CD36 N-terminal cytoplasmic domain is not required for the internalization of oxidized low-density lipoprotein.
The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.
McDermott-Roe
Chris
C
Functional Genomics and Atherothrombosis Unit, Thrombosis Research Institute, London SW3 6LR, UK. chris.mcdermott-roe@imperial.ac.uk
Martin
Juliette
J
Collot-Teixeira
Sophie
S
McGregor
John L
JL
D018955:CD36 Antigens / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D002232:Carbocyanines / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology
D018955:CD36 Antigens; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D011993:Recombinant Fusion Proteins; C099252:oxidized low density lipoprotein; C024286:3,3'-dioctadecylindocarbocyanine