The terminal six amino-acids of the carboxy cytoplasmic tail of CD36 contain a functional domain implicated in the binding and capture of oxidized low-density lipoprotein.
CD36, a major adhesion molecule expressed by monocytes/macrophages, plays a key role in the binding and internalization of oxidized low-density lipoprotein (OxLDL). This adhesion molecule, a member of an important scavenger receptor family, contains a very short C-terminal cytoplasmic tail that is known to induce intracellular signalling events. However, the domains on the cytoplasmic tail involved in such signal transduction are unknown. In this study, we have investigated the functional components of the cytoplasmic tail by site-directed mutagenesis coupled with functional OxLDL and monoclonal antibody (mAb) binding studies. Seven truncated or punctual CD36 constructs, localized in the cytoplasmic tail, were produced by site-directed mutagenesis. Each construct was stably expressed in HEK293 cells. We used a quantitative and a qualitative method, labelling OxLDL with either iodine or rhodamine, to determine the functional importance of the cytoplasmic domains in OxLDL internalization. Results indicate that: (1) a deletion of the last amino-acid (construct K472STOP) significantly reduces, compared with wild-type, the binding, internalization and degradation of OxLDL; (2) truncation of the last six amino-acids (construct R467STOP) significantly reduces OxLDL binding; (3) the above two constructs (K472STOP and R467STOP) showed a reduced rate of OxLDL internalization compared with wild-type; (4) the binding and rate of internalization of an anti-CD36 monoclonal antibody (10/5) was not affected by the above mentioned mutants (K472STOP and R467STOP), compared with wild-type. This study shows, for the first time, a specific site on the CD36 cytoplasmic tail that is critical for the binding, endocytosis and targeting of OxLDL.
Malaud
Eric
E
INSERM U331, Faculty of Medecine RTH Laënnec, 8 Rue Guillaume Paradin, 69372 Lyon Cedex 08, France.
Hourton
Delphine
D
Giroux
Louise Marie
LM
Ninio
Ewa
E
Buckland
Robin
R
McGregor
John L
JL
D000595:Amino Acid Sequence; D018955:CD36 Antigens / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D018955:CD36 Antigens; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein