Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei.

We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.

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Molecular and biochemical characterization of the human trk proto-oncogene.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.24 ◽

1989 ◽

Vol 9 (1) ◽

pp. 24-33 ◽

Cited By ~ 394

Author(s):

D Martin-Zanca ◽

R Oskam ◽

G Mitra ◽

T Copeland ◽

M Barbacid

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Tyrosine Kinase ◽

Biochemical Characterization ◽

Transmembrane Domain ◽

Cell Surface Receptor ◽

Kinase Gene ◽

Mature Form ◽

Amino Terminal

Molecular analysis of the human trk oncogene, a transforming gene isolated from a colon carcinoma biopsy, revealed the existence of a novel member of the tyrosine kinase gene family. This locus, which we now designate the trk proto-oncogene, codes for a protein of 790 amino acid residues that has several features characteristic of cell surface receptors. They include (i) a 32-amino-acid-long putative signal peptide, (ii) an amino-terminal moiety (residues 33 to 407) rich in consensus sites for N-glycosylation, (iii) a transmembrane domain, (iv) a kinase catalytic region highly related to that of other tyrosine kinases, and (v) a very short (15 residue) carboxy-terminal tail. Residues 1 to 392 were absent in the trk oncogene, as they were replaced by tropomyosin sequences. However, no other differences were found between the transforming and nontransforming trk alleles (residues 392 to 790), suggesting that no additional mutations are required to activate the transforming potential of this gene. The human trk proto-oncogene codes for a 140,000-dalton glycoprotein, designated gp140proto-trk. However, its primary translational product is a 110,000-dalton glycoprotein which becomes immediately glycosylated, presumably during its translocation into the endoplasmic reticulum. This molecule, designated gp110proto-trk, is further glycosylated to yield the mature form, gp140proto-trk. Both gp110proto-trk and gp140proto-trk proteins possess in vitro kinase activity specific for tyrosine residues. Finally, iodination of intact NIH 3T3 cells expressing trk proto-oncogene products indicated that only the mature form, gp140proto-trk, cross the plasma membrane, becoming exposed to the outside of the cell. These results indicate that the product of the human trk locus is a novel tyrosine kinase cell surface receptor for an as yet unknown ligand.

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Molecular and biochemical characterization of the human trk proto-oncogene

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.24-33.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 24-33

Author(s):

D Martin-Zanca ◽

R Oskam ◽

G Mitra ◽

T Copeland ◽

M Barbacid

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Tyrosine Kinase ◽

Biochemical Characterization ◽

Transmembrane Domain ◽

Cell Surface Receptor ◽

Kinase Gene ◽

Mature Form ◽

Amino Terminal

Molecular analysis of the human trk oncogene, a transforming gene isolated from a colon carcinoma biopsy, revealed the existence of a novel member of the tyrosine kinase gene family. This locus, which we now designate the trk proto-oncogene, codes for a protein of 790 amino acid residues that has several features characteristic of cell surface receptors. They include (i) a 32-amino-acid-long putative signal peptide, (ii) an amino-terminal moiety (residues 33 to 407) rich in consensus sites for N-glycosylation, (iii) a transmembrane domain, (iv) a kinase catalytic region highly related to that of other tyrosine kinases, and (v) a very short (15 residue) carboxy-terminal tail. Residues 1 to 392 were absent in the trk oncogene, as they were replaced by tropomyosin sequences. However, no other differences were found between the transforming and nontransforming trk alleles (residues 392 to 790), suggesting that no additional mutations are required to activate the transforming potential of this gene. The human trk proto-oncogene codes for a 140,000-dalton glycoprotein, designated gp140proto-trk. However, its primary translational product is a 110,000-dalton glycoprotein which becomes immediately glycosylated, presumably during its translocation into the endoplasmic reticulum. This molecule, designated gp110proto-trk, is further glycosylated to yield the mature form, gp140proto-trk. Both gp110proto-trk and gp140proto-trk proteins possess in vitro kinase activity specific for tyrosine residues. Finally, iodination of intact NIH 3T3 cells expressing trk proto-oncogene products indicated that only the mature form, gp140proto-trk, cross the plasma membrane, becoming exposed to the outside of the cell. These results indicate that the product of the human trk locus is a novel tyrosine kinase cell surface receptor for an as yet unknown ligand.

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Phosphorylation of a major GPI-anchored surface protein of Trypanosoma brucei during transport to the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.112.11.1785 ◽

1999 ◽

Vol 112 (11) ◽

pp. 1785-1795 ◽

Cited By ~ 4

Author(s):

P. Butikofer ◽

E. Vassella ◽

S. Ruepp ◽

M. Boschung ◽

G. Civenni ◽

...

