RESUMEN
Epithelial polarity and tight junction formation limit the ability of adenovirus, retrovirus and adeno-associated virus (AAV) to deliver and express virally encoded genes. Using an extended half-life luciferase assay and high-throughput luminometry, we screened 23 000 compounds and natural product extracts as potentiators to overcome this barrier. Seven strong activators were discovered (up to several hundred fold above control) and two of these exhibited spectrum of activity in multiple cell types (HeLa (human cervical carcinoma), cystic fibrosis bronchial epithelial (human bronchial), HT29 (human colonic carcinoma), Calu3 (airway serous glandular)). Enhanced transduction by unrelated gene transfer vectors (adenovirus, lentivirus, AAV, liposomal) was also observed. These results establish a strategy for identifying compounds that improve viral gene transfer to resistant cell types, and provide new tools for examining epithelial defense against viral infection. The compounds should have broad usefulness in experimental therapies for cancer and genetic diseases.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Células Epiteliales/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Virus/genética , Adenoviridae/genética , Línea Celular , Terapia Combinada , Dependovirus/genética , Expresión Génica/efectos de los fármacos , Genes Virales , Ingeniería Genética , Vectores Genéticos/farmacología , Células HeLa , Humanos , Lentivirus/genética , Luciferasas/genética , PlásmidosRESUMEN
The biological functions of immunoglobulin (Ig)A antibodies depend primarily on their interaction with cell surface receptors. Four IgA receptors are presently characterized. The FcalphaRI (CD89) expressed by myeloid cells selectively binds IgA1 and IgA2 antibodies, whereas the poly-IgR, Fcalpha/muR, and asialoglycoprotein receptors bind other ligands in addition to IgA. IgA binding by mesangial cells, epithelial cells, and proliferating lymphocytes is also well documented, but the nature of the IgA receptors on these cells remains elusive. A monoclonal antibody (A24) is described here that specifically blocks IgA binding to epithelial and B lymphocyte cell lines. Both the A24 antibody and IgA1 myelomas bind a cell surface protein that is identified as the transferrin receptor (CD71). The transferrin receptor selectively binds IgA1 antibodies, monomeric better than polymeric forms, and the IgA1 binding is inhibitable by transferrin. Transferrin receptor expression is upregulated on cultured mesangial cells as well as on glomerular mesangial cells in patients with IgA nephropathy. The characterization of transferrin receptor as a novel IgA1 receptor on renal mesangial cells suggests its potential involvement in the pathogenesis of IgA nephropathy.
Asunto(s)
Mesangio Glomerular/metabolismo , Glomerulonefritis por IGA/metabolismo , Receptores Fc/metabolismo , Receptores de Transferrina/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Masculino , Ratas , Regulación hacia ArribaRESUMEN
A biopanning process designed to find peptide epitopes specific for cell surface receptors has been used in this study to select seven- and 12-amino-acid peptides capable of binding to and internalizing with the human transferrin receptor (hTfR). Through sequential rounds of negative and positive selection, two peptide sequences were identified that specifically bind to the hTfR. Phage containing the sequences HAIYPRH or THRPPMWSPVWP were inhibited from binding the hTfR in a dose-dependent fashion when peptides of the same sequence were present in a competition assay. Interestingly, transferrin did not compete with either of these sequences for receptor binding, suggesting that these peptides bind a site on the hTfR distinct from the transferrin binding site. When either of these sequences was expressed as a fusion to green fluorescent protein (GFP), the recombinant GFP molecule was internalized in cells expressing the hTfR. These studies suggest that the two peptides can be used to target other proteins into the endosomal pathway. Further, they provide a strategy for identifying peptides that bind to other cell surface receptors that can be used for both diagnostic and therapeutic purposes.
