Abstract
The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.
Highlights
For more than a decade, adenovirus has been used as vector to transfer genes of interest into several tissues in both animals and humans
The complete intracellular tail of Coxsackievirus and adenovirus receptor (CAR)-1 was expressed as a fusion protein with the DNA binding domain (DBD) of the yeast transcription factor Gal4 by cloning into the yeast expression vector pGBT9
Yeast transformed with cDNA only that was able to grow on high-stringency selection plates, as well as yeast transformed with cDNAs that expressed proteins that bound to the Gal4 DNA binding domain, were eliminated from the screen
Summary
Yeast Expression Plasmids—pGBT9 and pACT-2 were purchased from Clontech. pGal4DBD—mCARic (DBD/CAR) (bait) harbors the Gal DNA binding domain fused to the complete intracellular tail of mouse CAR-1 and was constructed by PCR amplification of pBKCMVmCAR using primers 104 and 103. PHA/Wt p80 contains a double HA-tag and was constructed by transfer of an HA-Wt p80 fragment from the corresponding mammalian expression vector by EcoRI/Klenow/XhoI treatment into pACT-2 vector treated likewise. PHA/Wt p70 was constructed by transfer of a Wt p70 fragment from the mammalian expression vector pWt p70 treated with EcoRI/mungbean nuclease/XhoI into pACT-2 treated likewise. PHA/Wt p70 was cloned by excision of Wt p70 from pWt p70 by EcoRI/XhoI/mungbean nuclease treatment and ligation downstream of an HA-tag in a pcDNA3.1derived vector plasmid. To construct pWt p70, RT-nested PCR was performed using total RNA from mouse lung with the following primer pairs, A, Nos. 17 and 16; B, Nos. 122 and 91. The bait pGal4DBD—mCARic construct and the library were transformed sequentially into the yeast strain AH109 (Clontech), and the transformation mixture was plated on medium-stringency selection plates that lacked amino acids His, Leu, and Trp. In total, 3 ϫ 106 clones were screened. LipofectAMINE 2000 reagent (Invitrogen) was used for plasmid transfection of cells using conditions that were recommended by the manufacturer
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