Keyword(s):

Cell Surface ◽

Trypanosoma Brucei ◽

Surface Protein ◽

Surface Expression ◽

Surface Coat ◽

Placental Alkaline Phosphatase ◽

Flagellar Pocket ◽

Phosphorylated Form ◽

Human Placental Alkaline Phosphatase

The surface coat of procyclic forms of Trypanosoma brucei consists of related, internally repetitive glycoproteins known as EP and GPEET procyclins. Previously we showed that the extracellular domain of GPEET is phosphorylated. We now show that phosphorylation of this glycosylphosphatidylinositol-anchored surface protein can be induced in vitro using a procyclic membrane extract. Using antibodies that recognize either the phosphorylated or unphosphorylated form of GPEET, we analyzed their expression during differentiation of bloodstream forms to procyclic forms. Unphosphorylated GPEET, together with EP, was detected in cell lysates 2–4 hours after initiating differentiation whereas phosphorylated GPEET only appeared after 24 hours. Surface expression of EP and both forms of GPEET occurred after 24–48 hours and correlated with the detection of phosphorylated GPEET on immuno-blots. Electron micrographs showed that unphosphorylated GPEET was predominantly in the flagellar pocket whereas the phosphorylated form was distributed over the cell surface. In contrast, expression of a membrane-bound human placental alkaline phosphatase in procyclic forms caused the accumulation of dephosphorylated GPEET on the cell surface, while the phosphorylated form was restricted to the flagellar pocket. A GPEET-Fc fusion protein, which was retained intracellularly, was not phosphorylated. We propose that unphosphorylated GPEET procyclin is transported to a location close to or at the cell surface, most probably the flagellar pocket, where it becomes phosphorylated. To the best of our knowledge, this study represents the first localization of phosphorylated and unphosphorylated forms of a GPI-anchored protein within a cell.

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Sequence Requirements for Trafficking of the CRAM Transmembrane Protein to the Flagellar Pocket of African Trypanosomes

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.5149-5163.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 5149-5163 ◽

Cited By ~ 10

Author(s):

Hong Yang ◽

David G. Russell ◽

Baijing Zheng ◽

Manami Eiki ◽

Mary Gwo-Shu Lee

Keyword(s):

Amino Acids ◽

Cell Surface ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Surface Glycoprotein ◽

Cell Surface Expression ◽

Flagellar Pocket ◽

African Trypanosomes ◽

Cytoplasmic Extension ◽

C Terminus

ABSTRACT CRAM is a cysteine-rich acidic transmembrane protein, highly expressed in the procyclic form of Trypanosoma brucei. Cell surface expression of CRAM is restricted to the flagellar pocket of trypanosomes, the only place where receptor mediated endocytosis takes place in the parasite. CRAM can function as a receptor and was hypothesized to be a lipoprotein receptor of trypanosomes. We study mechanisms involved in the presentation and routing of CRAM to the flagellar pocket of insect- and bloodstream-form trypanosomes. By deletional mutagenesis, we found that deleting up to four amino acids from the C terminus of CRAM did not affect the localization of CRAM at the flagellar pocket. Shortening the CRAM protein by 8 and 19 amino acids from the C terminus resulted in the distribution of the CRAM protein in the endoplasmic reticulum (ER) (the CRAM protein is no longer uniquely sequestered at the flagellar pocket). This result indicates that the truncation of the CRAM C terminus affected the transport efficiency of CRAM from the ER to the flagellar pocket. However, when CRAM was truncated between 29 and 40 amino acids from the C terminus, CRAM was not only distributed in the ER but also located to the flagellar pocket and spread to the cell surface and the flagellum. Replacing the CRAM transmembrane domain with the invariant surface glycoprotein 65-derived transmembrane region did not affect the flagellar pocket location of CRAM. These results indicate that the CRAM cytoplasmic extension may exhibit two functional domains: one domain near the C terminus is important for efficient export of CRAM from the ER, while the second domain is of importance for confining CRAM to the flagellar pocket membrane.

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Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

Molecular Biology of the Cell ◽

10.1091/mbc.11.8.2643 ◽

2000 ◽

Vol 11 (8) ◽

pp. 2643-2655 ◽

Cited By ~ 37

Author(s):

Lolita Zaliauskiene ◽

Sunghyun Kang ◽

Christie G. Brouillette ◽

Jacob Lebowitz ◽

Ramin B. Arani ◽

...

Keyword(s):

Cell Surface ◽

Transmembrane Domain ◽

Gradient Centrifugation ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Surface Proteins ◽

Down Regulation ◽

Surface Receptors ◽

Receptor Complexes ◽

Polar Residues

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 Thr11Gln17Thr19 (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 (∼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.

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Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter

Nature ◽

10.1038/352729a0 ◽

1991 ◽

Vol 352 (6337) ◽

pp. 729-731 ◽

Cited By ~ 279

Author(s):

Hao Wang ◽

Michael P. Kavanaugh ◽

R. Alan North ◽

David Kabat

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Basic Amino Acid ◽

Amino Acid Transporter ◽

Cell Surface Receptor ◽

Surface Receptor ◽

Acid Transporter

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Divergent Traits and Ligand-Binding Features of the Cytomegalovirus CD48 Gene Family

Proceedings ◽

10.3390/proceedings2020050023 ◽

2020 ◽

Vol 50 (1) ◽

pp. 23

Author(s):

Pablo Martinez-Vicente ◽

Domènec Farré ◽

Elena Gracia-Latorre ◽

Pablo Engel ◽

Ana Angulo

Keyword(s):

Cell Surface ◽

Nk Cell ◽

Viral Evolution ◽

Costimulatory Molecule ◽

Surface Protein ◽

Gene Families ◽

Biochemical Properties ◽

Cell Surface Receptor ◽

Type I ◽

Independent Functions

The genesis of gene families through the capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a cell surface protein with an ectodomain composed of two immunoglobulin (Ig) domains. Via its N-terminal Ig domain, CD48 interacts with the cell surface receptor 2B4, triggering signal transduction events that regulate leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, derived from a common host CD48 ancestor gene acquired by retrotranscription. Recently, we examined one member of this family, A43, showing that it acts as a functional viral decoy receptor, binding with high affinity and stability to 2B4 and impairing NK-cell cytotoxicity. Here, we have characterized the rest of the vCD48s. We show that they are highly glycosylated type I transmembrane proteins that display remarkably distinct features: dissimilar structures (e.g., different size stalks and cytoplasmic tails), biochemical properties, locations (cell surface/soluble), and temporal kinetic classes. We found that, in contrast to A43, none of them interacts with 2B4. Consistent with this, the molecular modeling of the N-terminal Ig domains of these vCD48s evidences significant changes in the numbers and lengths of their β-strands and inter-sheet loops that participate in the interaction of CD48 with 2B4. This suggests that these vCD48s have diverged to perform new 2B4-independent functions. Interestingly, we determined that one of them, S30, tightly binds CD2, a T- and NK-cell adhesion and costimulatory molecule whose primary ligand is CD58. Thus, altogether, our results show how a key host immune receptor captured by a virus can be subsequently remodeled during viral evolution to yield new molecules with improved affinities to their cognate receptors or with shifted binding specificities to additional immune targets, expanding the repertoire of viral immunoevasins.

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Determinants of Tetherin Antagonism in the Transmembrane Domain of the Human Immunodeficiency Virus Type 1 Vpu Protein

Journal of Virology ◽

10.1128/jvi.01699-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12958-12970 ◽

Cited By ~ 97

Author(s):

Raphaël Vigan ◽

Stuart J. D. Neil

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Cell Surface ◽

Human Immunodeficiency Virus Type ◽

Transmembrane Domain ◽

Human Tetherin ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Tetherin Antagonism

ABSTRACT Tetherin (BST2/CD317) potently restricts the particle release of human immunodeficiency virus type 1 (HIV-1) mutants defective in the accessory gene vpu. Vpu antagonizes tetherin activity and induces its cell surface downregulation and degradation in a manner dependent on the transmembrane (TM) domains of both proteins. We have carried out extensive mutagenesis of the HIV-1 NL4.3 Vpu TM domain to identify three amino acid positions, A14, W22, and, to a lesser extent, A18, that are required for tetherin antagonism. Despite the mutants localizing indistinguishably from the wild-type (wt) protein and maintaining the ability to multimerize, mutation of these positions rendered Vpu incapable of coimmunoprecipitating tetherin or mediating its cell surface downregulation. Interestingly, these amino acid positions are predicted to form one face of the Vpu transmembrane alpha helix and therefore potentially contribute to an interacting surface with the transmembrane domain of tetherin either directly or by modulating the conformation of Vpu oligomers. While the equivalent of W22 is invariant in HIV-1/SIVcpz Vpu proteins, the positions of A14 and A18 are highly conserved among Vpu alleles from HIV-1 groups M and N, but not those from group O or SIVcpz that lack human tetherin (huTetherin)-antagonizing activity, suggesting that they may have contributed to the adaption of HIV-1 to human tetherin.

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Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Journal of Bacteriology ◽

10.1128/jb.00893-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7549-7555 ◽

Cited By ~ 28

Author(s):

Dustin L. Johnson ◽

James B. Mahony

Keyword(s):

Amino Acid ◽

Type Iii Secretion ◽

Transmembrane Domain ◽

Integral Membrane Protein ◽

Kinase Domain ◽

Chlamydophila Pneumoniae ◽

Structural Protein ◽

E Coli ◽

Type Iii ◽

Layer Chromatography

ABSTRACT Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.

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