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PubMed1996

Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport.

The product of the c-abl protooncogene is a nonreceptor tyrosine kinase found in both the cytoplasm and the nucleus. We report herein that cell adhesion regulates the kinase activity and subcellular localization of c-Abl. When fibroblastic cells are detached from the extracellular matrix, kinase activity of both cytoplasmic and nuclear c-Abl decreases, but there is no detectable alteration in the subcellular distribution. Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of a subset of c-Abl to early focal contacts is observed coincident with the export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating closely with its return to the nucleus and suggesting that the active nuclear c-Abl originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not activated upon cell attachment. These results show that c-Abl activation requires cell adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus where it may function to integrate adhesion and cell cycle signals.

3T3 Cells↗
PubMed1997

The ultrastructural localization of 60-kDa Ro protein and human cytoplasmic RNAs: association with novel electron-dense bodies.

The 60-kDa Ro ribonucleoprotein is an important target of humoral autoimmune responses. However, the ultrastructural locations of the 60-kDa Ro protein and its associated small cytoplasmic RNAs (Y RNAs) have not been previously determined, and the functions of the Ro protein and RNAs are not known. In this study, the cellular locations of the 60-kDa Ro protein and the Ro Y1 and Y4 RNAs are determined by immunoelectron microscopy and in situ hybridization electron microscopy, respectively. Both Ro protein and Y RNAs are concentrated in discrete areas of the nucleoplasm, nucleolus, and cytoplasm of cultured cells and human skin sections. The 60-kDa Ro protein and Y RNAs are also present diffusely in the cytoplasm, where they occur in ribosome-rich regions, and in the nucleus. The presence of Ro ribonucleoprotein components in nucleoli and in ribosome-rich cytoplasmic areas suggests a potential for the involvement of Y RNAs and/or 60-kDa Ro protein in ribosome synthesis, assembly, or transport. Double labeling experiments show that Ro protein and Y RNAs colocalize in the nucleoplasm, nucleolus, and cytoplasm. In addition, aggregates of Y RNA occur unassociated with 60-kDa Ro protein, and aggregates of 60-kDa Ro protein occur unassociated with Y RNA. Aggregates of both Ro protein and Y RNAs label previously unreported nuclear and cytoplasmic electron-dense bodies. We propose that these distinctive Ro-associated electron-dense bodies may represent structure(s) important for cellular transport and/or Ro function.

Cell Line↗
PubMed1995

Influence of second and third cytoplasmic loops on binding, internalization, and coupling of chimeric bombesin/m3 muscarinic receptors.

In order to investigate the molecular basis for differences in the characteristics of bombesin (Bn) and m3 muscarinic cholinergic (m3 ACh) receptors, chimeric Bn receptors possessing cytoplasmic domains from the m3 ACh receptor were produced. The receptors were expressed in CHO-K1 cells and binding, structural, and signal transduction characteristics were analyzed. Cell lines bearing chimeric Bn receptors possessing m3 ACh receptor domains in place of either the second cytoplasmic loop (BM2L), the third cytoplasmic loop (BM3L), or both loops (BM23L) each bound 125I-bombesin with a single affinity that was approximately the same as that of the Bn receptor (5-10 nM). However, Bn receptors possessing the m3 ACh third cytoplasmic loop were severely affected in other respects. Internalization of ligand in Bn and BM2L cells was rapid and extensive (> 80% of bound 125I-bombesin was acid-resistant). In contrast, internalization was dramatically reduced in BM3L and BM23L cells (approximately 20% of bound 125I-bombesin was acid-resistant). In Bn or BM2L cells 10 nM bombesin stimulated approximately 10-fold increases in phosphatidylinositol hydrolysis. Activation of Bn receptors also induced an increase in arachidonic acid release (478 +/- 32% of control, n = 3) and large increases in intracellular Ca2+. In contrast, in BM3L or BM23L cells, bombesin had no significant effect on phosphatidylinositol hydrolysis. Furthermore, BM3L receptor activation did not increase arachidonic acid release. However, BM3L and BM23L cells showed a small increase in intracellular Ca2+ at high concentrations of bombesin. These data indicate that the third cytoplasmic loop alone, or together with the second cytoplasmic loop, was not sufficient to transfer the characteristics of G protein interaction between m3 ACh and bombesin receptors. Furthermore, for the Bn receptor, ligand internalization does, whereas formation of the high affinity binding state does not, appear to require activation of G proteins.

