The coxsackie B virus and adenovirus receptor resides in a distinct membrane microdomain

Abstract

The coxsackie B virus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily. In addition to activity as a viral receptor, it may play a role in cellular adhesion. We asked what determines the cell membrane microdomain of CAR. We found that CAR is localized to a novel lipid-rich microdomain similar to that of the low-density lipoprotein receptor (LDLR) but distinct from that of a CAR variant that exhibited traditional lipid raft localization via fusion to a glycosylphosphatidylinositol (GPI) tail. The cytoplasmic tail determines its membrane localization, since deletion of this domain resulted in mislocalization. Results indicate that CAR, CAR-LDLR, and LDLR reside in a novel lipid raft that is distinct from caveolin-1-containing caveolae and GPI-linked proteins. Residence in a lipid-rich domain provides a mechanism that allows CAR to interact with other cell adhesion proteins and yet function as an adenovirus receptor.

FIG. 1.

TEM of Ad bound to COS cells. Ad was allowed to bind to COS cells for 1 h on ice followed by 5 min (panels A and B) or 20 min (panels C to G) of incubation at 37°C. Cells were then fixed and processed for TEM. Ad entering via CCP as well as caveolae can be observed. Arrows identify Ad associated with clathrin-containing structures, while arrowheads indicate nonclathrin structures. Size bar, 100 nm.

FIG. 2.

(A) Percent Ad-β-Gal infection in COS cells treated with 10 mM cyclodextrin (CD) or 100 μM chlorpromazine (CH) or left untreated (Ad Control). *, P < 0.01, n = 3 to 4 experiments. Confocal microscopy of endocytic events after coincubation of A488-transferrin (green) and Cy3-Ad (red) (B) or A488-cholera toxin (green) and Cy3-Ad (red) (C). Regions of green (marker alone), red (Ad alone), and yellow (both marker and Ad) were visible after treatment with either transferrin or cholera toxin. The lower insets in panels B and C show an XZ projection of the Ad particles associated with the cell. (D) Confocal image of wt CAR (red) patched at 12°C with FLAG Ab and anti-mouse A568 followed by fixation and staining with A488-cholera toxin as a marker for the cholera toxin receptor GM1 (green).

FIG. 3.

Western blotting results for clathrin, wt CAR, and caveolin-1 after nondetergent Na₂CO₃ (A) or Triton X-100 (B) extraction followed by sucrose gradient fractionation. Blots were routinely divided into three sections prior to immunoblotting. Additional flotillin and TR controls were analyzed on separate blots. (C) Endogenous CAR in control COS-7 cells analyzed by both nondetergent (i) and detergent (ii) extraction. (D) CAR in primary polarized human airway epithelia analyzed by both nondetergent (i) and detergent (ii) extraction.

FIG. 4.

Comparison of immunofluorescence of wt CAR (panels A and E), GPI-CAR (panels B and F), CAR-LDLR (panels C and G) and Tailless-CAR (panels D and H) in transiently transfected unpermeabilized (panels A to D) and Triton X-100-permeabilized (panels E to H) COS-7 cells. Three additional representative fields are included for each condition. A schematic diagram of each of the variants of CAR is inserted in the top right corner of panels A to D.

FIG. 5.

Extraction and ultracentrifugation of the variants of CAR by nondetergent Na₂CO₃ extraction (A) or detergent Triton X-100 extraction (B). Panel C shows a comparison of transfected human LDLR after nondetergent and detergent extractions.

FIG. 6.

Comparison of Ad infections in CAR-deficient CHO cells complemented with wt CAR or CAR-LDLR. (A) CAR-deficient cells complemented with wt CAR or CAR-LDLR result in identical Ad infections. Transfected cells were infected with Ad-GFP at an MOI of 5, immunostained for CAR or CAR-LDLR, and analyzed by FACS. Data shown are the numbers of events testing positive in both of the green and red channels out of 10,000 events (n = 3). (B) A representative experiment showing that CAR-deficient cells complemented with wt CAR-eGFP or CAR-LDLR-eGFP result in similar Ad infections. Cells were infected with Ad-RFP at various MOI and analyzed by FACS. The regions shown in green represent cells expressing CAR-GFP or CAR-LDLR-GFP and not infected with Ad-RFP. The regions in red show cells expressing CAR-GFP or CAR-LDLR-GFP and infected with Ad-RFP. The regions in black represent untransfected, uninfected cells. (C) Graphical depiction of data averaged from six experiments as represented by panel B. The y axis represents the percentage of cells testing positive in both of the green and red channels out of total transfected (green) cells.