The recent identification of two novel missense mutations, S112F and T175A in human SR-BI, prompted us to characterize the functionality of these two variants of SR-BI that underlie elevated HDL-cholesterol levels in humans. Our data revealed that both mutant SR-BI receptors had significantly impaired abilities to bind HDL and mediate the selective uptake of HDL-COE. In addition to displaying defective efflux of FC to HDL, both mutants were also unable to redistribute the plasma membrane pools of FC. The impaired functions of these mutant receptors were not due to changes in oligomeric status. However, decreased cell surface expression and defective functions of T175A-SR-BI could be explained, in part, by an altered glycosylation status as compared to wild-type SR-BI.
The accumulation of HDL-cholesterol levels upon deletion of the SR-BI gene in mice 
strongly suggests that the athero-protective role of SR-BI 
is primarily due to its ability to mediate the selective uptake of CE from circulating HDL particles 
. Based on our in vitro studies, we suggest that inefficient clearance of HDL-C from circulation may explain the 37% increases in HDL-C (with no significant differences in triglycerides, LDL-cholesterol or body mass index) in the two individuals heterozygous for mutations at Ser112 or Thr175 compared to family member controls 
. As carriers of P297S-SR-BI, the first human SR-BI mutation, displayed similar increases in HDL-C levels 
, our data lend support to the notion that human SR-BI does indeed play a role in hepatic HDL-CE uptake.
The athero-protective effects of SR-BI can also be explained, in part, by its role in mediating FC efflux from lipid-laden macrophages to HDL, although this remains controversial 
. The reduced ability of S112F- and T175A-SR-BI mutant receptors to efflux FC to HDL mirrors similar trends observed with the P297S-SR-BI mutant receptor in mice 
. At least in the case of T175A-SR-BI, lower levels of FC efflux may correlate with its lower levels of expression at the cell surface 
. The inability of both mutant receptors to efficiently mediate cholesterol-transport functions may be associated, in part, with their inability to mediate plasma membrane reorganization of cholesterol, as we have previously observed with other SR-BI receptors harboring mutations in this region of the extracellular domain 
. More in-depth cholesterol-transport and membrane re-organization studies in macrophage or hepatocyte/hepatoma cell lines warrant further investigation and can provide sharper insight as to how these mutant SR-BI receptors behave when all efflux machinery (i.e. ABCA1 and ABCG1) is present.
Mutation of Thr175 to alanine appears to disrupt N-linked glycosylation at the evolutionarily-conserved Asn173 residue as judged by its faster migration as compared to the wild-type or S112F-SR-BI receptors following SDS-PAGE. It was previously demonstrated that N-linked oligosaccharide chains at Asn173 are a critical determinant of cell surface expression and efficient lipid uptake 
. In support of these observations, reduced cell surface expression of T175-SR-BI similarly correlates with reduced selective HDL-CE uptake efficiency. Although oligosaccharides on glycoproteins have been shown to participate in protein-protein interactions in other systems 
, our analyses indicated that SR-BI oligomerization is not affected by altered glycosylation status. Rather, it is likely that altered glycosylation in T175A-SR-BI and subsequent changes in cell surface expression result from either improper folding in the ER and consequent transport through the Golgi, or decreased receptor stability 
Mutation of Ser112 to Phe did not affect cell surface receptor expression, although it did significantly affect HDL-CE selective uptake efficiency. Interestingly, Ser112 lies adjacent to another N-linked glycosylation site (Asn108) that is critical for cell surface expression and function 
. It is certainly possible that introduction of a large amino acid like phenylalanine could impede the addition of oligosaccharides at Asn108, however an altered glycosylation status S112F-SR-BI was not evident from our immunoblot analyses. Alternatively, proper receptor-ligand interactions may be compromised by (i) altered secondary or tertiary structure of the area surrounding Ser112, (ii) the large nature of the phenyl side chain 
, or (iii) the inability of phenylalanine to participate in potential hydrogen bonding with residues on HDL proteins due to the lack of a hydroxyl group 
. Remarkably, both the S112F and T175A point mutations occur in conserved regions of hydrophobicity in the N-terminal half of the extracellular domain of SR-BI. We have previously shown that the hydrophobicity of this region is critical for SR-BI-mediated cholesterol transport function 
and hypothesized that this region may interact with the plasma membrane as has been reported for other integral membrane proteins 
. While mutation of Thr to Ala does not impose major effects on hydrophobicity, mutation of Ser to Phe (hydrophobic index of −0.8 and 2.8, respectively 
) significantly increases the hydrophobicity of the region surrounding residue 112. This leads us to speculate that the region surrounding the Phe residue becomes less solvent-exposed, and perhaps even more ‘buried’ in the membrane, thus preventing key conformational changes that may be required to support efficient selective uptake of HDL-CE. These ideas are currently being investigated.
Although human and rodent SR-BI share similar expression patterns, tissue abundance and in vitro receptor activities 
, the role of human SR-BI in hepatic clearance of HDL-CE and its relevance to human physiology has remained a mystery until recently. Not surprisingly, our functional characterization of these two newest human SR-BI mutations raises new questions. Due to inefficient clearance of plasma HDL particles, do carriers of the S112F or T175A mutations possess larger-sized HDL particles, similar to carriers of the P297S mutation 
, and SR-BI knock-out mice 
? Has the oxidation status and efflux capacity of circulating HDL (i.e. HDL ‘functionality’) been altered? Importantly, carriers of SR-BI mutations at Ser112, Thr175 or Pro297 do not currently show clinical signs of atherosclerosis. Therefore, identification and investigation of additional subjects with the same or new SR-BI mutations will be invaluable as we address the on-going debate as to whether higher HDL-C levels are truly athero-protective 
. Answers to these lingering questions are particularly relevant in light of current efforts directed at developing HDL-raising therapeutics to lower cardiovascular risk