Coding

Part:BBa_K1470000

Designed by: Pascal Sartor   Group: iGEM14_Freiburg   (2014-10-06)

Ecotropic murine leukemia virus (MuLV) receptor / Cationic amino acid transporter 1 (CAT-1)

Natural Function

The cationic amino acid transporter 1 (CAT-1) is part of the CAT family which is a subfamily of the solute carrier family 7 (SLC7). These solute carriers are expressed ubiquitously and build the main entry gate for amino acids such as histidine, arginine or ornithin in mammalian cells. They enable the influx of their substrate independent of Na+. Additionally it was shown that the absence of CAT-1 leads to non-viable mice pubs [1][2].

Structure and virus recognition

CAT-1 is a 66 kDa membrane protein. It is built out of up to 622 amino acids contains 14 transmembrane domains which resolutes in seven extracellulare and eight intracellulare domains. There are two sites for N-glycosylation in the third extracellulare loop. The glycosyled position is rather significant for the entering by the virus. The murine leukeamia virus is only able to enter the cell, when it detects the CAT-1 sugar-bound moieties[3].


Scheme of mCAT-1. Members of the CAT family are predicted to have
14 transmembrane domains with intracellular N- and C-termini.
Two asparagine residues in the third extracellular loop (indicated as branched lines)
have been shown to be glycosylated [7].

More information

A comparison between CAT-1 sequences from different species like rats and hamsters shows as well that this region does not include conserved amino acids making a virus infection impossible [4]. The mouse CAT-1 was originally identified by Albritton in 1989 as the receptor for murine ecotropic leukemia viruses (MuLV) [5]. It was shown that these cells could be infected by the MuLV in the presence of mCAT-1 on the surface of mouse cells. However, human cells acquire the susceptibility to infection by MuLV only if the cells express mCAT-1 ectopically. Studies by Albritton et al. have shown that amino acids in the extracellular loop three of mCAT-1 are critical for virus binding [6].


Receptor expression

To provide optimal conditions for viral infection, the best point in time for transduction with the largest number of receptors present on the cell surface was determined. On that account HEK-293T cells had been transfected with CAT-1 fused with a HA-tag. Cells expressing CAT-1 were analyzed after distinct incubation times.


Expression time of the receptor that was transfected into HEK-293T cells. After transfection with mCAT-1-HA cells were lysed with RIPA buffer at distinct points of time. A western blot was performed using an anti-HA antibody.


We found out that the expression of the receptor peaked at 24 hours after transfection. In later experiments we performed viral infections past that timeframe based on these results.

Localisation of CAT-1

We wanted to detect the localisation of CAT-1 and fused mCherry to its N-terminus. Using confocal microscopy we varified not only the existance of the protein on the cell surface but showed in a spatial way via several sectional planes how the receptore is expressed by HEK-293T cells.

HEK-293T cells transfected with mCAT-1-mCherry (p14rz_005). Confocal pictures were taken with an 100x oil immersion objective (NA 1.40). Nuclear staining (DAPI) is shown in blue and the mCAT-1-mCherry in red.

More information

Confocal pictures were taken with a 20x plan apo objective. Nuclear staining (DAPI) is shown in blue and the mCAT-1-mCherry in red.

More information

Please click on the link below to view a spatial resolution of CAT-1 in a HEK-293T cell observing the presence of CAT-1 on the surface or inside the cells, like Golgi apparatus or endoplasmic reticulum



References

[1] Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. J. W. Kim, E. I. Closs, L. M. Albritton, J. M. Cunningham Nature. 1991 August 22; 352(6337): 725–728.
[2] Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1. Perkins CP, Mar V, Shutter JR, del Castillo J, Danilenko DM, Medlock ES, Ponting IL, Graham M, Stark KL, Zuo Y, Cunningham JM, Bosselman RA. Genes Dev. 1997 Apr 1;11(7):914-25.
[3] Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses. Albritton LM, Kim JW, Tseng L, Cunningham JM. J Virol. 1993 Apr;67(4):2091-6.
[4] Second site mutation in the virus envelope expands the host range of a cytopathic variant of Moloney murine leukemia virus, John Ferrarone, Ryan C. Knoper, Randolph Li, Christine A. Kozak, Virology. 2012 November 10; 433(1): 7–11
[5] Naturally Occurring Polymorphisms of the Mouse Gammaretrovirus Receptors CAT-1 and XPR1 Alter Virus Tropism and Pathogenicity,Christine A. Kozak, Adv Virol. 2011
[6] Albritton LM, Tseng L, Scadden D, Cunningham JM (1989). A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666.
[7] Louis J. Ignarro, Nitric Oxide: Biology and Pathobiology (2009).

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 77
    Illegal NgoMIV site found at 238
    Illegal NgoMIV site found at 1477
    Illegal AgeI site found at 1386
  • 1000
    COMPATIBLE WITH RFC[1000]


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