Abstract
Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (∼ 2-fold). Gly(18) and Gly(25) were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G(18)X(3)AX(2)G(25) in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a "lattice effect" (interligand steric interference accompanying binding to adjacent receptors).
Highlights
SR-BI is a member of the CD36 superfamily of proteins in metazoans (ϳ25–30% sequence identities)
We identified a conserved glycine dimerization motif, G18X3AX2G25, in the N-terminal transmembrane (N-TM) domain of SR-BI that plays a major role in mediating receptor homo-oligomerization
The N-TM domain of SR-BI might contribute to homo-oligomerization by interacting directly with the N-TM domains on other SR-BI molecules, with its C-TM domain, or with both
Summary
Lipoproteins—Human HDL was isolated as eight separate density fractions using KBr gradient centrifugation from freshly harvested, EDTA-treated plasma as described previously [32, 55, 56] (see supplemental materials for additional details). Co-immunoprecipitation—COS M6 cells in 6-well dishes were grown and transfected (4 g of total plasmid DNA/well) as described above using either the empty vector pcDNA4/TO or wild-type SR-BI or triple glycine mutant G15L/G18L/G25L expression vectors encoding receptors in which the C termini bear either rho or myc epitope tag extensions (see above). Specific activities of SR-BI mutants were expressed as the percentage of wild-type SR-BI activity measured in the same experiment These values were corrected by the relative levels of surface expression determined using flow cytometry and the KKB-1 anti-SR-BI antibody as described above. Cells transfected with the empty vector only were used to determine background staining and to set the receptor-specific surface expression gate in which mean values of DiI fluorescence intensity were measured These values, defined as DiI uptake activity, are expressed as the percentage of wild-type SR-BI activity (100%) measured in the same experiment. Statistical analysis for the comparison between multiple SR-BI mutants/chimeras was performed with one-way analysis of variance and Tukey post-test at 95% confidence intervals
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