Animals↗
PubMed1996

The cytoplasmic tail of FcgammaRIIIAalpha is involved in signaling by the low affinity receptor for immunoglobulin G.

The low affinity receptor for IgG, FcgammaRIIIA, is a multimeric receptor composed of the ligand binding subunit FcgammaRIIIAalpha (CD16) in association with the signal-transducing subunits zeta or gamma. Previous studies suggested that the cytoplasmic tail of FcgammaRIIIAalpha was not required for FcgammaRIIIAalpha-zeta association or signaling by FcgammaRIIIA. However, in these studies, the truncated FcgammaRIIIAalpha chains still expressed the four most membrane-proximal amino acids of the cytoplasmic tail (amino acids 230-233). By successive truncations from the C terminus of FcgammaRIIIAalpha, we have studied the role played by the membrane-proximal amino acids of the cytoplasmic tail of FcgammaRIIIAalpha in (i) FcgammaRIIIA expression, (ii) FcgammaRIIIAalpha-zeta association, and (iii) signal transduction. We provide evidence that this region is not required for FcgammaRIIIA expression or FcgammaRIIIAalpha-zeta association. However, signaling by FcgammaRIIIA is strictly dependent on the membrane-proximal amino acids in the cytoplasmic tail of FcgammaRIIIAalpha. Thus, total deletion of the cytoplasmic tail of FcgammaRIIIAalpha results in a severely impaired tyrosine phosphorylation of phospholipase C-gamma1, zap, and syk and rise in intracellular free Ca2+ following receptor ligation with specific anti-CD16 monoclonal antibody or Ig-anti-Ig complexes, suggesting that FcgammaRIIIAalpha-zeta association per se is not sufficient to establish the signal function of FcgammaRIIIA. In conclusion, the present findings demonstrate that the most membrane-proximal amino acids of the FcgammaRIIIAalpha cytoplasmic tail play a critical role in ligand-induced signal transduction by the FcgammaRIIIAalpha-zeta complex.

Amino Acid Sequence↗
PubMed1997

Nuclear cotransport mechanism of cytoplasmic human MxB protein.

Interferon-alpha/beta-inducible Mx proteins belong to the family of large GTPases and share high sequence homology with dynamins in their N-terminal GTP-binding domains. In addition, Mx proteins have a conserved C-terminal leucine zipper element that is involved in their oligomerization. Cytoplasmic human MxA protein mediates resistance to multiple RNA viruses, whereas no antiviral activity has been found for human MxB protein. We have previously shown that MxB protein exists as a nuclear 78-kDa and as a cytoplasmic 76-kDa form in interferon-alpha-induced human cells. Using various influenza hemagglutinin epitope-tagged MxB gene constructs in transient transfection experiments in COS-1 cells, we show that the cytoplasmic 76-kDa MxB protein forms hetero-oligomers with the nuclear 78-kDa MxB protein via the C-terminal leucine zipper element. This enables the cytoplasmic form of MxB protein to be translocated into the nucleus together with the nuclear form of MxB protein. This finding was confirmed in interferon-alpha-induced HEp-2 and T98G cells transfected with various MxB gene constructs. Cell fractionation studies also suggest that a considerable amount of the cytoplasmic MxB protein is also found in the nucleus. Using confocal laser microscopy, we also demonstrate that the cytoplasmic MxA and the nuclear MxB proteins do not colocalize/oligomerize with each other, and both of these proteins are retained in their specific cellular compartments.

Animals↗
PubMed1998

Integrin beta cytoplasmic domains differentially bind to cytoskeletal proteins.