Asunto(s)
Péptidos/metabolismo , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , Embrión de Pollo , Electroforesis en Gel de Poliacrilamida , Endocitosis , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia MolecularRESUMEN
How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the "signal" within the TM necessary and sufficient for down-regulation is Thr(11)Gln(17)Thr(19) (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well ( approximately 79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/química , Endocitosis/fisiología , Antígenos de Histocompatibilidad Clase II/química , Receptores de Superficie Celular/metabolismo , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos B/efectos de los fármacos , Antígenos de Diferenciación de Linfocitos B/fisiología , Embrión de Pollo , Reactivos de Enlaces Cruzados/farmacología , Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Fibroblastos , Semivida , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/fisiología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Transferrina/química , Receptores de Transferrina/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaAsunto(s)
Sistema Nervioso Central/inmunología , Regulación de la Expresión Génica , Genes MHC Clase II/genética , Proteínas Nucleares , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/patología , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Enfermedades Autoinmunes Desmielinizantes SNC/inmunología , Enfermedades Autoinmunes Desmielinizantes SNC/metabolismo , Enfermedades Autoinmunes Desmielinizantes SNC/patología , Genes MHC Clase II/inmunología , Humanos , Neuroglía/citología , Neuroglía/inmunología , Neuroglía/metabolismo , Neuroglía/patología , Regiones Promotoras Genéticas/genética , Transactivadores/metabolismoRESUMEN
The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.
Asunto(s)
Endocitosis/fisiología , Inhibidores Enzimáticos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Transferrina/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Quinasa de la Caseína II , Embrión de Pollo , Secuencia de Consenso , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endocitosis/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/síntesis química , Péptidos/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Receptores de Transferrina/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192-5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2-78) and carboxyl-terminal tail regions (residues 1391-1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2-78) and CFTR-(1391-1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440-1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had approximately 40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Endocitosis , Señales de Clasificación de Proteína/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Cartilla de ADN , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/química , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein-protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl- currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant DeltaF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.
Asunto(s)
Antígenos de Superficie/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Proteínas del Tejido Nervioso/fisiología , Animales , Antígenos de Superficie/metabolismo , Células COS , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Células LLC-PK1 , Mutación , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Porcinos , Sintaxina 1 , XenopusRESUMEN
The invariant chain (Ii) targets newly synthesized major histocompatibility complex class II complexes to a lysosome-like compartment. Previously, we demonstrated that both the cytoplasmic tail (CT) and transmembrane (TM) domains of Ii were sufficient for this targeting and that the CT contains two di-leucine signals, 3DQRDLI8 and 12EQLPML17 (Odorizzi, C. G., Trowbridge, I. S., Xue, L., Hopkins, C. R., Davis, C. D., and Collawn, J. F. (1994) J. Cell Biol. 126, 317-330). In the present study, we examined the relationship between signals required for endocytosis and those required for lysosomal targeting by analyzing Ii-transferrin receptor chimeras in quantitative transport assays. Analysis of the Ii CT signals indicates that although 3DQRDLI8 is necessary and sufficient for endocytosis, either di-leucine signal is sufficient for lysosomal targeting. Deletions between the two signals reduced endocytosis without affecting lysosomal targeting. Transplantation of the DQRDLI sequence in place of the EQLPML signal produced a chimera that trafficked normally, suggesting that this di-leucine sequence coded for an independent structural motif. Structure-function analysis of the Ii TM region showed that when Ii TM residues 11-19 and 20-29 were individually substituted for the corresponding regions in the wild-type transferrin receptor, lysosomal targeting was dramatically enhanced, whereas endocytosis remained unchanged. Our results therefore demonstrate that the structural requirements for Ii endocytosis and lysosomal targeting are different.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/química , Endocitosis/fisiología , Antígenos de Histocompatibilidad Clase II/química , Lisosomas/fisiología , Secuencia de Aminoácidos , Cloruro de Amonio/farmacología , Animales , Embrión de Pollo , Humanos , Cinética , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutación/genética , Receptores de Transferrina/química , Proteínas Recombinantes de Fusión/química , Relación Estructura-ActividadRESUMEN
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). Each chimera was expressed on the cell surface and rapidly internalized. Metabolic pulse-chase experiments showed that the CD3 gamma-chain and lysosomal acid phosphatase chimeras, unlike the lysosomal-associated membrane glycoprotein chimera, were rapidly degraded in a post-Golgi compartment following normal glycosylation. Transplantation of signals from CD3 gamma-chain and lysosomal acid phosphatase into the TR cytoplasmic tail in place of the native signal, Y20TRF23, indicated that each signal was sufficient to promote endocytosis but not lysosomal targeting of the resulting mutant. Transplantation of two CD3 signals at specific sites in the TR cytoplasmic tail or a single tyrosine-based signal in a truncated TR tail, however, was sufficient to promote lysosomal targeting. Our results therefore suggest that the relative position of the signal within the cytoplasmic tail is a critical feature that distinguishes lysosomal targeting signals from internalization signals.
