the selective sorting of a-2 macroglobulin in chick embryo fibroblasts, we have shown that the concentration of 60 nm diam tubules which surrounds the centrioles represents a distal compartment on the recycling pathway. In migrating cells transferrin receptor tracers can be loaded into this compartment and then chased to the cell surface. When they emerge the recycling transferrin receptors are distributed over the surface of the leading lamella.

CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development.

CD45 is one of the most abundant leukocyte cell surface glycoproteins and is expressed exclusively upon cells of the hematopoietic system. Different isoforms of CD45 are generated by alternative splicing and are expressed in cell type-specific patterns on functional subpopulations of lymphocytes. In a major advance, CD45 was identified as one of the first members of a novel class of enzymes, the protein tyrosine phosphatases (PTPs).

YTRF is the conserved internalization signal of the transferrin receptor, and a second YTRF signal at position 31-34 enhances endocytosis.

By functional analysis of mutant human transferrin receptors (TR) expressed in chicken embryo fibroblasts, we previously identified a tetrapeptide sequence, Y20TRF23, within the 61-residue cytoplasmic tail as the signal for high-efficiency endocytosis (Collawn, J. F., Stangel, M.

Ligand-induced internalization of the epidermal growth factor receptor is mediated by multiple endocytic codes analogous to the tyrosine motif found in constitutively internalized receptors.

Ligand-induced internalization of epidermal growth factor (EGF) receptors via a high affinity saturable pathway requires sequences located in the carboxyl terminus distal to the tyrosine kinase domain. Three regions were found to contain endocytic motifs as defined by their ability to restore internalization function to EGF receptors truncated at the distal border of the kinase domain at residue 958. Deletional analysis identified the sequence 996QQGFF as essential for function of the region encompassing residues 993-1022.

Rate of 59Fe uptake into brain and cerebrospinal fluid and the influence thereon of antibodies against the transferrin receptor.

Uptake of 59Fe from blood into brains of anaesthetized rats and mice has been studied by intravenous infusion of [59Fe]ferrous ascorbate or of 59Fe-transferrin, the results not being significantly different. Uptakes in the rat were linear with time, but increased at longer times in the mouse. Transfer constants, K(in) (in ml/g/h x 10(3)), for cerebral hemispheres were 5.

Structural requirements for high efficiency endocytosis of the human transferrin receptor.

Wild-type and mutant human transferrin receptors (TR) have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. By functional studies of the mutant TRs, we have identified the tetrapeptide sequence, YXRF, in the cytoplasmic tail of the receptor as the internalization signal required for high efficiency endocytosis and shown that transplanted internalization signals from the low density lipoprotein receptor (LDLR) and the cation-independent mannose-6-phosphate receptor (Man-6-PR) are able to promote rapid internalization of the human TR. A six-residue LDLR signal, FDNPVY, is required for activity in TR, whereas a four-residue Man-6-PR signal, YSKV, is sufficient.

Monoclonal antibodies against defined epitopes of the human transferrin receptor cytoplasmic tail.

Three murine monoclonal antibodies (mAbs) against the 61-residue amino-terminal cytoplasmic tail of the human transferrin receptor (TR) have been produced by immunization of mice with recombinant human TR produced in a baculovirus expression system. Mutant human TRs expressed in chick embryo fibroblasts (CEFs) with point mutations or deletions in their cytoplasmic tails have been used to map the epitopes defined by each of the mAbs. One mAb, H68.