Integrin cytoplasmic domains connect these receptors to the cytoskeleton. Furthermore, integrin-cytoskeletal interactions involve ligand binding (occupancy) to the integrin extracellular domain and clustering of the integrin. To construct mimics of the cytoplasmic face of an occupied and clustered integrin, we fused the cytoplasmic domains of integrin beta subunits to an N-terminal sequence containing four heptad repeat sequences. The heptad repeats form coiled coil dimers in which the cytoplasmic domains are parallel dimerized and held in an appropriate vertical stagger. In these mimics we found 1) that both conformation and protein binding properties are altered by insertion of Gly spacers C-terminal to the heptad repeat sequences; 2) that the cytoskeletal proteins talin and filamin are among the polypeptides that bind to the integrin beta1A tail. Filamin, but not talin binding, is enhanced by the insertion of Gly spacers; 3) binding of both cytoskeletal proteins to beta1A is direct and specific, since it occurs with purified talin and filamin and is inhibited in a point mutant (beta1A(Y788A)) or in splice variants (beta1B, beta1C) known to disrupt cytoskeletal associations of beta1 integrins; 4) that the muscle-specific splice variant, beta1D, binds talin more tightly than beta1A and is therefore predicted to form more stable cytoskeletal associations; and 5) that the beta7 cytoplasmic domain binds filamin better than beta1A. The structural specificity of these associations suggests that these mimics offer a useful approach for the analysis of the interactions and structure of the integrin cytoplasmic face.

Amino Acid Sequence↗
PubMed1998

Cooperation of a single lysine mutation and a C-terminal domain in the cytoplasmic sequestration of the p53 protein.

Cytoplasmic sequestration of the p53 tumor suppresser protein has been proposed as a mechanism involved in abolishing p53 function. However, the mechanisms regulating p53 subcellular localization remain unclear. In this report, we analyzed the possible existence of cis-acting sequences involved in intracellular trafficking of the p53 protein. To study p53 trafficking, the jellyfish green fluorescent protein (GFP) was fused to the wild-type or mutated p53 proteins for fast and sensitive analysis of protein localization in human MCF-7 breast cancer, RKO colon cancer, and SAOS-2 sarcoma cells. The wild-type p53/GFP fusion protein was localized in the cytoplasm, the nucleus, or both compartments in a subset of the cells. Mutagenesis analysis demonstrated that a single amino acid mutation of Lys-305 (mt p53) caused cytoplasmic sequestration of the p53 protein in the MCF-7 and RKO cells, whereas the fusion protein was distributed in both the cytoplasm and the nucleus of SAOS-2 cells. In SAOS-2 cells, the mutant p53 was a less efficient inducer of p21/CIP1/WAF1 expression. Cytoplasmic sequestration of the mt p53 was dependent upon the C-terminal region (residues 326-355) of the protein. These results indicated the involvement of cis-acting sequences in the regulation of p53 subcellular localization. Lys-305 is needed for nuclear import of p53 protein, and amino acid residues 326-355 can sequester mt p53 in the cytoplasm.

Amino Acid Sequence↗
PubMed1998

Filamin binds to the cytoplasmic domain of the beta1-integrin. Identification of amino acids responsible for this interaction.

Integrins play an important role in regulating cell adhesion, motility, and activation. In an effort to identify intracellular proteins expressed by activated T cells that interact with the cytoplasmic domain of beta1-integrin (CD29), we used the beta1-integrin cytoplasmic domain as bait in the yeast two-hybrid system. Here we report that the cytoplasmic domain of beta1-integrin specifically interacts with the cytoskeletal protein filamin. This interaction required all but the most carboxyl-terminal three residues of the cytoplasmic domain of beta1, and the carboxyl-terminal 477 residues of filamin containing the terminal 4. 5 approximately 96-residue tandem repeats of filamin. To verify this interaction in vivo, we showed that filamin specifically coprecipitated with beta1 in mammalian cells. We also showed that recombinant filamin chimeric proteins were able to bind to the beta1 cytoplasmic domain in vitro. We observed that a subset of single point mutations in the cytoplasmic domain of beta1, which had been previously reported to impair its function, disrupt the interaction between beta1 and filamin. Taken together, these findings suggest that the interaction between beta1 and filamin, which in turn can bind actin, provides a mechanism for the interaction of this cell surface receptor with cytoskeletal proteins and that this interaction plays a role in normal receptor function.