Asunto(s)
Lisosomas/fisiología , Receptores de Transferrina/química , Proteínas Recombinantes de Fusión/química , Fosfatasa Ácida/química , Secuencia de Aminoácidos , Cloruro de Amonio/farmacología , Animales , Antígenos CD/química , Complejo CD3/química , Embrión de Pollo , Endocitosis/fisiología , Fibroblastos , Humanos , Inmunohistoquímica , Hierro/farmacocinética , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Tirosina/metabolismoRESUMEN
Poliovirus genomes have been constructed in which the capsid genes have been substituted with the murine gene encoding interleukin-2 (IL-2) (referred to as replicons). One replicon contained the gene for IL-2 in place of the poliovirus capsid VP2 and VP3 genes, and a second replicon was constructed that contained the murine IL-2 substituted for the poliovirus VP3 and VP1 genes. The IL-2 genes were cloned into the replicon so as to maintain the translational reading frame with the remaining poliovirus proteins. Transfection of either replicon into cells resulted in the expression of replicon-encoded proteins and replication of replicon RNA. Using a procedure developed in this laboratory, we have encapsidated these replicons into authentic polio virions by passaging the replicons in the presence of a recombinant vaccinia virus, VVP1, which expresses the capsid precursor, P1, protein. Using a quantitative immunoassay, we determined that the majority of the IL-2 produced remained intracellular, with approximately 1%-2% released from the infected cells, and that the IL-2 was biologically active. The results of these studies demonstrate the utility of poliovirus replicons for expression of small bioactive molecules and are discussed with respect to future applications as immune adjuvants as well as potential new tumor therapies.
Asunto(s)
Cápside/genética , Regulación Viral de la Expresión Génica/fisiología , Genoma Viral , Interleucina-2/genética , Poliovirus/genética , Replicón/genética , Animales , ADN Recombinante/genética , Glicoproteínas/genética , Células HeLa , Humanos , Ratones , Señales de Clasificación de Proteína/genéticaRESUMEN
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. Four of 16 chimeric human/chicken transferrin receptors expressed in chick embryo fibroblasts were efficiently transported to the plasma membrane and displayed on the cell surface. Studies of the four chimeric receptors indicated that binding of human transferrin was abolished if the carboxy terminal 192 amino acids of the human transferrin receptor (residues 569-760) were replaced with the corresponding region from the chicken transferrin receptor. Further, a chimeric receptor in which the carboxy-terminal 72 residues were derived from the chicken transferrin receptor exhibited a 16-fold decrease in binding affinity for human transferrin. In contrast, analysis of the other two chimeric receptors showed that 340 amino acids of the human transferrin receptor external domain more proximal to the transmembrane region (residues 151-490) could be replaced with the corresponding region from the chicken transferrin receptor without loss of high-affinity ligand binding. In contrast, two mAbs against the human transferrin receptor external domain, B3/25 and D65.3, that do not compete with transferrin binding, do not bind the chimeric transferrin receptors in which the membrane proximal part is replaced by chicken sequences, while they do bind the two other chimeric transferrin receptors with high affinity. These data indicate that sequence differences in the carboxy-terminal region of human and chicken transferrin receptor external domains are important for the species specificity of transferrin binding and imply that this portion of the human transferrin receptor is critical for ligand binding.
