Abstract

Lysosomal Integral Membrane Protein II (LIMP II) is structurally related to the cell surface glycoprotein CD36. We recently defined a CD36 domain that binds adhesive glycoprotein thrombospondin 1 (TSP1). The CD36-TSP interaction plays a role in cell adhesion and ligand intrenalization. In solid phase assays, ¹²⁵I-TSP1 bound to LIMP II fusion protein LFP75-155 (aa75-155) corresponding to the CD36 TSP1 binding domain (TBD), but not to another region (aa156-243). Time-dependent binding was saturable, with an apparent K_d of ∼80nM, was blocked by TSP1 LFP75-155, or anti-L75-155, and was partially inhibited by EDTA, consistent with divalent cation-dependent TSP1 activity. Native CD36 and CD36 TBD fusion proteins competed with LFP75-15, confirming that LIMP II shares CD36 TSP1 receptor activity. Additionally, LFP75-155 bound ¹²⁵I-oxLDL, half maximal at ≈250[special characters omitted]g/ml, and was blocked by oxLDL, LFP75-155, or CD36 TBD. Inhibition by acLDL, but not native LDL, suggests that LIMP II may share CD36 scavenger receptor specificity. We conclude that structural homology correlates with functional similarity for at least two CD36 gene family members.

Expression of native LIMP II as an activation-dependent platelet surface markers was demonstrated by flow cytometry, consistent with our previous findings for Lysosome-Associated Membrane Protein-1 and -2 (LAMPs). Binding of exogenous ¹²⁵I-TSP1 to activated platelets was blocked by LFP75-155, suggesting that LIMP II could compete with other platelet TSP1 receptors, and provide additional binding sites. TSP1 receptor function was shown in melanoma cells expressing chimeric LIMP II/CD36, containing the extracytoplasmic domain of LIMP II coupled to the C-terminal cytoplasmic tail of CD36, targeted to the plasma membrane. LIMP II transfectants bound ¹²⁵I-TSP1, and adhered to TSP1-coated surfaces and activated platelets. Analysis of metastatic potential for LIMP II transfectants revealed accelerated growth, increased cell motility, and rapid spreading on TSP1-coated surfaces or activated platelets. These data suggest that surface LIMP II could serve as an authentic adhesion receptor, and may potentiate metastatic behavior.

Comparative sequence analysis of the TBD among CD36 gene family members identified a bipartite motif within CD36 exon V. Homology searches matched sequences in several known TSP receptors, and in numerous classes of other proteins. From this second group, ICAM-1 and HIV1 env were tested for TSP1 receptor activity. ¹²⁵I-TSP binding to ICAM-1 transfected fibroblasts was blocked by LFP75-155, and by anti-ICAM-1. HIV1 infectivity decreased when preincubated with TSP1, and inhibition was abrogated by LFP75-155. These results suggest that the structural domain defined by the CD36/LIMP II TBD constitutes a putative protein-protein recognition motif that may elucidate novel TSP-mediated interactions relevant to vascular, developmental, and tumor biology.