Amino Acid Sequence↗
PubMed2001

Binding of Paxillin to the alpha 9 Integrin Cytoplasmic Domain Inhibits Cell Spreading.

alpha(9)beta(1) integrin is a member of the beta(1) integrin family, plays an important role in extravasation of neutrophils at sites of acute inflammation, and is required for the normal development of the lymphatic system. The alpha(9) and alpha(4) integrin subunits are most closely related and form a subfamily of integrin alpha subunits. Previously, we have reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule. This interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion, including stimulation of cell migration and inhibition of cell spreading and focal adhesion formation. In the current studies, we have examined the interaction between the alpha(9) cytoplasmic domain and paxillin. Here we report that the alpha(9) cytoplasmic domain binds paxillin directly and tightly and that the alpha(9)-paxillin association inhibits cell spreading. We have identified amino acid residues in the alpha(9) cytoplasmic domain, Trp(999) and Trp(1001), that are critical for paxillin binding, and alanine substitution of either Trp(999) or Trp(1001) blocks paxillin binding. Furthermore, these mutations also reverse the effect of the alpha(9) cytoplasmic domain on cell spreading. Thus, the alpha(9) and alpha(4) integrin subunits form a paxillin-binding subfamily of integrin alpha subunits, and direct binding of paxillin to the alpha(9) cytoplasmic domain mediates some of the biological activities of the alpha(9)beta(1) integrin.

Amino Acid Sequence↗
PubMed2001

Phosphorylation-dependent interactions between ADAM15 cytoplasmic domain and Src family protein-tyrosine kinases.

The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are proline-rich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family protein-tyrosine kinases (PTKs) and the adaptor protein Grb2 in hematopoietic cells (Jurkat, THP-1, U937, and K562 cell lines). Src homology 3 domains from several Src family PTKs including Lck, Fyn, Abl, and Src associate with ADAM15 in vitro. Dephosphorylation of cell extracts resulted in decreased association of ADAM15 with Src family PTK SH3 domains, indicating that phosphorylation influences ADAM15 interactions with its binding partners. This was confirmed in vitro for Hck, Lck, and Grb2, which showed enhanced association with tyrosine-phosphorylated glutathione S-transferase-ADAM15 cytoplasmic domain compared with unphosphorylated protein. In contrast, binding of MAD2 to ADAM15 was slightly reduced by phosphorylation of the ADAM. Immunoprecipitation of ADAM15 from Jurkat cells confirmed the association with Lck in vivo, and upon PMA stimulation, the phosphorylation level of ADAM15 was increased. Cotransfection of ADAM15 and Hck showed Hck-dependent phosphorylation of ADAM15 in vivo. Hck, and to a lesser extent Lck, phosphorylated the ADAM15 cytoplasmic domain in vitro in immune complex kinase assays. Binding of ADAM15 cytoplasmic domain to Hck and Lck was also shown by Far Western analysis. In contrast to Hck, Lck activity was not required for binding to ADAM15, as shown by treatment of cells with PP1. Deletion and point mutation analysis of the ADAM15 cytoplasmic domain confirmed the importance of the proline-rich motifs for Grb2 and Lck binding and indicated the regulatory nature of Tyr(715) and Tyr(735). These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.

ADAM Proteins↗
PubMed2002

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3.

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.

Amino Acid Sequence↗
PubMed2003

Cytoplasmic p21Cip1 is involved in Ras-induced inhibition of the ROCK/LIMK/cofilin pathway.

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21(Cip1) was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21(Cip1) expression accompanied by restoration of phosphocofilin, which was not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. Our findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility thereby contributing to invasion and metastasis.

Actin Depolymerizing Factors↗
PubMed2003

Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells.

Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural beta-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47-57), as well as a similarly labeled 12-residue beta-peptide, beta-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47-57)) or nucleolus (beta-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47-57) and beta-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.

Active Transport, Cell Nucleus↗
PubMed2004

A dileucine motif and a cluster of acidic amino acids in the second cytoplasmic domain of the batten disease-related CLN3 protein are required for efficient lysosomal targeting.

The juvenile form of ceroid lipofuscinosis (Batten disease) is a neurodegenerative lysosomal storage disorder caused by mutations in the CLN3 gene. CLN3 encodes a multimembrane-spanning protein of unknown function, which is mainly localized in lysosomes in non-neuronal cells and in endosomes in neuronal cells. For this study we constructed chimeric proteins of three CLN3 cytoplasmic domains fused to the lumenal and transmembrane domains of the reporter proteins LAMP-1 and lysosomal acid phosphatase to identify lysosomal targeting motifs and to determine the intracellular transport and subcellular localization of the chimera in transfected cell lines. We report that a novel type of dileucine-based sorting motif, EEEX(8)LI, present in the second cytoplasmic domain of CLN3, is sufficient for proper targeting to lysosomes. The first cytoplasmic domain of CLN3 and the mutation of the dileucine motif resulted in a partial missorting of chimeric proteins to the plasma membrane. At equilibrium, 4-13% of the different chimera are present at the cell surface. Analysis of lysosome-specific proteolytic processing revealed that lysosomal acid phosphatase chimera containing the second cytoplasmic domain of CLN3 showed the highest rate of lysosomal delivery, whereas the C terminus of CLN3 was found to be less efficient in lysosomal targeting. However, none of these cytosolic CLN3 domains was able to interact with AP-1, AP-3, or GGA3 adaptor complexes. These data revealed that lysosomal sorting motifs located in an intramolecular cytoplasmic domain of a multimembrane-spanning protein have different structural requirements for adaptor binding than sorting signals found in the C-terminal cytoplasmic domains of single- or dual-spanning lysosomal membrane proteins.

Acid Phosphatase↗
PubMed2006

Cytoplasmic function of mutant promyelocytic leukemia (PML) and PML-retinoic acid receptor-alpha.

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) regulates major apoptotic and growth-suppressive pathways. In APL, PML is involved in a chromosomal translocation generating the PML-retinoic acid receptor-alpha (RARalpha) fusion protein. Two missense mutations in the remaining PML alleles have been identified, which give rise to a truncated cytoplasmic PML protein (Mut PML). APL patients carrying these mutations display resistance to retinoic acid (RA) and very poor prognosis. Here we show that Mut PML associates with the cytoplasmic regions we refer to as PML-cytoplasmic bodies (PML-CBs). Mut PML interacts with PML-RARalpha in PML-CB and potentiates PML-RARalpha-mediated inhibition of RA-dependent transcription. Remarkably, Mut PML stabilizes PML-RARalpha and inhibits differentiation induced by pharmacological doses of RA. A mutant form of PML-RARalpha that accumulates in the cytoplasm inhibits RA-dependent transcription and differentiation, thus suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we show that the bcr3 PML-RARalpha form is predominantly cytoplasmic and accumulates in PML-CBs. Taken together, these findings reveal novel insights into the molecular mechanisms contributing to APL.

Animals↗
PubMed1990

Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization.

Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is approximately 10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be approximately 20% as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.

Amino Acid Sequence↗
PubMed1992

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways.

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Cytoplasmic retention of Xenopus nuclear factor 7 before the mid blastula transition uses a unique anchoring mechanism involving a retention domain and several phosphorylation sites.

Xenopus nuclear factor 7 (xnf7) is a maternally expressed protein that belongs to the B-box zinc finger gene family consisting of transcription factors, protooncogenes, and ribonucleoproteins. Its function is regulated by retention in the cytoplasm from oocyte maturation until the mid blastula transition (MBT) when it reenters the nucleus. We defined a 22-amino acid cytoplasmic retention domain (CRD) in xnf7 that functioned cooperatively with two phosphorylation sites within the xnf7 molecule to retain the protein in the cytoplasm until the MBT. Deletion of this region or mutations in the phosphorylation sites resulted in the early entry of xnf7 into the nucleus. A mutation changing one of the phosphorylation sites to a glutamic acid resulted in the prolonged retention of the xnf7 protein in the cytoplasm until stages 9-10, well past the MBT. Additionally, a mutant form of xnf7 possessing a second nuclear localization signal at the COOH terminus was retained in the cytoplasm. This suggests that retention of xnf7 was not due to the masking of its NLS as is the case with NFkB and dorsal but was due to a novel anchoring mechanism in which the CRD interacts with an anchor protein. The CRD sequence is also found in another B-box zinc finger protein that is also retained in the cytoplasm until the MBT in the newt. Therefore, we believe that this may be an important mechanism whereby the function of a number of nuclear proteins is regulated during development.

Amino Acid Sequence↗