Asunto(s)
Receptores de Transferrina/metabolismo , Animales , Anticuerpos Monoclonales , Sitios de Unión , Células Cultivadas , Embrión de Pollo , Pollos , Humanos , Inmunoquímica , Técnicas In Vitro , Ligandos , Reacción en Cadena de la Polimerasa , Receptores de Transferrina/química , Receptores de Transferrina/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Transferrina/metabolismoRESUMEN
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. Leu7 and Ile8 were required for the activity of the signal most distal to the cell membrane whereas Pro15 Met16 Leu17 were important for the membrane-proximal signal. The same or overlapping non-tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80% of the Ii-TR chimera containing the Ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the Ii sorting signals are efficiently recognized by sorting machinery located in the trans-Golgi.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B , Proteínas Aviares , Endocitosis/fisiología , Aparato de Golgi/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Señales de Clasificación de Proteína/fisiología , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Membrana Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Citoplasma/metabolismo , Fibroblastos , Antígenos de Histocompatibilidad Clase II/análisis , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Hierro/metabolismo , Glicoproteínas de Membrana/análisis , Datos de Secuencia Molecular , Receptores de Transferrina/análisis , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia/genética , Eliminación de Secuencia/fisiologíaRESUMEN
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., Kuhn, L. A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J.A. (1990) Cell 63, 1061-1072). It has been inferred from other studies, however, that the TR internalization signal was localized to a much larger region, residues 7 through 26 (Girones, N., Alvarez, E., Seth, A., Lin, I-M., Latour, D.A., and Davis, R.J. (1991) J. Biol. Chem. 266, 19006-19012). Additionally, Tyr20 was reported to not be conserved in the Chinese hamster cytoplasmic tail sequence (Alvarez, E., Girones, N., and Davis, R.J. (1990) Biochem. J. 267, 31-35). In the studies reported here, we examined the effect of insertion of an extra copy of a YTRF sequence at three different locations within the human TR cytoplasmic domain and show that the insertion of another YTRF signal at position 31-34 in the wild-type TR, but not the other two locations, increases the rate of endocytosis 2-fold. Furthermore, introduction of YTRF at position 31-34 in an internalization-defective mutant receptor restores endocytosis to wild-type levels, indicating that YTRF signals at either positions 20-23 or 31-34 are necessary and sufficient to promote TR internalization and function in an independent and additive manner. We also report the complete primary structure of the Chinese hamster TR deduced from its cDNA sequence and show that the Tyr20 as well as the complete YTRF motif is conserved.
Asunto(s)
Endocitosis , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Embrión de Pollo , Secuencia Conservada , Cricetinae , Cartilla de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/metabolismo , Receptores de Transferrina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
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. QQGFF and the deduced sequence of the region encompassing residues 973-991 (973FYRAL) could effectively replace the endogenous endocytic code of the transferrin receptor (YTRF). FYRAL and YTRF were less active than QQGFF when substituted into region 993-1022 of the EGF receptor, but a synthetic sequence (NNAYF), predicted to have structural features of a tight turn, effectively replaced QQGFF for EGF receptor internalization. Whereas EGF receptor sequences functioned effectively in the transferrin receptor, function of these sequences in the EGF receptor was strictly dependent on intrinsic tyrosine kinase activity as demonstrated kinetically and by immunofluorescence using semithin cryosections. Ligand-dependent endocytosis and down-regulation of the EGF receptor thus require multiple sequence motifs that are exchangeable between ligand-dependent and -independent receptors, but that require intrinsic tyrosine kinase activity for function in the context of the EGF receptor.
Asunto(s)
Endocitosis , Receptores ErbB/metabolismo , Tirosina , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/efectos de los fármacos , Receptores ErbB/genética , Técnica del Anticuerpo Fluorescente , Humanos , Cinética , Células L , Ligandos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , TransfecciónAsunto(s)
Endocitosis , Proteínas de la Membrana/metabolismo , Orgánulos/fisiología , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Clatrina/fisiología , Aparato de Golgi/fisiología , Lisosomas/fisiología , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/fisiologíaRESUMEN
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. These data indicate that internalization signals are interchangeable self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. Putative internalization signals in the cytoplasmic tails of other receptors and membrane proteins can be identified based on the sequence patterns of the LDLR, Man-6-PR, and TR signals. Two such putative four-residue internalization signals, one from the poly-Ig receptor and one from the asialoglycoprotein receptor, were tested for activity by transplantation into TR and were found to promote high efficiency internalization. These results suggest that an exposed tight turn is the conformational motif for high efficiency endocytosis.
Asunto(s)
Endocitosis , Receptores de Transferrina/química , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Fibroblastos/metabolismo , Humanos , Hierro/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Transferrina/genética , Receptores de Transferrina/fisiología , Relación Estructura-Actividad , TransfecciónRESUMEN
The internalization signals of several constitutively recycling receptors have recently been identified as regions of four or six amino acids that include an aromatic residue, usually tyrosine. Here, we show that transplanted signals from the low density lipoprotein receptor (LDLR) and cation-independent mannose-6-phosphate receptor (Man-6-PR) promote rapid internalization of the transferrin receptor (TR), directly establishing that recognition signals are interchangeable, self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. We also show that the chemical and spatial patterns of critical residues in both four- and six-residue internalization motifs are consistent with a tight turn structure. A six-residue LDLR signal is needed for activity in TR, suggesting that an amino-terminal aromatic side chain is obligatory. In contrast, the carboxy-terminal aromatic side chain in the TR signal can be replaced by a large hydrophobic residue. Thus, internalization signals apparently require an aromatic amino-terminal residue and either an aromatic or large hydrophobic carboxy-terminal residue rather than a conserved tyrosine per se. Consistent with this conclusion, predicted internalization signals from the poly-Ig receptor, YSAF, and asialoglycoprotein receptor (ASGPR) subunit H1, YQDL, also promote internalization of TR.
Asunto(s)
Endocitosis , Manosafosfatos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de LDL/metabolismo , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Receptor de Asialoglicoproteína , Humanos , Hierro/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Receptor IGF Tipo 2 , Receptores Inmunológicos/metabolismo , Receptores de Transferrina/genética , Transferrina/metabolismoRESUMEN
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.
Asunto(s)
Endocitosis , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Embrión de Pollo , Deleción Cromosómica , Citoplasma/metabolismo , Fibroblastos/metabolismo , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores de Transferrina/genética , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
The conformations of two 17-residue peptide analogues derived from the C-terminal sequence of pigeon cytochrome c (native sequence = KAERADLIAYLKQATAK) were examined in aqueous and lipid environments by CD spectroscopy. The two analogues, KKLLKKLIAYLKQATAK (K peptide) and EELLEELIAYLKQATAK (E peptide), were made amphipathic with respect to helical segregation by substituting a 6-residue sequence at the N-terminus of the native peptide. Their structures were compared to the native peptide under aqueous conditions of varying pH and temperature, and in the presence of liposomes composed of phosphatidylcholine and phosphatidylserine in the ratio of 9:1. The results indicated that the native peptide remains unstructured under all the conditions examined even though this region of the native molecule is surface exposed and helical. The E peptide, however, was helical under aqueous conditions at 25 degrees C from pH 2-10 with a maximum helicity at pH 4 (54% helix from analysis of CD data). The ellipticity of the E peptide at pH 4 and 8 was concentration dependent, indicating an aggregation phenomenon. In studies in which the CD spectrum was measured at different temperatures, the E peptide became more helical at lower temperatures at pH 4 but not at pH 8. Upon interaction with a lipid membrane in the form of liposomes, there appeared to be a slight destabilization in the structure of the E peptide. The K peptide in an aqueous environment behaved like the native peptide in that it was structureless at all pHs and temperatures examined. In the presence of liposomes, however, this peptide had a high helical content (75% helix from analysis of CD data). These findings suggest that while stabilization of the helix dipole with negative charges at the N-terminus are important in inducing helical conformation in the E peptide, hydrophobic interactions created during aggregation appear to provide the principal stabilizing force. The results with the K peptide demonstrate that the positive N-terminal sequence of this peptide is able to interact with the negatively charged head groups in the phospholipid membrane in such a fashion as to stabilize a helical structure that is not apparent in an aqueous environment alone.