MOLECULAR BIOLOGY OF THE HUMAN CD36 GENE

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Glycoprotein IV (CD36) has been identified as a platelet receptor for collagen and thrombospondin. CD36 also mediates cytoadherence of Plasmodium falciparum-parasitized erythrocytes, indicating a role in malariasequestration. In addition, CD36 deficiency has been correlated to a clinically observed transfusion phenotype designated Naka-negative. This diversity of function constitutes a basis for investigation of the CD36 gene, its expression, and its products. The principal goal of this proposal is to elucidate the coding and processing of CD36 on the molecular level. There are four specific aims: 1. Isolate and sequence the human CD36 gene. CD36 cDNA will be used to screen genomic libraries and purify clones corresponding to the CD36 coding sequence and its neighboring regions. Homologies to known exonintron boundaries and regulatory elements will be delineated by complete sequencing and sequence analysis. 2. Identify RNA processing intermediates and alternatively spliced products of the CD36 gene. cDNAs will be isolated, sequenced, and analyzed for evidence of precursor and alternatively spliced CD36 mRNAs. Rare mRNAs will be amplified by PCR to allow identification. S1 nuclease digestion of mRNA/DNA hybrids will be used to confirm alternative splicing events in CD36 expressing cell types. 3. Characterize promoter and other regulatory sequences for the CD36 gene. Transcriptional start sites will be determined by primer extension and S1 nuclease analysis coupled with sequence analysis. Suspected regulatory regions will be evaluated in reporter gene constructs transfected into CD36-expressing cell lines. Trans-acting factors will be detected by gel mobility shift assay and DNase I footprinting of the CD36 gene combined with nuclear extracts from CD36-expressing cell types. 4. Identify the molecular basis of the Naka-negative phenotype. CD36 appears deficient in Naka-negative platelets, although related lower molecular weight species are detected as well as CD36 mRNA. The origin of the deficiency will be correlated with the regulatory pathways defined for expression of normal CD36 protein. These studies will elucidate mechanisms of CD36 expression in normal megakaryocytes and other cells, identify any CD36 variants and define the molecular basis of the Naka-negative phenotype.